Search

Purpose In case of a mass-casualty radiological event, there would be a need for networking to overcome surge limitations and to quickly obtain homogeneous results (reported aberration frequencies or estimated doses) among biodosimetry laboratories. These results must be consistent within such network. Inter-laboratory comparisons (ILCs) are widely accepted to achieve this homogeneity. At the European level, a great effort has been made to harmonize biological dosimetry laboratories, notably during the MULTIBIODOSE and RENEB projects. In order to continue the harmonization efforts, the RENEB consortium launched this intercomparison which is larger than the RENEB network, as it involves 38 laboratories from 21 countries. In this ILC all steps of the process were monitored, from blood shipment to dose estimation. This exercise also aimed to evaluate the statistical tools used to compare laboratory performance. Materials and methods Blood samples were irradiated at three different doses, 1.8, 0.4 and 0 Gy (samples A, C and B) with 4-MV X-rays at 0.5 Gy min-1, and sent to the participant laboratories. Each laboratory was requested to blindly analyze 500 cells per sample and to report the observed frequency of dicentric chromosomes per metaphase and the corresponding estimated dose. Results This ILC demonstrates that blood samples can be successfully distributed among laboratories worldwide to perform biological dosimetry in case of a mass casualty event. Having achieved a substantial harmonization in multiple areas among the RENEB laboratories issues were identified with the available statistical tools, which are not capable to advantageously exploit the richness of results of a large ILCs. Even though Z- and U-tests are accepted methods for biodosimetry ILCs, setting the number of analyzed metaphases to 500 and establishing a tests' common threshold for all studied doses is inappropriate for evaluating laboratory performance. Another problem highlighted by this ILC is the issue of the dose-effect curve diversity. It clearly appears that, despite the initial advantage of including the scoring specificities of each laboratory, the lack of defined criteria for assessing the robustness of each laboratory's curve is a disadvantage for the 'one curve per laboratory' model. Conclusions Based on our study, it seems relevant to develop tools better adapted to the collection and processing of results produced by the participant laboratories. We are confident that, after an initial harmonization phase reached by the RENEB laboratories, a new step toward a better optimization of the laboratory networks in biological dosimetry and associated ILC is on the way.

PURPOSE The development of an exposure apparatus for in situ α-irradiation studies of cells. The construction of the apparatus is simple and the apparatus is maintenance free, easy to use and of low cost. This small device can be placed in an incubator, where the exposure environment is controlled. Moreover the vapor saturated incubator protects the cells from drying out, allowing long irradiation intervals. MATERIALS AND METHODS The system includes a 234U alpha (α)-source of total activity 0.77 ± 0.03 MBq in the form of a thin disk deposited on an aluminum substrate. The α-particles emitted in the air have a mean energy of 4.9 MeV at the disk surface. Source homogeneity has been studied via Rutherford Backscattering Spectrometry. Using SRIM 2013 and Monte Carlo (MC) simulations via the MCNP6.1 code, LET and energy deposition values have been calculated for various filling gasses. Furthermore, based on these simulations, the assembly's dimensions and equivalent irradiation rate have been determined. With respect to the aforementioned dimensions, the experimental setup is constructed in a way to provide uniform irradiation of the sample. Using Sacalc3v1.4 irradiation radial homogeneity has been studied. In order to evaluate biologically our apparatus, a well-established chromosomal aberration assay has been utilized, applied in exponentially growing hamster (CHO) cells. Furthermore, immunofluorescence gamma-H2AX/53BP1 foci assay has been performed as a 'biological detector', in order to validate α-particles surface density. RESULTS Source surface homogeneity: emission deviations do not exceed 10-15%. The optimal distance between the source and the cells for irradiation is determined to be 14.8 mm. Irradiation radial homogeneity: a deviation of 5% occurs at the first 8 mm from the center of the irradiation area, and a 10% deviation occurs after 12 mm. Chromosomal aberrations were found in good agreement with the corresponding in bibliography. CONCLUSIONS The current technical report describes analytically the development and evaluation stages of this experimental housing; from MC simulations to the irradiation of mammalian cells and data analysis. Moreover, guidance is provided as well as a report of the variables on which critical parameters are depended, so as to make this work useful to anyone who wants to construct a similar in-house α-irradiation apparatus for radiobiological studies using mammalian cells.

Purpose: Depending on the exposure scenario, different biological and/or physical assays will be required to recover the exposure situation and to obtain reliable dose estimates to aid medical decision making. Inter-laboratory comparisons (ILCs) are a central tool to validate and improve the performance of methods and laboratories. In most cases such ILCs are performed under laboratory conditions where the influence of parameters contributing to uncertainties can be minimized. In a reallife scenario, the situation is much more complicated, and it is therefore crucial to validate methods and laboratories’ abilities to carry out these methods under more realistic conditions. In 2019, EURADOS (The European Radiation Dosimetry Group) Working Group 10 (Retrospective Dosimetry) and RENEB (Running the European Network of Biological and retrospective Physical dosimetry) organized a field exercise in Lund, Sweden, to validate different methods for biological and physical retrospective dosimetry in parallel by simulating real- life exposure scenarios. Materials and methods: For the dicentric chromosome assay (DCA), 18 blood tubes were distributed across 8 thermos flasks filled with heated water and positioned at the hips and shoulders of anthropomorphic phantoms in different geometries. Two irradiations were performed with a 1.36 TBq 192Ir-source, each with two phantoms, to simulate close, distant, lateral or partially shielded exposures. For each of the blood tubes DCA dose estimates were provided by at least one laboratory and for tubes from four thermos flasks by 17 participating RENEB laboratories. In addition, blood from the different thermos flasks on each phantom was mixed to simulate heterogeneous exposures. Three radio-photoluminescence (RPL) glass dosimeters (GD) were placed at each tube to assess reference doses and the inter- and intra-tube variability. Results: The results from the DCA were homogeneous between participants and matched well with RPL GD reference doses (≥95% of estimates within ±0.5 Gy of the reference). For samples close to the source, exposed to doses >1 Gy, systematic underestimation could be corrected by accounting for exposure time. Heterogeneity within and between tubes was detected for RPL GD reference doses as well as for doses estimates from the DCA. The sample simulating a heterogeneous exposure with two different doses was detected by ~50% of the participants. Conclusions: The participants were able to successfully estimate the doses and to provide important information on the exposure scenarios under conditions closely resembling a real-life situation. The reliability of the results emphasizes the value of the DCA for retrospective dosimetry under field conditions.

For precision cancer radiotherapy, high linear energy transfer (LET) particle irradiation offers a substantial advantage over photon-based irradiation. In contrast to the sparse deposition of low-density energy by &chi, or &gamma, rays, particle irradiation causes focal DNA damage through high-density energy deposition along the particle tracks. This is characterized by the formation of multiple damage sites, comprising localized clustered patterns of DNA single- and double-strand breaks as well as base damage. These clustered DNA lesions are key determinants of the enhanced relative biological effectiveness (RBE) of energetic nuclei. However, the search for a fingerprint of particle exposure remains open, while the mechanisms underlying the induction of chromothripsis-like chromosomal rearrangements by high-LET radiation (resembling chromothripsis in tumors) await to be elucidated. In this work, we investigate the transformation of clustered DNA lesions into chromosome fragmentation, as indicated by the induction and post-irradiation repair of chromosomal damage under the dynamics of premature chromosome condensation in G0 human lymphocytes. Specifically, this study provides, for the first time, experimental evidence that particle irradiation induces localized shattering of targeted chromosome domains. Yields of chromosome fragments and shattered domains are compared with those generated by &gamma, rays, and the RBE values obtained are up to 28.6 for &alpha, particles (92 keV/&mu, m), 10.5 for C-ions (295 keV/&mu, m), and 4.9 for protons (28.5 keV/&mu, m). Furthermore, we test the hypothesis that particle radiation-induced persistent clustered DNA lesions and chromatin decompaction at damage sites evolve into localized chromosome shattering by subsequent chromatin condensation in a single catastrophic event&mdash, posing a critical risk for random rejoining, chromothripsis, and carcinogenesis. Consistent with this hypothesis, our results highlight the potential use of shattered chromosome domains as a fingerprint of high-LET exposure, while conforming to the new model we propose for the mechanistic origin of chromothripsis-like rearrangements.

The discovery of chromothripsis in cancer genomes challenges the long-standing concept of carcinogenesis as the result of progressive genetic events. Despite recent advances in describing chromothripsis, its mechanistic origin remains elusive. The prevailing conception is that it arises from a massive accumulation of fragmented DNA inside micronuclei (MN), whose defective nuclear envelope ruptures or leads to aberrant DNA replication, before main nuclei enter mitosis. An alternative hypothesis is that the premature chromosome condensation (PCC) dynamics in asynchronous micronucleated cells underlie chromosome shattering in a single catastrophic event, a hallmark of chromothripsis. Specifically, when main nuclei enter mitosis, premature chromatin condensation provokes the shattering of chromosomes entrapped inside MN, if they are still undergoing DNA replication. To test this hypothesis, the agent RO-3306, a selective ATP-competitive inhibitor of CDK1 that promotes cell cycle arrest at the G2/M boundary, was used in this study to control the degree of cell cycle asynchrony between main nuclei and MN. By delaying the entrance of main nuclei into mitosis, additional time was allowed for the completion of DNA replication and duplication of chromosomes inside MN. We performed interphase cytogenetic analysis using asynchronous micronucleated cells generated by exposure of human lymphocytes to &gamma, rays, and heterophasic multinucleated Chinese hamster ovary (CHO) cells generated by cell fusion procedures. Our results demonstrate that the PCC dynamics during asynchronous mitosis in micronucleated or multinucleated cells are an important determinant of chromosome shattering and may underlie the mechanistic origin of chromothripsis.

A sensitive biodosimetry tool is required for rapid individualized dose estimation and risk assessment in the case of radiological or nuclear mass casualty scenarios to prioritize exposed humans for immediate medical countermeasures to reduce radiation related injuries or morbidity risks. Unlike the conventional Dicentric Chromosome Assay (DCA), which takes about 3-4 days for radiation dose estimation, cell fusion mediated Premature Chromosome Condensation (PCC) technique in G0 lymphocytes can be rapidly performed for radiation dose assessment within 6-8 hrs of sample receipt by alleviating the need for ex vivo lymphocyte proliferation for 48 hrs. Despite this advantage, the PCC technique has not yet been fully exploited for radiation biodosimetry. Realizing the advantage of G0 PCC technique that can be instantaneously applied to unstimulated lymphocytes, we evaluated the utility of G0 PCC technique in detecting ionizing radiation (IR) induced stable and unstable chromosomal aberrations for biodosimetry purposes. Our study demonstrates that PCC coupled with mFISH and mBAND techniques can efficiently detect both numerical and structural chromosome aberrations at the intra- and inter-chromosomal levels in unstimulated T- and B-lymphocytes. Collectively, we demonstrate that the G0 PCC technique has the potential for development as a biodosimetry tool for detecting unstable chromosome aberrations (chromosome fragments and dicentric chromosomes) for early radiation dose estimation and stable chromosome exchange events (translocations) for retrospective monitoring of individualized health risks in unstimulated lymphocytes.

In radiation accidents and large-scale radiological emergencies, a fast and reliable triage of individuals according to their degree of exposure is important for accident management and identification of those who need medical assistance. In this work, the applicability of cell-fusion-mediated premature chromosome condensation (PCC) in G0-lymphocytes is examined for the development of a rapid, minimally invasive and automatable micro-PCC assay, which requires blood volumes of only 100 μl and can be performed in 96-well plates, towards risk assessments and categorization of individuals based on dose estimates. Chromosomal aberrations are visualized for dose-estimation analysis within two hours, without the need of blood culturing for two days, as required by conventional cytogenetics. The various steps of the standard-PCC procedure were adapted and, for the first time, lymphocytes in blood volumes of 100 μl were successfully fused with CHO-mitotics in 96-well plates of 2 ml/well. The plates are advantageous for high-throughput analysis since the various steps required are applied to all 96-wells simultaneously. Interestingly, the use of only 1.5 ml hypotonic and Carnoy’s fixative per well offers high quality PCC-images, and the morphology of lymphocyte PCCs is identical to that obtained using the conventional PCC-assay, which requires much larger blood volumes and 15 ml tubes. For dose assessments, appropriate calibration curves were constructed and for PCC analysis specialized software (MetaSystems) was used. The micro-PCC assay can be combined with fluorescence in situ hybridization (FISH), using simultaneously centromeric/telomeric (C/T) peptide nucleic acid (PNA) probes. This allows dose assessments on the basis of accurate scoring of dicentric and centric ring chromosomes in G0-lymphocyte PCCs, which is particularly helpful when further evaluation into treatment-level categories of exposed individuals is needed. The micro-PCC assay has significant advantages for early triage biodosimetry when compared to other cytogenetic biodosimetry assays. It is rapid, cost-effective, and could pave the way to its subsequent automation.

Curcumin and trans-resveratrol are well-known antioxidant polyphenols with radiomodulatory properties, radioprotecting non-cancerous cells while radiosensitizing tumor cells. This dual action may be the result of their radical scavenging properties and their effects on cell-cycle checkpoints that are activated in response to radiation-induced chromosomal damage. It could be also caused by their effect on regulatory pathways with impact on detoxification enzymes, the up-regulation of endogenous protective systems, and cell-cycle-dependent processes of DNA damage. This work aims to elucidate the mechanisms underlying the dual action of these polyphenols and investigates under which conditions they exhibit radioprotecting or radiosensitizing properties. The peripheral blood lymphocyte test system was used, applying concentrations ranging from 1.4 to 140 mu M curcumin and 2.2 to 220 mu M trans-resveratrol. The experimental design focuses first on their radioprotective effects in non-cycling lymphocytes, as uniquely visualized using cell fusion-mediated premature chromosome condensation, excluding, thus, cell-cycle interference to repair processes and activation of checkpoints. Second, the radiosensitizing potential of these chemicals on the induction of chromatid breaks in cultured lymphocytes following G2-phase irradiation was evaluated by a standardized G2-chromosomal radiosensitivity predictive assay. This assay uses caffeine for G2-checkpoint abrogation and it was applied to obtain an internal control for radiosensitivity testing, which simulates conditions similar to those of the highly radiosensitive lymphocytes of AT patients. The results demonstrate for the first time the cell-cycle-dependent action of these polyphenols. When non-cycling cells are irradiated, the radioprotective properties of curcumin and trans-resveratrol are more prominent. However, when cycling cells are irradiated during G2-phase, the radiosensitizing features of these compounds are more pronounced. This observation offers a new biological basis for the mechanisms underlying the action of these polyphenols in cancer radiotherapy. (C) 2014 Elsevier B.V. All rights reserved.

Dose assessment intercomparisons within the RENEB network were performed for triage biodosimetry analyzing GComparative analysis among different partners for dose assessment included shipment of PCC-slides and captured images to construct dose-response curves for up to 6 Gy γ-rays. Accident simulation exercises were performed to assess the suitability of the PCC assay by detecting speed of analysis and minimum number of cells required for categorization of potentially exposed individuals.Calibration data based on Giemsa-stained fragments in excess of 46 PCC were obtained by different partners using galleries of PCC images for each dose-point. Mean values derived from all scores yielded a linear dose-response with approximately 4 excess-fragments/cell/Gy. To unify scoring criteria, exercises were carried out using coded PCC-slides and/or coded irradiated blood samples. Analysis of samples received 24 h post-exposure was successfully performed using Giemsa staining (1 excess-fragment/cell/Gy) or centromere/telomere FISH-staining for dicentrics.Dose assessments by RENEB partners using appropriate calibration curves were mostly in good agreement. The PCC assay is quick and reliable for whole- or partial-body triage biodosimetry by scoring excess-fragments or dicentrics in G

Detrimental effects of ionizing radiation (IR) are correlated to the varying efficiency of IR to induce complex DNA damage. A double strand break (DSB) can be considered the simpler form of complex DNA damage. These types of damage can consist of DSBs, single strand breaks (SSBs) and/or non-DSB lesions such as base damages and apurinic/apyrimidinic (AP; abasic) sites in different combinations. Enthralling theoretical (Monte Carlo simulations) and experimental evidence suggests an increase in the complexity of DNA damage and therefore repair resistance with linear energy transfer (LET). In this study, we have measured the induction and processing of DSB and non-DSB oxidative clusters using adaptations of immunofluorescence. Specifically, we applied foci colocalization approaches as the most current methodologies for the in situ detection of clustered DNA lesions in a variety of human normal (FEP18-11-T1) and cancerous cell lines of varying repair efficiency (MCF7, HepG2, A549, MO59K/J) and radiation qualities of increasing LET, that is γ-, X-rays 0.3–1 keV/μm, α-particles 116 keV/μm and 36Ar ions 270 keV/μm. Using γ-H2AX or 53BP1 foci staining as DSB probes, we calculated a DSB apparent rate of 5–16 DSBs/cell/Gy decreasing with LET. A similar trend was measured for non-DSB oxidized base lesions detected using antibodies against the human repair enzymes 8-oxoguanine-DNA glycosylase (OGG1) or AP endonuclease (APE1), that is damage foci as probes for oxidized purines or abasic sites, respectively. In addition, using colocalization parameters previously introduced by our groups, we detected an increasing clustering of damage for DSBs and non-DSBs. We also make correlations of damage complexity with the repair efficiency of each cell line and we discuss the biological importance of these new findings with regard to the severity of IR due to the complex nature of its DNA damage.

The effect of cocaine and crack on the ploidy status of Feulgen-stained Tetrahymena pyriformis macronuclei using computerized DNA image analysis system was tested. For this purpose, selected doses of 5, 10 and 20 μg (per mL culture) of both drugs were administered for 2, 5 and 20 h to protozoa cultures and DNA image analysis of T. pyriformis nuclei was performed. The analysis was based on the measurement of the following parameters: Ploidy Balance (PB), Degree of Aneuploidy (DA), skewness and kurtosis. The results have shown a positive effect of both cocaine and crack on PB and on DA of T. pyriformis macronuclei. In particular, our results reveal that the aneugenic effect (which is expressed as a decrease in PB and an increase in DA) of cocaine on T. pyriformis macronuclei follows a dose-dependent manner, while crack induces aneuploidy in a dose-independent manner. Changes in the PB and DA values would induce a disturbance in the cellular density and heterogeneity of chromatin and the increase in skewness and kurtosis values after exposure of T. pyriformis to both drugs, did confirm this hypothesis. These observations were further correlated with alterations in the chromosomal segregation and with damage in mitotic spindle microtubules observed previously. In this study the impact of cocaine and crack on genomic instability and carcinogenesis was further supported and T. pyriformis can be proposed as a model organism to test the nuclear ploidy status after exposure to harmful chemicals and drugs.

Purpose To combine telomere and centromere (TC) staining of premature chromosome condensation (PCC) fusions to identify dicentrics, centric rings, and acentric chromosomes, making possible the realization of a dose–response curve and automation of the process. Methods and Materials Blood samples from healthy donors were exposed to 60 Co irradiation at varying doses up to 8 Gy, followed by a repair period of 8 hours. Premature chromosome condensation fusions were carried out, and TC staining using peptide nucleic acid probes was performed. Chromosomal aberration (CA) scoring was carried out manually and automatically using PCC-TCScore software, developed in our laboratory. Results We successfully optimized the hybridization conditions and image capture parameters, to increase the sensitivity and effectiveness of CA scoring. Dicentrics, centric rings, and acentric chromosomes were rapidly and accurately detected, leading to a linear-quadratic dose–response curve by manual scoring at up to 8 Gy. Using PCC-TCScore software for automatic scoring, we were able to detect 95% of dicentrics and centric rings. Conclusion The introduction of TC staining to the PCC fusion technique has made possible the rapid scoring of unstable CAs, including dicentrics, with a level of accuracy and ease not previously possible. This new approach can be used for biological dosimetry in radiation emergency medicine, where the rapid and accurate detection of dicentrics is a high priority using automated scoring. Because there is no culture time, this new approach can also be used for the follow-up of patients treated by genotoxic therapy, creating the possibility to perform the estimation of induced chromosomal aberrations immediately after the blood draw.

Checkpoint abrogation in G2 compromises repair of DNA double-strand breaks (DSB) and confers enhanced G2 chromosomal radiosensitivity in ataxia telangiectasia (AT) cells. To directly test this hypothesis, we combined calyculin A–induced premature chromosome condensation with conventional cytogenetics to evaluate chromosome damage before and after the G2 checkpoint in irradiated primary AT and normal human lymphocytes and their lymphoblastoid derivatives. Direct analysis of radiation damage in G2 by premature chromosome condensation reveals practically indistinguishable levels of chromosomal breaks in AT and normal cells. Yet a 4-fold increase in metaphase chromosome damage is observed in AT cells as compared with normal cells which, in contrast to AT cells, exhibit a strong G2 arrest manifest as an abrupt reduction in the mitotic index. Thus, an active checkpoint facilitates repair of chromosomal breaks in normal cells. Treatment with caffeine that abrogates the G2 checkpoint without significantly affecting DSB rejoining increases metaphase chromosome damage of normal cells to the AT level but leaves unchanged interphase chromosome damage in G2. Caffeine has no effect on any of these end points in AT cells. These observations represent the first direct evidence that the G2 checkpoint facilitates repair of chromosome damage, presumably by supporting repair of DNA DSBs. Failure to arrest will lead to chromatin condensation and conversion of unrepaired DNA DSBs to chromosomal breaks during G2-to-M phase transition. (Cancer Res 2005; 65(24): 11292-6)

The aim of the present study was to develop and standardize a cytogenetic approach for evaluation of the mutagenic potential of chemicals that induce cell cycle arrest in the G2 phase. Even though cytogenetic end-points such as sister chromatid exchange (SCE) have been extensively used to indirectly assess the DNA-damaging potential of various chemicals, they are based on metaphase chromosome analysis. Cells delayed in G2 phase after chemical exposure are not included in conventional SCE analysis. The yield of SCEs obtained, therefore, can be biased, since predominantly undamaged cells proceed to metaphase without delay. To overcome this shortcoming of conventional SCE analysis, the use of a new cytogenetic approach for genotoxic studies is presented that enables the analysis of SCEs directly in G2 phase using drug-induced premature chromosome condensation in cultured peripheral blood lymphocytes. By means of this method, firstly, the possibility that SCE analysis in metaphase chromosomes underestimates the mutagenic potential of various chemicals was tested. Secondly, whether the genotoxic potential of suspected carcinogens could be evaluated using SCE analysis in G2 phase, even at exposures that arrest cells in G2 phase, was examined. Thirdly, whether an important part of the background variation in SCE frequency among individuals is due to the delay of affected cells in G2 phase, rather than to a true biological variation in the cytogenetic end-point used, was tested. The results showed that a higher SCE frequency was scored in G2 phase than in metaphase. Subsequently, the mutagenic potential of chemicals that temporarily arrest cells in G2 phase could now be evaluated more accurately. In addition, it may be of interest to further examine the involvement of cell cycle kinetics in the baseline SCE variation among individuals since a lesser SCE variability was observed when the analysis was carried out in G2 phase rather than at metaphase.

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.

In Europe, a network for biological dosimetry has been created to strengthen the emergency preparedness and response capabilities in case of a large-scale nuclear accident or radiological emergency. Through the RENEB (Realising the European Network of Biodosimetry) project, 23 experienced laboratories from 16 European countries will establish a sustainable network for rapid, comprehensive and standardised biodosimetry provision that would be urgently required in an emergency situation on European ground. The foundation of the network is formed by five main pillars: (1) the ad hoc operational basis, (2) a basis of future developments, (3) an effective quality-management system, (4) arrangements to guarantee long-term sustainability and (5) awareness of the existence of RENEB. RENEB will thus provide a mechanism for quick, efficient and reliable support within the European radiation emergency management. The scientific basis of RENEB will concurrently contribute to increased safety in the field of radiation protection.

The established dogma in radiation sciences that underlies radiation protection and therapeutic applications is that radiation effects require induction of DNA damage only in cells that are directly hit by the radiation. However, extensive work during the last decade demonstrates that DNA damage responses can be detected in cells that are only bystanders. Such effects include cell killing and responses associated with DNA and chromosome damage. Here, we developed a strategy for investigating bystander effects on chromosomal integrity by premature chromosome condensation using hybrid cell formation between nontargeted human lymphocytes and targeted CHO cells or vice versa. We reasoned that signaling molecules generated in the targeted component of the hybrid will transfer to the nontargeted cell, inducing damage detectable at the chromosomal level. The results indicate that bystander cytogenetic effects between CHO and human lymphocytes cannot be detected under the experimental conditions used. This may be due either to the lack of communication of such responses between the components of the hybrid or to their abrogation by the experimental manipulations. These observations and the methodology developed should be useful in the further development of protocols for investigating bystander responses and for elucidating the underlying mechanisms.

Efforts to identify chemicals that are likely to pose a potential cancer threat to humans have intensified in recent years. Since radiation is a well known carcinogenic and leukemogenic agent, the chemically-induced radiosensitization may have important implications for human health. In this study, we propose an experimental methodology to evaluate in vitro the G2-chromosomal radiosensitization induced by chemicals at non-clastogenic doses and elucidate the mechanism involved. The proposed approach is based on a combination of cytogenetic methods, such as the G2chromosomal radiosensitivity assay (G2-assay) and the premature chromosome condensation (PCC) via calyculin-A induction or cell fusion. The constantly growing research regarding the clastogenic and carcinogenic potential of a variety of harmful physical and chemical agents includes the monitoring of cellular alterations linked with carcinogenesis such as (i) induction of chromosomal damage, (ii) disturbances of the DNA-repair machinery, and (iii) alterations in cell-cycle control mechanisms (Luch 2002; Sakata et al. 2007). However, most assessments of possible deleterious outcomes from environmental and occupational exposures are directed mainly towards single agents (either chemicals or radiation) and thus combined effects that may cause synergism often remain unclear. Since ionizing radiation has been associated with cancer (Finch 2007), chemically-induced-chromosomal radiosensitization may escalate the risk of genetic diseases and carcinogenesis (Scott et al. 1996). In some occupational environment (e.g., diagnostic radiology laboratories), the extensive exposure to both chemicals and radiation increases

Oxidatively-induced clustered DNA lesions are considered the signature of any ionizing radiation like the ones human beings are exposed daily from various environmental sources (medical X-rays, radon, etc.). To evaluate the role of BRCA1 deficiencies in the mitigation of radiation-induced toxicity and chromosomal instability we have used two human breast cancer cell lines, the BRCA1 deficient HCC1937 cells and as a control the BRCA1 wild-type MCF-7 cells. As an additional control for the DNA damage repair measurements, the HCC1937 cells with partially reconstituted BRCA1 expression were used. Since clustered DNA damage is considered the signature of ionizing radiation, we have measured the repair of double strand breaks (DSBs), non-DSB bistranded oxidative clustered DNA lesions (OCDLs) as well as single strand breaks (SSBs) in cells exposed to radiotherapy-relevant γ-ray doses. Parallel measurements were performed in the accumulation of chromatid and isochromatid breaks. For the measurement of OCDL repair, we have used a novel adaptation of the denaturing single cell gel electrophoresis (Comet assay) and pulsed field gel electrophoresis with Escherichia coli repair enzymes as DNA damage probes. Independent monitoring of the γ-H2AX foci was also performed while metaphase chromatid lesions were measured as an indicator of chromosomal instability. HCC1937 cells showed a significant accumulation of all types of DNA damage and chromatid breaks compared to MCF-7 while BRCA1 partial expression contributed significantly in the overall repair of OCDLs. These results further support the biological significance of repair resistant clustered DNA damage leading to chromosomal instability. The current results combined with previous findings on the minimized ability of base clusters to induce cell death (mainly induced by DSBs), enhance the potential association of OCDLs with breast cancer development especially in the case of a BRCA1 deficiency leading to the survival of breast cells carrying a high load of unrepaired DNA damage clusters.

Increased yield of chromatid breaks, following in vitro G2-phase lymphocyte irradiation, can be a marker of individual radiosensitivity and cancer predisposing genes whose role is to respond to DNA damage. Mutations or polymorphisms of genes encoding DNA repair pathways may underlie the increased chromosomal radiosensitivity. However, genes that facilitate DNA damage recognition, using signal transduction pathways to activate cell cycle arrest and preserve genomic integrity, are perhaps the most important determinant. Based on the latter hypothesis, an individual radiosensitivity parameter (IRP) is introduced, which expresses, at individual level, the G2-checkpoint potential to facilitate DNA damage recognition and repair of radiation-induced chromosomal damage during G2 to M-phase transition. Based on this parameter a new methodology for assessment of individual radiosensitivity is proposed, which involves G2-checkpoint abrogation by caffeine to obtain the IRP values. To evaluate the proposed methodology, blood samples from 52 healthy donors were taken for inter-individual radiosensitivity analysis using both the conventional G2 chromosomal radiosensitivity assay as well as the new approach using caffeine-induced G2-checkpoint abrogation. The two assays were compared in experiments using samples from 5 hypersensitive patients, 3 AT-homozygotes, 3 AT-heterozygotes, and the GM15786, GM03188A, GM09899, HCC1937 and MCF-7 cell lines. Using the G2 chromosomal radiosensitivity assay, donors are predicted as G2 radiosensitive or normal, while according to the new approach, individuals can be classified as highly radiosensitive, radiosensitive, normal, radioresistant and highly radioresistant. Overall, the new approach provides better individual radiosensitivity discrimination and intra-experimental reproducibility. Therefore, the proposed methodology using IRP values may provide a clinically applicable predictive assay for individual radiosensitivity and predisposition to cancer.

Glutaraldehyde (GA) is a high production volume chemical that is very reactive with a wide spectrum of medical, scientific and industrial applications. Since human exposure in anthropogenic and occupational environment occurs frequently, GA has been extensively tested for genotoxic activity in vitro and in vivo. However, there are conflicting results in the literature and there is a lack of information concerning the combined effects of exposure to both GA and ionizing radiation in human cells. In the present study, the results obtained using conventional cytogenetic analysis do not suggest a statistically significant clastogenic or genotoxic activity of GA when concentrations in the range of 10 -6 to 10 -2 mM were applied. However, a 24-h pre-irradiation exposure of human peripheral blood lymphocytes (PBLs) to non-genotoxic doses of GA showed a statistically significant (P > 0.05) increase in chromosomal radiosensitivity. The observed increase may be an effect of GA-induced alterations in the cell-cycle and feedback control mechanisms during the cell-cycle transition points or it may be a consequence of an effect of GA either on the DNA repair capacity of the cells after irradiation or on the initial induction of radiation-induced chromosomal damage. To elucidate the mechanism underlying the obtained radiosensitization, conventional cytogenetics, the G2 chromosomal radiosensitivity assay and premature chromosome condensation methodologies were applied. The results support the hypothesis that pre-irradiation exposure of PBLs to GA induces radiosensitization by increasing the initial yield of chromosomal aberrations following irradiation.

Measurement of dicentric chromosomes in human lymphocytes has been applied to assess dose received by potentially overexposed people and estimate risk for health effects. Since the dicentrics in exposed people decrease with time, the introduction of fluorescent in situ hybridization (FISH) enables to measure stable translocations for biodosimetry and address old or long-term exposures. In addition, premature chromosome condensation (PCC), which enables analysis in interphase, offers several advantages for biodosimetry. However, dose and risk estimates derived using cytogenetics and adequate calibration curves are based on the assumption that all individuals respond equally to radiation. Since increased radiosensitivity has been associated with cancer proneness, there is particular interest for risk assessment at the individual level. Towards this end, the efficiency of dynamics that govern DNA repair and apoptosis, as well as the conserved cellular processes that have evolved to facilitate DNA damage recognition using signal transduction pathways to activate cell cycle arrest and preserve genomic integrity, are being investigated. Recent work in cancer cytogenetics and on the modulation of radiation effects at the chromosome level using changes in gene expression associated with proteins or factors such as caffeine, amifostine, or heat treatment during G2 to M-phase transition, reconfirmed the importance of G2-chekpoint in determining radiosensitivity and of the cdk1/cyclin-B activity in the conversion of DNA damage into chromatid breaks. G2-chromosomal radiosensitivity offers therefore a basis for the identification or testing of key genetic targets for modulation of radiation effects, and a new screening method to detect intrinsic radiosensitivity is presented.

Cells of higher eukaryotes process double strand breaks (DSBs) in their genome using a non-homologous end joining apparatus that utilizes DNA-PK and other well characterized factors (D-NHEJ). Cells with defects in D-NHEJ, repair the majority of DSBs using a slow-repair pathway which is independent of genes of the RAD52 epistasis group and functions as a backup (B-NHEJ). Recent studies implicate DNA ligase III, PARP-1 and histone H1 in this pathway of NHEJ. The present study investigates the operation of B-NHEJ in the repair of interphase chromosome breaks visualized in irradiated G0 human lymphocytes by premature chromosome condensation (PCC). Chromosome breaks are effectively repaired in human lymphocytes, but repair is significantly compromised after treatment with wortmannin, a DNA-PK inhibitor. Despite slower kinetics, cells exposed to wortmannin rejoin the majority of IR induced chromosome breaks suggesting that B-NHEJ is also functional at the chromosome level. Complementation of D-NHEJ defect in wortmannin-treated lymphocytes by newly made DNA-PK is only possible under conditions of nuclear envelope break down and premature chromosome condensation, suggesting that in interphase cells the shunting of chromosome breaks from D-NHEJ to B-NHEJ is irreversible. The understanding of chromosomal aberration formation allows mechanistic explanations for the carcinogenic potential of D-NHEJ defects.

The hypothesis tested is that a 24-h pre-irradiation-exposure of peripheral blood lymphocytes (PBL) to the benzene metabolite hydroquinone (HQ), at doses that are non-acutely toxic (5 microM), induces a less efficient G2-M-checkpoint and enhances the G2-chromosomal radiosensitivity in a statistically significant manner (p

Cell fusion, premature chromosome condensation (PCC) and conventional cytogenetics were used to test whether the biochemical process of chromatin condensation-decondensation throughout the cell cycle, which depends on cyclin-regulated histone H1 kinase activity, affects the conversion of DNA damage into chromosome damage and determines intrinsic cell cycle-stage radiosensitivity. Results from three sets of experiments are presented. Irradiated G 0 human lymphocytes were fused to exponentially growing hamster cells and time allowed for repair, while following the hamster cells in their progress towards mitosis. Severe fragmentation was observed in the induced lymphocyte PCCs when hamster cells entered mitosis 13 h after irradiation, suggesting conversion of DNA damage into non-repairable chromosome damage during G 1 / S transition. When PCC was used to analyse chromosome damage directly in G 0 and G 2 phase lymphocytes, the induction of breaks per cell per chromatid per Gy was found to be similar, suggesting that G 2 increased radiosensitivity is related to chromatin condensation occurring during G 2 /M transition and not to an inherent chromatin structure at this phase. When chromatin condensation-decondensation at the G 1 /S and G 2 /M transitions was modified after irradiation by using conditioned media or elevated temperature (40°C), a dramatic change in the yield and the type of chromosomal aberrations was observed. All results obtained were consistent with the proposed hypothesis. They may be also helpful in the characterization of a DNA-chromosome damage conversion process which could give a biochemical explanation of the variability in radiosensitivity observed at the various stages of the cell cycle as well as among mutant cells and cells of different origin. The proposed conversion process is cell cycle-regulated and, therefore, subject to up-regulation or down-regulation following mutagen exposure and genetic alterations.