Multidisciplinary European Low Dose Initiative (MELODI): strategic research agenda for low dose radiation risk research.

MELODI (Multidisciplinary European Low Dose Initiative) is a European radiation protection research platform with focus on research on health risks after exposure to low-dose ionising radiation. It was founded in 2010 and currently includes 44 members from 18 countries. A major activity of MELODI is the continuous development of a long-term European Strategic Research Agenda (SRA) on low-dose risk for radiation protection. The SRA is intended to identify priorities for national and European radiation protection research programs as a basis for the preparation of competitive calls at the European level. Among those key priorities is the improvement of health risk estimates for exposures close to the dose limits for workers and to reference levels for the population in emergency situations. Another activity of MELODI is to ensure the availability of European key infrastructures for research activities, and the long-term maintenance of competences in radiation research via an integrated European approach for training and education. The MELODI SRA identifies three key research topics in low dose or low dose-rate radiation risk research: (1) dose and dose rate dependence of cancer risk, (2) radiation-induced non-cancer effects and (3) individual radiation sensitivity. The research required to improve the evidence base for each of the three key topics relates to three research lines: (1) research to improve understanding of the mechanisms contributing to radiogenic diseases, (2) epidemiological research to improve health risk evaluation of radiation exposure and (3) research to address the effects and risks associated with internal exposures, differing radiation qualities and inhomogeneous exposures. The full SRA and associated documents can be downloaded from the MELODI website ( http://www.melodi-online.eu/sra.html ).

Genotoxic risk of ethyl-paraben could be related to telomere shortening.

The ability of parabens to promote the appearance of multiple cancer hallmarks in breast epithelium cells provides grounds for regulatory review of the implication of the presence of parabens in human breast tissue. It is well documented that telomere dysfunction plays a significant role in the initiation of genomic instability during carcinogenesis in human breast cancer. In the present study, we evaluated the genotoxic effect of ethyl 4-hydroxybenzoate (ethyl-paraben), with and without metabolic activation (S9), in studies following OECD guidelines. We observed a significant increase in genotoxic damage using the Mouse Lymphoma Assay and in vitro micronucleus (MN) tests in the L5178Y cell line in the presence of S9 only after a short exposure. A high frequency of MN was observed in the TK6 cells after a short exposure (3 h) in the presence of S9 and a long exposure (26 h) without S9. We found significant increases in the MN frequency and induced chromosomal aberrations in the lymphocytes of only one donor after ethyl-paraben exposure in the presence of S9 after a short exposure. Cytogenetic characterization of the paraben-treated cells demonstrated telomere shortening associated with telomere loss and telomere deletions in L5178Y and TK6 cells and lymphocytes of the paraben sensitive-donor. In a control cohort of 68 human lymphocytes, telomere length and telomere aberrations were age-dependent and showed high inter-individual variation. This study is the first to link telomere shortening and the genotoxic effect of ethyl paraben in the presence of S9 and raises the possibility that telomere shortening may be a proxy for underlying inter-individual sensitivity to ethyl-paraben. Copyright © 2016 John Wiley & Sons, Ltd.

RENEB Inter-Laboratory Comparison 2021: The Dicentric Chromosome Assay.

After large-scale radiation accidents where many individuals are suspected to be exposed to ionizing radiation, biological and physical retrospective dosimetry assays are important tools to aid clinical decision making by categorizing individuals into unexposed/minimally, moderately or highly exposed groups. Quality-controlled inter-laboratory comparisons of simulated accident scenarios are regularly performed in the frame of the European legal association RENEB (Running the European Network of Biological and Physical retrospective Dosimetry) to optimize international networking and emergency readiness in case of large-scale radiation events. In total 33 laboratories from 22 countries around the world participated in the current RENEB inter-laboratory comparison 2021 for the dicentric chromosome assay. Blood was irradiated in vitro with X rays (240 kVp, 13 mA, ∼75 keV, 1 Gy/min) to simulate an acute, homogeneous whole-body exposure. Three blood samples (no. 1: 0 Gy, no. 2: 1.2 Gy, no. 3: 3.5 Gy) were sent to each participant and the task was to culture samples, to prepare slides and to assess radiation doses based on the observed dicentric yields from 50 manually or 150 semi-automatically scored metaphases (triage mode scoring). Approximately two-thirds of the participants applied calibration curves from irradiations with γ rays and about 1/3 from irradiations with X rays with varying energies. The categorization of the samples in clinically relevant groups corresponding to individuals that were unexposed/minimally (0-1 Gy), moderately (1-2 Gy) or highly exposed (>2 Gy) was successfully performed by all participants for sample no. 1 and no. 3 and by ≥74% for sample no. 2. However, while most participants estimated a dose of exactly 0 Gy for the sham-irradiated sample, the precise dose estimates of the samples irradiated with doses >0 Gy were systematically higher than the corresponding reference doses and showed a median deviation of 0.5 Gy (sample no. 2) and 0.95 Gy (sample no. 3) for manual scoring. By converting doses estimated based on γ-ray calibration curves to X-ray doses of a comparable mean photon energy as used in this exercise, the median deviation decreased to 0.27 Gy (sample no. 2) and 0.6 Gy (sample no. 3). The main aim of biological dosimetry in the case of a large-scale event is the categorization of individuals into clinically relevant groups, to aid clinical decision making. This task was successfully performed by all participants for the 0 Gy and 3.5 Gy samples and by 74% (manual scoring) and 80% (semiautomatic scoring) for the 1.2 Gy sample. Due to the accuracy of the dicentric chromosome assay and the high number of participating laboratories, a systematic shift of the dose estimates could be revealed. Differences in radiation quality (X ray vs. γ ray) between the test samples and the applied dose effect curves can partly explain the systematic shift. There might be several additional reasons for the observed bias (e.g., donor effects, transport, experimental conditions or the irradiation setup) and the analysis of these reasons provides great opportunities for future research. The participation of laboratories from countries around the world gave the opportunity to compare the results on an international level.

Retrospective biodosimetry techniques: Focus on cytogenetics assays for individuals exposed to ionizing radiation.

In the absence of physical data, biodosimetry tools are required for fast dose and risk assessment in the event of radiological or nuclear mass accidents or attacks to triage exposed humans and take immediate medical countermeasures. Biodosimetry tools have mostly been developed for retrospective dose assessment and the follow-up of victims of irradiation. Among them, cytogenetics analyses, to reveal chromosome damage, are the most developed and allow the determination of doses from blood samples as low as 100 mGy. Various cytogenetic tests have already allowed retrospective dose assessment of Chernobyl liquidators and military personnel exposed to nuclear tests after decades. In this review, we discuss the properties of various biodosimetry techniques, such as their sensitivity and limitations as a function of the time from exposure, using multiple examples of nuclear catastrophes or working exposure. Among them, chromosome FISH hybridization, which reveals chromosome translocations, is the most reliable due to the persistence of translocations for decades, whereas dicentric chromosome and micronuclei assays allow rapid and accurate dose assessment a short time after exposure. Both need to be adjusted through mathematical algorithms for retrospective analyses, accounting for the time since exposure and the victims' age. The goal for the future will be to better model chromosome damage, reduce the time to result, and develop new complementary biodosimetry approaches, such as mutation signatures.

Research plans in Europe for radiation health hazard assessment in exploratory space missions.

The European Space Agency (ESA) is currently expanding its efforts in identifying requirements and promoting research towards optimizing radiation protection of astronauts. Space agencies use common limits for tissue (deterministic) effects on the International Space Station. However, the agencies have in place different career radiation exposure limits (for stochastic effects) for astronauts in low-Earth orbit missions. Moreover, no specific limits for interplanetary missions are issued. Harmonization of risk models and dose limits for exploratory-class missions are now operational priorities, in view of the short-term plans for international exploratory-class human missions. The purpose of this paper is to report on the activity of the ESA Topical Team on space radiation research, whose task was to identify the most pertinent research requirements for improved space radiation protection and to develop a European space radiation risk model, to contribute to the efforts to reach international consensus on dose limits for deep space. The Topical Team recommended ESA to promote the development of a space radiation risk model based on European-specific expertise in: transport codes, radiobiological modelling, risk assessment, and uncertainty analysis. The model should provide cancer and non-cancer radiation risks for crews implementing exploratory missions. ESA should then support the International Commission on Radiological Protection to harmonize international models and dose limits in deep space, and guarantee continuous support in Europe for accelerator-based research configured to improve the models and develop risk mitigation strategies.

Telomere shortening is correlated with the DNA damage response and telomeric protein down-regulation in colorectal preneoplastic lesions.

BACKGROUND: A relation between telomere attrition in early carcinogenesis and activation of DNA damage response (DDR) has been proposed. We explored telomere length and its link with DDR in colorectal multistep carcinogenesis. PATIENTS AND METHODS: We studied normal mucosa, low-grade dysplasia (LGD) and high-grade dysplasia (HGD) and invasive carcinoma (IC) in matched human colon specimens by evaluating p-ataxia telangiectasia mutated (ATM), p-checkpoint kinase 2 (Chk2), c-H2AX, TRF1 and TRF2 expressions by immunohistochemistry. FISH was used to assess telomere length. RESULTS: Telomeres shortened significantly from normal (N) to LGD and HGD (P < 0.0001; P = 0.012), then increased in length in IC (P = 0.006). TRF1 and TRF2 expressions were diminished from N to LGD and HGD (P = 0.004, P < 0.0001, ns) and were reexpressed at the invasive stage (P = 0.053 and P = 0.046). Phosphorylated ATM, Chk2 and H2AX appeared already in LGD (respectively, P = 0.001, P = 0.002 and P = 0.02). Their expression decreased from HGD to IC (respectively, P = 0.03, P = 0.02 and P = 0.37). These activating phosphorylations were inversely correlated with telomere length and TRF1/2 expression. CONCLUSION: In a model of colon multistep carcinogenesis, our data indicate that telomeric length and protein expression levels are inversely correlated with the activation of the DDR pathway.

Increased genomic alteration complexity and telomere shortening in B-CLL cells resistant to radiation-induced apoptosis.

B-cell chronic lymphocytic leukemia (B-CLL) results in an accumulation of mature CD5(+)/CD23(+) B cells due to an uncharacterized defect in apoptotic cell death. B-CLL is not characterized by a unique recurrent genomic alteration but rather by genomic instability giving rise frequently to several chromosomal aberrations. Besides we reported that approximately 15% of B-CLL patients present malignant B-cells resistant to irradiation-induced apoptosis, contrary to approximately 85% of patients and normal human lymphocytes. Telomere length shortening is observed in radioresistant B-CLL cells. Using fluorescence in situ hybridization (FISH) and multicolour FISH, we tested whether specific chromosomal aberrations might be associated with the radioresistance of a subset of B-CLL cells and whether they are correlated with telomere shortening. In a cohort of 30 B-CLL patients, all of the radioresistant B-CLL cell samples exhibited homozygous or heterozygous deletion of 13q14.3 in contrast to 52% of the radiosensitive samples. In addition to the 13q14.3 deletion, ten out of the 11 radioresistant B-cell samples had another clonal genomic alteration such as trisomy 12, deletion 17p13.1, mutation of the p53 gene or translocations in contrast to only three out of 19 radiosensitive samples. Telomere fusions and non-reciprocal translocations, hallmarks of telomere dysfunction, are not increased in radioresistant B-CLL cells. These findings suggest (i) that the 13q14.3 deletion accompanied by another chromosomal aberration is associated with radioresistance of B-CLL cells and (ii) that telomere shortening is not causative of increased clonal chromosomal aberrations in radioresistant B-CLL cells.

hTERT: a novel endogenous inhibitor of the mitochondrial cell death pathway.

hTERT is the catalytic subunit of the telomerase and is hence required for telomerase maintenance activity and cancer cell immortalization. Here, we show that acute hTERT depletion has no adverse effects on the viability or proliferation of cervical and colon carcinoma cell lines, as evaluated within 72 h after transfection with hTERT-specific small interfering RNAs (siRNAs). Within the same time frame, hTERT depletion facilitated the induction of apoptotic cell death by cisplatin, etoposide, mitomycin C and reactive oxygen species, yet failed to sensitize cells to death induction via the CD95 death receptor. Experiments performed with p53 knockout cells or chemical p53 inhibitors revealed that p53 was not involved in the chemosensitizing effect of hTERT knockdown. However, the proapoptotic Bcl-2 family protein Bax was involved in cell death induction by hTERT siRNAs. Depletion of hTERT facilitated the conformational activation of Bax induced by genotoxic agents. Moreover, Bax knockout abolished the chemosensitizing effect of hTERT siRNAs. Inhibition of mitochondrial membrane permeabilization by overexpression of Bcl-2 or expression of the cytomegalovirus-encoded protein vMIA (viral mitochondrial inhibitor of apoptosis), which acts as a specific Bax inhibitor, prevented the induction of cell death by the combination of hTERT depletion and chemotherapeutic agents. Altogether, our data indicate that hTERT inhibition may constitute a promising strategy for facilitating the induction of the mitochondrial pathway of apoptosis.

TRF2 inhibition promotes anchorage-independent growth of telomerase-positive human fibroblasts.

Although telomere instability is observed in human tumors and is associated with the development of cancers in mice, it has yet to be established that it can contribute to the malignant transformation of human cells. We show here that in checkpoint-compromised telomerase-positive human fibroblasts an episode of TRF2 inhibition promotes heritable changes that increase the ability to grow in soft agar, but not tumor growth in nude mice. This transforming activity is associated to a burst of telomere instability but is independent of an altered control of telomere length. Moreover, it cannot be recapitulated by an increase in chromosome breaks induced by an exposure to gamma-radiations. Since it can be revealed in the context of telomerase-proficient human cells, telomere dysfunction might contribute to cancer progression even at late stages of the oncogenesis process, after the telomerase reactivation step.

Telomere dynamics, end-to-end fusions and telomerase activation during the human fibroblast immortalization process.

Loss of telomeric repeats during cell proliferation could play a role in senescence. It has been generally assumed that activation of telomerase prevents further telomere shortening and is essential for cell immortalization. In this study, we performed a detailed cytogenetic and molecular characterization of four SV40 transformed human fibroblastic cell lines by regularly monitoring the size distribution of terminal restriction fragments, telomerase activity and the associated chromosomal instability throughout immortalization. The mean TRF lengths progressively decreased in pre-crisis cells during the lifespan of the cultures. At crisis, telomeres reached a critical size, different among the cell lines, contributing to the peak of dicentric chromosomes, which resulted mostly from telomeric associations. We observed a direct correlation between short telomere length at crisis and chromosomal instability. In two immortal cell lines, although telomerase was detected, mean telomere length still continued to decrease whereas the number of dicentric chromosomes associated was stabilized. Thus telomerase could protect specifically telomeres which have reached a critical size against end-to-end dicentrics, while long telomeres continue to decrease, although at a slower rate as before crisis. This suggests a balance between elongation by telomerase and telomere shortening, towards a stabilized 'optimal' length.

p-Butyroxybenzenediazonium fluoroborate, substrate of acetylcholinesterase and butyrylcholinesterase, discriminates between the two enzymes by a specific affinity labelling.

p-Butyroxybenzenediazonium fluoroborate 1 was shown to be a substrate of both acetylcholinesterase (AcChE) and butyrylcholinesterase (BuChE) with Michaelis constants of 6.10(-5) M and 1.3. 10(-4)M, respectively. Upon incubation in the dark, 1 was able to discriminate between the two enzymes AcChE was efficiently inactivated in a time-dependent manner while BuChE remained unaffected. Kinetic analysis of the inactivation of AcChE (i) by various concentrations of 1 indicated that it behaves as an affinity label, (ii) at three different pH levels suggested that the pKa of the labelled residue was higher than 7 and (iii) in the presence of different selective ligands for either the active site (edrophonium) or the peripheral site (propidium) indicated that 1 alkylated the active site rather than the peripheral one. Differences of reactivity between AcChE and BuChE suggest a different positioning and/or a different chemical environment of the substrate within two active sites.

Molecular detection of telomerase-positive circulating epithelial cells in metastatic breast cancer patients.

The detection of circulating tumor cells and micrometastases may have important therapeutic and prognostic implications. Telomerase is a hallmark of cancer and is absent from normal epithelial cells. The aim of this study was to use telomerase activity as a molecular marker for the detection of cancer cells in blood of patients with breast cancer. Blood samples were collected from 25 women with stage IV breast cancer and 9 healthy volunteers. Peripheral blood mononuclear cells were isolated by using Ficoll/Hypaque. Immunomagnetic beads coated with an epithelial-specific antibody (BerEP4) were used to harvest epithelial cells from peripheral blood mononuclear cells. Telomerase activity was detected in harvested epithelial cells (HECs) using two different telomerase-PCR-ELISA methods. HECs from blood samples of 21 of 25 (84%) patients with breast cancer were telomerase positive. Telomerase activity was undetectable in HECs from the nine healthy volunteers, demonstrating the specificity of the association between telomerase activity in HECs and stage IV breast cancer. Thus, determination of telomerase activity in HECs appears to be a sensitive, specific, and noninvasive approach for detecting circulating epithelial cancer cells in patients with metastatic breast cancer. This method could be of great value in monitoring the cancer cell proliferation during chemotherapy. This study should be now extended to patients with early-stage breast cancer to investigate the role of telomerase expression by HECs and to evaluate its prognostic value.

Telomere shortening: a new prognostic factor for cardiovascular disease post-radiation exposure.

Telomere length has been proposed as a marker of mitotic cell age and as a general index of human organism aging. Telomere shortening in peripheral blood lymphocytes has been linked to cardiovascular-related morbidity and mortality. The authors investigated the potential correlation of conventional risk factors, radiation dose and telomere shortening with the development of coronary artery disease (CAD) following radiation therapy in a large cohort of Hodgkin lymphoma (HL) patients. Multivariate analysis demonstrated that hypertension and telomere length were the only independent risk factors. This is the first study in a large cohort of patients that demonstrates significant telomere shortening in patients treated by radiation therapy who developed cardiovascular disease. Telomere length appears to be an independent prognostic factor that could help determine patients at high risk of developing CAD after exposure in order to implement early detection and prevention.

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.

The relationship between spontaneous telomere loss and chromosome instability in a human tumor cell line.

Chromosome instability plays an important role in cancer by promoting the alterations in the genome required for tumor cell progression. The loss of telomeres that protect the ends of chromosomes and prevent chromosome fusion has been proposed as one mechanism for chromosome instability in cancer cells, however, there is little direct evidence to support this hypothesis. To investigate the relationship between spontaneous telomere loss and chromosome instability in human cancer cells, clones of the EJ-30 tumor cell line were isolated in which a herpes simplex virus thymidine kinase (HSV-tk) gene was integrated immediately adjacent to a telomere. Selection for HSV-tk-deficient cells with ganciclovir demonstrated a high rate of loss of the end these "marked" chromosomes (10-4 events/cell per generation). DNA sequence and cytogenetic analysis suggests that the loss of function of the HSV-tk gene most often involves telomere loss, sister chromatid fusion, and prolonged periods of chromosome instability. In some HSV-tk-deficient cells, telomeric repeat sequences were added on to the end of the truncated HSV-tk gene at a new location, whereas in others, no telomere was detected on the end of the marked chromosome. These results suggest that spontaneous telomere loss is a mechanism for chromosome instability in human cancer cells.

Chromosomal instability and alteration of telomere repeat sequences.

The very end of the chromosome is called the telomere and is composed of DNA repeat sequences and associated proteins. Genetic and biochemical analyses of this complex, the telosome, lead to the hypothesis that transcription and DNA replication are submitted to position effects mediated by the telomere proximity. Telomere length reduction and alterations of the telomeric chromatin assembly might explain the chromosome instability which occurs during the senescence and the immortalization process in vitro. A particular polymerase, the telomerase, is able to lengthen the telomeres. A telomerase activity was characterized in yeast, Tetrahymena, but also in transformed and in germline cells. We reviewed the involvement of telomeres in the aging process. We proposed that the short size of the telomere repeat at each chromosome could direct the loss of heterozygosity, thus telomere length could play a role in individual and tissular susceptibility to develop cancer. Antitelomerase strategy for cancer therapy is attractive but limited by the short decrease of the telomere length at each cell division.

Interstitial telomeric repeats are not preferentially involved in radiation-induced chromosome aberrations in human cells.

Telomeric repeat sequences, located at the end of eukaryotic chromosomes, have been detected at intrachromosomal locations in many species. Large blocks of telomeric sequences are located near the centromeres in hamster cells, and have been reported to break spontaneously or after exposure to ionizing radiation, leading to chromosome aberrations. In human cells, interstitial telomeric sequences (ITS) can be composed of short tracts of telomeric repeats (less than twenty), or of longer stretches of exact and degenerated hexanucleotides, mainly localized at subtelomeres. In this paper, we analyzed the radiation sensitivity of a naturally occurring short ITS localized in 2q31 and we found that this region is not a hot spot of radiation-induced chromosome breaks. We then selected a human cell line in which approximately 800 bp of telomeric DNA had been introduced by transfection into an internal euchromatic chromosomal region in chromosome 4q. In parallel, a cell line containing the plasmid without telomeric sequences was also analyzed. Both regions containing the transfected plasmids showed a higher frequency of radiation-induced breaks than expected, indicating that the instability of the regions containing the transfected sequences is not due to the presence of telomeric sequences. Taken together, our data show that ITS themselves do not enhance the formation of radiation-induced chromosome rearrangements in these human cell lines.

Human fibroblasts expressing hTERT show remarkable karyotype stability even after exposure to ionizing radiation.

Ectopic expression of telomerase results in an immortal phenotype in various types of normal cells, including primary human fibroblasts. In addition to its role in telomere lengthening, telomerase has now been found to have various functions, including the control of DNA repair, chromatin modification, and the control of expression of genes involved in cell cycle regulation. The investigations on the long-term effects of telomerase expression in normal human fibroblast highlighted that these cells show low frequencies of chromosomal aberrations. In this paper, we describe the karyotypic stability of human fibroblasts immortalized by expression of hTERT. The ectopic overexpression of telomerase is associated with unusual spontaneous as well as radiation-induced chromosome stability. In addition, we found that irradiation did not enhance plasmid integration in cells expressing hTERT, as has been reported for other cell types. Long-term studies illustrated that human fibroblasts immortalized by telomerase show an unusual stability for chromosomes and for plasmid integration sites, both with and without exposure to ionizing radiation. These results confirm a role for telomerase in genome stabilisation by a telomere-independent mechanism and point to the possibility for utilizing hTERT-immortalized normal human cells for the study of gene targeting.

Dosimetric and cytogenetic studies of multiple radiation-induced meningiomas for a single patient.

No criteria are currently available to determine the spontaneous or radiation-induced origin of a malignant tumor occurring in a previously irradiated area. This study presents the dosimetric and cytogenetic analysis of meningiomas diagnosed in irradiated brain areas from a single patient and a discussion of the karyotypes of spontaneous meningiomas and radiation-induced tumors published in the literature.

Expression of thymidylate synthase gene in human colorectal carcinomas by northern blotting and reflectance in-situ hybridization (rish) - correlation with the number of chromosome-18.

The thymidylate synthase (TYMS) gene expression was analysed by Northern-blotting and by reflectance in situ hybridization (RISH) in human histological colorectal cancer. Scattering reflectance signals from 1-nm colloidal-gold particles after in situ hybridization, using digoxigenin-labeled probe, were quantified by confocal scanning laser microscopy. The importance of the RISH method is demonstrated by results obtained on colorectal cancer specimens. This study demonstrates a quantitative difference between cancer cells and normal colon epithelial cells in situ. In addition, TYMS expression varies from turner to tumor, in relation with karyotypic patterns. When the number of copies of chromosome 18, which carries the TYMS gene, are considered, there is a significant correlation with TYMS mRNA amounts, suggesting the existence of a gene dosage effect in colorectal cancer cells.