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Byline: A. Vral, H. Thierens, A. Baeyens, L. De Ridder The development of biomarkers of susceptibility or sensitivity towards ionising radiation is important for the identification of individuals who may be at increased risk for the development of cancer after occupational, environmental or medical exposures. In this study we investigated the inter- and intra-individual variation of the MN assay and the G2 assay in irradiated lymphocytes to assess their suitability as biomarkers of susceptibility. For this, the G2 assay and the MN assay were performed on blood samples of 15 healthy individuals. For the MN assay Go lymphocytes were exposed to 3.5 Gy Co I[sup.3]-rays either at high dose-rate (HDR) or low dose-rate (LDR). For the G2 assay lymphocytes were irradiated with a dose of 0.4 Gy Co I[sup.3]-rays in G2 phase of the cell cycle. Two individuals were assayed nine times each in nine different experiments over a time period of one year. All samples were analysed by two scorers and no significant differences between them were observed using a paired t-test. The repeat experiments on blood samples of the same donor revealed that the inter-experimental/intra-individual coefficients of variation were not significantly different from the inter-individual coefficients of variation in both G2 and MN assay. As the intra-individual variability determines the assay reproducibility this would indicate that the assays are not able to detect real, reproducible differences in radiation sensitivity between normal individuals in the population. The repeat experiments further revealed that for some healthy donors a high value, defined as sensitive taking the 90 percentile as cut-off point to define sensitivity, is obtained only at one time point while the values obtained at the other time points were within the normal range (non-sensitive). Based on this one time point the individual would have been regarded as sensitive. Furthermore a donor identified as radiosensitive with the G2 assay in our initial experiments turned out to be normal when the assay was repeated. To conclude, our results show that a chromosomal aberration assay based on one blood sample may lead to erroneous conclusions with respect to the individual radiosensitivity of workers.

Byline: H. Thierens, B. Aousalah, A. Vral, L. De Ridder, M. Barbe The micronucleus (MN) assay already applied to the biomonitoring of radiation workers is a relatively easy and fast technique allowing assessment of the individual chromosomal radiosensitivity status of radiation workers on a large scale. A pilot study was set up to evaluate the chromosomal radiosensitivity in a group of 99 male radiation workers from the nuclear power plant Doel in Belgium. Heparinized blood samples of the workers were divided into three parts: one served for the assessment of a possible effect of occupational exposure on the number of micronuclei, the two others served for the assessment of individual chromosomal radiosensitivity by the increase of the number of micronuclei after 1 and 2 Gy in vitro I[sup.3]-irradiation. For each worker, date of birth, smoking habit and previous medical exposures were registered. Furthermore the external radiation burden accumulated over the last 10 years was calculated from the personnel dosimetry records. Concerning the MN frequency as a biomarker for effect, the present study confirms the strong increase of MN frequency with age and the tendency of an increase with an accumulated radiation burden, observed in previous studies. Using the MN frequency after in vitro irradiation as a biomarker for chromosomal radiosensitivity, the data of the present study do not support a lowered radiosensitivity in radiation workers due to the adaptive response mechanism induced by occupational exposure. As the overall in vitro radiation induced MN frequency distributions can be represented by a normal distribution without a high frequency tail, none of the workers can be considered as exceptionally by radiosensitive with the test. Further validation of the MN assay as a radiosensitivity assay for a population of workers is necessary.

As hematopoietic stem and progenitor cells (HSPCs) self-renew throughout life, accumulation of genomic alterations can potentially give rise to radiation carcinogenesis. In this study we examined DNA double-strand break (DSB) induction and repair as well as mutagenic effects of ionizing radiation in CD34+ cells and T lymphocytes from the umbilical cord of newborns. The age dependence of DNA damage repair end points was investigated by comparing newborn T lymphocytes with adult peripheral blood T lymphocytes. As umbilical cord blood (UCB) contains T lymphocytes that are practically all phenotypically immature, we examined the radiation response of separated naive (CD45RA+) and memory (CD45RO+) T lymphocytes. The number of DNA DSBs was assessed by microscopic scoring of γ-H2AX/53BP1 foci 0.5 h after low-dose radiation exposure, while DNA repair was studied by scoring the number of residual γ-H2AX/53BP1 foci 24 h after exposure. Mutagenic effects were studied by the cytokinesis block micronucleus (CBMN) assay. No significant differences in the number of DNA DSBs induced by low-dose (100-200 mGy) radiation were observed among the three different cell types. However, residual γ-H2AX/53BP1 foci levels 24 h postirradiation were significantly lower in CD34+ cells compared to newborn T lymphocytes, while newborn T lymphocytes showed significantly higher foci yields than adult T lymphocytes. No significant differences in the level of radiation-induced micronuclei at 2 Gy were observed between CD34+ cells and newborn T lymphocytes. However, newborn T lymphocytes showed a significantly higher number of micronuclei compared to adult T lymphocytes. These results confirm that CD34+ cell quiescence promotes mutagenesis after exposure. Furthermore, we can conclude that newborn peripheral T lymphocytes are significantly more radiosensitive than adult peripheral T lymphocytes. Using the results from the comparative study of radiation-induced DNA damage repair end points in naive (CD45RA+) and memory (CD45RO+) T lymphocytes, we could demonstrate that the observed differences between newborn and adult T lymphocytes can be explained by the immunophenotypic change of T lymphocytes with age, which is presumably linked with the remodeling of the closed chromatin structure of naive T lymphocytes. [ABSTRACT FROM AUTHOR]

In the event of a mass casualty radiation incident, the gamma-H2AX foci assay could be a useful tool to estimate radiation doses received by individuals. The rapid processing time of blood samples of just a few hours and the potential for batch processing, enabling high throughput, make the assay ideal for early triage categorisation to separate the 'worried well' from the low and critically exposed by quantifying radiation-induced foci in peripheral blood lymphocytes. Within the RENEB framework, 8 European laboratories have taken part in the first European gamma-H2AX biodosimetry exercise, which consisted of a telescoring comparison of 200 circulated foci images taken from 8 samples, and a comparison of 10 fresh blood lymphocyte samples that were shipped overnight to participating labs 4 or 24 h post-exposure. Despite large variations between laboratories in the dose- response relationship for foci induction, the obtained results indicate that the network should be able to use the gamma-H2AX assay for rapidly identifying the most severely exposed individuals within a cohort who could then be prioritised for accurate chromosome dosimetry. [ABSTRACT FROM AUTHOR]