High Resolution and Automatable Cytogenetic Biodosimetry Using In Situ Telomere and Centromere Hybridization for the Accurate Detection of DNA Damage: An Overview.

In the event of a radiological or nuclear accident, or when physical dosimetry is not available, the scoring of radiation-induced chromosomal aberrations in lymphocytes constitutes an essential tool for the estimation of the absorbed dose of the exposed individual and for effective triage. Cytogenetic biodosimetry employs different cytogenetic assays including the scoring of dicentrics, micronuclei, and translocations as well as analyses of induced premature chromosome condensation to define the frequency of chromosome aberrations. However, inherent challenges using these techniques include the considerable time span from sampling to result, the sensitivity and specificity of the various techniques, and the requirement of highly skilled personnel. Thus, techniques that obviate these challenges are needed. The introduction of telomere and centromere (TC) staining have successfully met these challenges and, in addition, greatly improved the efficiency of cytogenetic biodosimetry through the development of automated approaches, thus reducing the need for specialized personnel. Here, we review the role of the various cytogenetic dosimeters and their recent improvements in the management of populations exposed to genotoxic agents such as ionizing radiation. Finally, we discuss the emerging potentials to exploit these techniques in a wider spectrum of medical and biological applications, e.g., in cancer biology to identify prognostic biomarkers for the optimal triage and treatment of patients.

A Central Role of Telomere Dysfunction in the Formation of a Unique Translocation within the Sub-Telomere Region Resulting in Duplication and Partial Trisomy.

Telomeres play a major role in maintaining genome stability and integrity. Putative involvement of telomere dysfunction in the formation of various types of chromosomal aberrations is an area of active research. Here, we report a case of a six-month-old boy with a chromosomal gain encompassing the 11q22.3q25 region identified by SNP array analysis. The size of the duplication is 26.7 Mb and contains 170 genes (OMIM). The duplication results in partial trisomy of the region in question with clinical consequences, including bilateral renal dysplasia, delayed development, and a heart defect. Moreover, the karyotype determined by R-banding and chromosome painting as well as by hybridization with specific sub-telomere probes revealed the presence of an unbalanced t(9;11)(p24;q22.3) translocation with a unique breakpoint involving the sub-telomere region of the short arm of chromosome 9. The karyotypes of the parents were normal. Telomere integrity in circulating lymphocytes from the child and from his parents was assessed using an automated high-throughput method based on fluorescence in situ hybridization (FISH) with telomere- and centromere-specific PNA probes followed by M-FISH multicolor karyotyping. Very short telomeres, as well as an increased frequency of telomere loss and formation of telomere doublets, were detected in the child's cells. Interestingly, similar telomere profiles were found in the circulating lymphocytes of the father. Moreover, an assessment of clonal telomere aberrations identified chromosomes 9 and 11 with particularly high frequencies of such aberrations. These findings strongly suggest that telomere dysfunction plays a central role in the formation of this specific unbalanced chromosome rearrangement via chromosome end-to-end fusion and breakage-fusion-bridge cycles.

Telomere and Centromere Staining Followed by M-FISH Improves Diagnosis of Chromosomal Instability and Its Clinical Utility.

Dicentric chromosomes are a relevant marker of chromosomal instability. Their appearance is associated with telomere dysfunction, leading to cancer progression and a poor clinical outcome. Here, we present Telomere and Centromere staining followed by M-FISH (TC+M-FISH) for improved detection of telomere dysfunction and the identification of dicentric chromosomes in cancer patients and various genetic syndromes. Significant telomere length shortening and significantly higher frequencies of telomere loss and deletion were found in the peripheral lymphocytes of patients with cancer and genetic syndromes relative to similar age-matched healthy donors. We assessed our technique against conventional cytogenetics for the detection of dicentric chromosomes by subjecting metaphase preparations to both approaches. We identified dicentric chromosomes in 28/50 cancer patients and 21/44 genetic syndrome patients using our approach, but only 7/50 and 12/44, respectively, using standard cytogenetics. We ascribe this discrepancy to the identification of the unique configuration of dicentric chromosomes. We observed significantly higher frequencies of telomere loss and deletion in patients with dicentric chromosomes (p < 10-4). TC+M-FISH analysis is superior to classical cytogenetics for the detection of chromosomal instability. Our approach is a relatively simple but useful tool for documenting telomere dysfunction and chromosomal instability with the potential to become a standard additional diagnostic tool in medical genetics and the clinic.

A new tool for genotoxic risk assessment: Reevaluation of the cytokinesis-block micronucleus assay using semi-automated scoring following telomere and centromere staining.

BACKGROUND: The cytokinesis-block micronucleus (CBMN) assay is an internationally recognized method for measuring DNA damage after exposure to genotoxic agents, as well as a biomarker for DNA repair and chromosomal instability. The high baseline level of micronuclei (MN) in the healthy population has limited the sensitivity and application of the CBMN assay for the follow-up of exposed populations. We reevaluated the sensitivity of the CBNM assay using semi-automated MN scoring following telomere and centromere (TC) staining after in vitro exposure to genotoxic agents (mitomycin or radiation) or aneugenic agents (vinblastine). MATERIALS AND METHODS: Blood samples from 12 healthy donors were exposed to 137Cs at seven doses from 0.1-4 Gy and cultured for 72 h. Cytochalasin B was added at 46 h of culture. The exposure of chemical agents (mitomycin or vinblastine) was performed after 48 h of culture for 3 h. Cytochalasin B was added after treatment and slides were prepared 24 h after. MN was semi-automatically scored following TC staining. Nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) were assessed in a human cell line after TC staining. RESULTS: The introduction TC staining to the scoring of MN not only renders MN scoring more efficient and robust, but also permits discrimination between exposure to clastogenic (MN with only telomere signals) and aneugenic agents (MN with both TC signals). The resulting improvement of MN detection led to an increase in the sensitivity of the CBMN assay following low-dose radiation exposure (0.3 versus 0.1 Gy). Hyperradiosensitivity phenomenon was observed after low dose exposure. A dose-response curve was obtained for up to 4 Gy. In addition, TC staining permits assessment of the nature of NPBs and NBUDs as biomarkers for genotoxicity and chromosomal instability. CONCLUSION: These approaches can be potentially used to follow-up populations exposed to genotoxic agents and assess cancer risk.

Twenty years of FISH-based translocation analysis for retrospective ionizing radiation biodosimetry.

PURPOSE: The fluorescent in situ hybridization (FISH) technique, which easily detects reciprocal translocations, is currently used to estimate doses in retrospective biological dosimetry, after suspected accidental overexposure to ionizing radiation (IR). This study of 42 cases aimed to verify the appropriateness of this assay for radiation dose reconstruction, compared to the dicentric assay, and to evaluate other limitations. MATERIAL AND METHODS: We labeled chromosomes 2, 4, and 12 by 3-color FISH painting to detect translocations on lymphocytes of patients with suspected past IR overexposure. RESULT: Translocation dose estimation showed doses significantly different from 0 Gy in 25 of the 42 cases. The lowest positive dose measured was 0.3 Gy. Several months after IR exposure, the doses measured by translocation and dicentric assays are quite similar. For a year, dose estimation by translocation assay becomes more relevant as dicentric frequency starts to decrease, coming close to 0 for more than a year after the exposure. The persistence of translocations enabled us to corroborate an overexposure 44 years earlier. Interpretation of the observed translocation yield requires the knowledge of the patient's other radiation exposures. A dose assessment by this biomarker is relevant only if the radiation exposure is confirmed. CONCLUSIONS: This technique is appropriate for corroborating a former IR exposure of individuals. When the radiation dose is greater than 1 Gy, the translocations in complex exchanges must be considered. Another relevant point is the use of an appropriate background yield of translocations. The dose assessment, however, also depends on exposure to various genotoxic agents besides IR. If no evidence about the existence of radiation exposure is available, dose assessment is not useful. For this reason, report only the translocation frequency and its comparison with the background yield by age class is preferable.

Operational guidance for radiation emergency response organisations in Europe for using biodosimetric tools developed in EU MULTIBIODOSE project.

In the event of a large-scale radiological emergency, the triage of individuals according to their degree of exposure forms an important initial step of the accident management. Although clinical signs and symptoms of a serious exposure may be used for radiological triage, they are not necessarily radiation specific and can lead to a false diagnosis. Biodosimetry is a method based on the analysis of radiation-induced changes in cells of the human body or in portable electronic devices and enables the unequivocal identification of exposed people who should receive medical treatment. The MULTIBIODOSE (MBD) consortium developed and validated several biodosimetric assays and adapted and tested them as tools for biological dose assessment in a mass-casualty event. Different biodosimetric assays were validated against the 'gold standard' of biological dosimetry-the dicentric assay. The assays were harmonised in such a way that, in an emergency situation, they can be run in parallel in a network of European laboratories. The aim of this guidance is to give a concise overview of the developed biodosimetric tools as well as how and when they can be used in an emergency situation.

Detection of partial-body exposure to ionizing radiation by the automatic detection of dicentrics.

In accidental exposure to ionizing radiation, it is essential to estimate the dose received by the victims. Currently dicentric scoring is the best biological indicator of exposure. The standard biological dosimetry procedure (500 metaphases scored manually) is suitable for a few dose estimations, but the time needed for analysis can be problematic in the case of a large-scale accident. Recently, a new methodology using automatic detection of dicentrics has greatly decreased the time needed for dose estimation and preserves the accuracy of the estimation. However, the capability to detect nonhomogeneous partial-body exposures is an important advantage of dicentric scoring-based biodosimetry, and this remains to be tested with automatic scoring. Thus we analyzed the results obtained with in vitro blood dilutions and in real cases of accidental exposure (partial- or whole-body exposure) using manual scoring and automatic detection of dicentrics. We confirmed that automatic detection allows threefold quicker dicentric scoring than the manual procedure with similar dose estimations and uncertainty intervals. The results concerning partial-body exposures were particularly promising, and homogeneously exposed samples were correctly distinguished from heterogeneously exposed samples containing 5% to 75% of blood irradiated with 2 Gy. In addition, the results obtained for real accident cases were similar whatever the methodology used. This study demonstrates that automatic detection of dicentrics is a credible alternative for recent and acute cases of whole- and partial-body accidental exposures to ionizing radiation.

Quantification of gamma-H2AX foci in human lymphocytes: a method for biological dosimetry after ionizing radiation exposure.

Recent studies have suggested that visualization of gamma-H2AX nuclear foci can be used to estimate exposure to very low doses of ionizing radiation. Although this approach is widely used for various purposes, its suitability for individual human biodosimetry has not yet been assessed. We therefore conducted such an assessment with the help of available software for observing and automatically scoring gamma-H2AX foci. The presence of gamma-H2AX foci was evaluated in human peripheral blood lymphocytes exposed ex vivo to gamma rays in a dose range of 0.02 to 2 Gy. We analyzed the response of gamma-H2AX to ionizing radiation in relation to dose, time after exposure, and individual variability. We constructed dose-effect calibration curves at 0.5, 8 and 16 h after exposure and evaluated the threshold of detection of the technique. The results show the promise of automatic gamma-H2AX scoring for a reliable assessment of radiation doses in a dose range of 0.6 Gy to 2 Gy up to 16 h after exposure. This gamma-H2AX-based assay may be useful for biodosimetry, especially for triage to distinguish promptly among individuals the ones who have received negligible doses from those with significantly exposures who are in need of immediate medical attention. However, additional in vivo experiments are needed for validation.

Broad modulation of gene expression in CD4+ lymphocyte subpopulations in response to low doses of ionizing radiation.

To compare the responses of the different lymphocyte subtypes after an exposure of whole blood to low doses of ionizing radiation, we examined variations in gene expression in different lymphocyte subpopulations using microarray technology. Blood samples from five healthy donors were independently exposed to 0 (sham irradiation), 0.05 and 0.5 Gy of ionizing radiation. Three and 24 h after exposure, CD56+, CD4+ and CD8+ cells were negatively isolated. RNA from each set of experimental conditions was competitively hybridized on 25k oligonucleotide microarrays. Modifications of gene expression were measured after both intervals and in all cell types. Twenty-four hours after exposure to 0.5 Gy, we observed an induction of the expression of BAX, PCNA, GADD45, DDB2 and CDKN1A. However, the numbers of modulated genes greatly differed between cell types. In particular, 3 h after exposure to doses as low as 0.05 Gy, the number of down-modulated genes was 10 times greater for CD4+ cells than for all other cell types. Moreover, most of these repressed genes were taking part in the cell processes of protein biosynthesis and oxidative phosphorylation. The results suggest that several biological pathways in CD4+ cells could be sensitive to low doses of radiation. Therefore, specifically studying CD4+ cells could help to understand the mechanisms involved in low-dose response and allow their detection.

Neuro-inflammatory response in rats chronically exposed to (137)Cesium.

After the Chernobyl nuclear accident, behavioural disorders and central nervous system diseases were frequently observed in populations living in the areas contaminated by (137)Cs. Until now, these neurological disturbances were not elucidated, but the presence of a neuro-inflammatory response could be one explanation. Rats were exposed for 3 months to drinking water contaminated with (137)Cs at a dose of 400Bqkg(-1), which is similar to that ingested by the population living in contaminated areas in the former USSR countries. Pro-inflammatory and anti-inflammatory cytokine genes were assessed by real-time PCR in the frontal cortex and the hippocampus. At this level of exposure, gene expression of TNF-alpha and IL-6 increased in the hippocampus and gene expression of IL-10 increased in the frontal cortex. Concentration of TNF-alpha, measured by ELISA assays, was also increased in the hippocampus. The central NO-ergic pathway was also studied: iNOS gene expression and cNOS activity were significantly increased in the hippocampus. In conclusion, this study showed for the first time that sub-chronic exposure with post-accidental doses of (137)Cs leads to molecular modifications of pro- and anti-inflammatory cytokines and NO-ergic pathway in the brain. This neuro-inflammatory response could contribute to the electrophysiological and biochemical alterations observed after chronic exposure to (137)Cs.

Modifications of inflammatory pathways in rat intestine following chronic ingestion of depleted uranium.

The environmental contamination by dispersion of depleted uranium (DU) might result in its chronic ingestion of DU by local populations. The aim of this study was to determine if chronic ingestion of DU at low doses induces inflammatory reactions in intestine, first biological system exposed to uranium after ingestion. Experiments were performed with rats receiving uranium in drinking water (40 mg/l) during 3, 6, or 9 months. Several parameters referring to prostaglandin, histamine, cytokine, and nitric oxide (NO) pathways were assessed in ileum. Concerning the prostaglandin pathway, a twofold increase in gene expression of cyclooxygenase of type 2 was noted after 6 months, with no changes in prostaglandins levels. At the same time, a decrease in mast cell number was observed without any changes in histamine levels. Experiments on cytokines showed increased gene expression of interleukin (IL)-1beta and IL-10 at 6 months, and decreased messenger RNA level of CCL-2. This change was associated with decreased macrophage density. An opposite effect of DU was induced on neutrophils, since increased number was observed at 3 (x1.7) and 9 months (x3). The results obtained on NO pathway seemed to indicate that DU exposure inhibited this pathway (decreased endothelial NO synthase messenger RNA, inductive NO synthase activity and NO(2)(-)/NO(3)(-) levels) at 6 months. In conclusion, this study demonstrated that chronic ingestion of DU-induced time-dependent modifications of inflammatory pathways, notably in terms of immune cell content. The ultimate effects of DU contamination might be pathogenic by suppressing defense mechanisms or inducing hypersensitivity. Further experiments should be thus performed to determine real consequences on intestinal response to oral antigens.

Human keratinocyte radiosensitivity is linked to redox modulation.

BACKGROUND: Ionising radiation-induced reactive oxygen species (ROS) overproduction induces keratinocyte alterations and constitutes one of the most common effects after therapeutic gamma-irradiation. ROS production is controlled by a complex enzymatic system. OBJECTIVE: The aim of our study is to analyse the role of radiation-induced oxidative stress in keratinocytes death by apoptosis. We hypothesized that keratinocyte capacity to hamper radiation-induced ROS generation may control their radiosensitivity. METHODS: For this purpose, an original human skin explant model was developed and two types of human epidermal cells were used: primary keratinocytes NHEK and spontaneous non-tumourigenic cell line HaCaT. RESULTS: cDNA-arrays analysis was performed 24h after a 20Gy gamma-radiation and revealed down-regulation of genes involved in oxidative stress control and the apoptosis process. This was confirmed by alterations in catalase, GPx and SOD enzymatic activities. This redox modulation was concomitant to the down-regulation of anti-apoptotic genes and up-regulation of some pro-apoptotic genes (caspase 10, ubiquitin C). Interestingly TUNEL labelling revealed an increase in the number of apoptotic cells. We also demonstrated a differential inducibility of the cell antioxidant network in two keratinocyte lines, which results in a differential cellular level of ROS, explaining their different radiosensitivities. CONCLUSION: Keratinocytes apoptosis is partly dependent on ROS production after exposure to gamma-rays. In addition, the differential radiosensitivity of keratinocytes is linked to different oxidative stress responses.

Overview of low-level radiation exposure assessment: biodosimetry.

The capability to make diagnostic assessments of radiation exposure is needed to support triage of radiation casualties and medical treatment decisions in military operations. At the International Conference on Low-Level Radiation Injury and Medical Countermeasures session on biodosimetry in the military, participants reviewed the field of biomarkers, covering a wide range of biological endpoints. Participants evaluated early changes associated with exposure to ionizing radiation, including chromosomal and DNA damage, gene expression and associated proteins, and DNA mutations. The use and development of advanced monitoring and diagnostic technologies compatible with military operations was emphasized. Conventional radiation bioassays require a substantial amount of time between when the sample is taken and when the data can be provided for decision making. These "reach back" bioassays are evaluated in laboratories that are not in the field; these laboratories routinely measure exposures of 25 cGy (photon equivalent levels). Detection thresholds can be reduced approximately fivefold by the addition of significant and tiresome scoring efforts. Alternative real-time biomarkers that can be measured in field laboratories or with handheld detection devices show promise as screening and clinical diagnostic tools, but they require further development and validation studies.