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The dicentric chromosome assay (DCA) is a well-established biodosimetry test to estimate exposure to ionizing radiation. The Korea Institute of Radiological and Medical Sciences (KIRAMS) established a DCA protocol as a medical response to radiation emergencies in South Korea. To maintain its accuracy and performance, intercomparison exercises with Health Canada (HC) have been conducted; herein, we aimed to validate our capacity of DCA analysis based on those results. Blood samples irradiated at HC were shipped to KIRAMS to assess the irradiation dose to blinded samples using conventional DCA full scoring and triage-based techniques (conventional DCA scoring in triage mode and DCA QuickScan method). Actual doses fell within the 95% confidence intervals of dose estimates for 70-100% of the blinded samples in 2015-2018. All methods discriminated binary dose categories, reflecting clinical significance. This DCA can be used as a reliable radiation biodosimetry tool in preparation for radiation accidents in South Korea., (© The Author(s) 2019. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.)

Purified microtubules have been shown to align along the static magnetic field (SMF) in vitro because of their diamagnetic anisotropy. However, whether mitotic spindle in cells can be aligned by magnetic field has not been experimentally proved. In particular, the biological effects of SMF of above 20 T (Tesla) have never been reported. Here we found that in both CNE-2Z and RPE1 human cells spindle orients in 27 T SMF. The direction of spindle alignment depended on the extent to which chromosomes were aligned to form a planar metaphase plate. Our results show that the magnetic torque acts on both microtubules and chromosomes, and the preferred direction of spindle alignment relative to the field depends more on chromosome alignment than microtubules. In addition, spindle morphology was also perturbed by 27 T SMF. This is the first reported study that investigated the cellular responses to ultra-high magnetic field of above 20 T. Our study not only found that ultra-high magnetic field can change the orientation and morphology of mitotic spindles, but also provided a tool to probe the role of spindle orientation and perturbation in developmental and cancer biology.

Background: Breast cancer risk increases drastically in individuals carrying a germline BRCA1 mutation. The exposure to ionizing radiation for diagnostic or therapeutic purposes of BRCA1 mutation carriers is counterintuitive, since BRCA1 is active in the DNA damage response pathway. The aim of this study was to investigate whether healthy BRCA1 mutations carriers demonstrate an increased radiosensitivity compared with healthy individuals., Methods: We defined a novel radiosensitivity indicator (RIND) based on two endpoints measured by the G2 micronucleus assay, reflecting defects in DNA repair and G2 arrest capacity after exposure to doses of 2 or 4 Gy. We investigated if a correlation between the RIND score and nonsense-mediated decay (NMD) could be established., Results: We found significantly increased radiosensitivity in the cohort of healthy BRCA1 mutation carriers compared with healthy controls. In addition, our analysis showed a significantly different distribution over the RIND scores (p = 0.034, Fisher's exact test) for healthy BRCA1 mutation carriers compared with non-carriers: 72 % of mutation carriers showed a radiosensitive phenotype (RIND score 1-4), whereas 72 % of the healthy volunteers showed no radiosensitivity (RIND score 0). Furthermore, 28 % of BRCA1 mutation carriers had a RIND score of 3 or 4 (not observed in control subjects). The radiosensitive phenotype was similar for relatives within several families, but not for unrelated individuals carrying the same mutation. The median RIND score was higher in patients with a mutation leading to a premature termination codon (PTC) located in the central part of the gene than in patients with a germline mutation in the 5' end of the gene., Conclusions: We show that BRCA1 mutations are associated with a radiosensitive phenotype related to a compromised DNA repair and G2 arrest capacity after exposure to either 2 or 4 Gy. Our study confirms that haploinsufficiency is the mechanism involved in radiosensitivity in patients with a PTC allele, but it suggests that further research is needed to evaluate alternative mechanisms for mutations not subjected to NMD.

Purpose: The purpose of this study was to determine whether radiation treatment induces chromosomal aberrations in uveal melanoma (UM) and to evaluate which tumor features determine success of karyotyping and FISH., Methods: Material from 327 UM-containing enucleated eyes was submitted for karyotyping, while FISH for chromosome 3 was performed in 248 samples. Thirty-six UMs had previously undergone irradiation. Karyotypes were analyzed, and the success rate of karyotyping/FISH was evaluated and compared with clinicopathologic tumor characteristics and prior irradiation., Results: Aberrations were observed in all chromosomes, with chromosomes 1, 3, 6, 8, 13, 15, 16, and Y being altered in at least 15% of the tumors. Aberrations were more common and more complex in previously irradiated tumors (significant for chromosomes 5 [P = 0.004] and 13 [P = 0.04]). Karyotyping and FISH failed significantly more often in irradiated tumors (both P < 0.001). In nonirradiated cases, successful karyotyping was related to a large tumor prominence (P = 0.004) and a high mitotic count (P = 0.007). The success of FISH in these tumors was not associated with any of the studied parameters. In irradiated tumors, karyotyping succeeded more frequently in cases with a high mitotic count (P = 0.03), whereas FISH was more often successful in tumors with a high mitotic count (P = 0.001), a large diameter (P = 0.009) and large prominence (P = 0.008)., Conclusions: Karyotyping and FISH are more often successful in UMs with features characteristic of high tumor aggressiveness, whereas prior irradiation leads to multiple chromosome aberrations and to unsuccessful tests. It will be interesting to determine whether other techniques can provide reliable information on the chromosome status of previously irradiated UMs.

Human exposure to high-linear energy transfer α-particles includes environmental (e.g. radon gas and its decay progeny), medical (e.g. radiopharmaceuticals) and occupational (nuclear industry) sources. The associated health risks of α-particle exposure for lung cancer are well documented however the risk estimates for leukaemia remain uncertain. To further our understanding of α-particle effects in target cells for leukaemogenesis and also to seek general markers of individual exposure to α-particles, this study assessed the transmission of chromosomal damage initially-induced in human haemopoietic stem and progenitor cells after exposure to high-LET α-particles. Cells surviving exposure were differentiated into mature T-cells by extra-thymic T-cell differentiation in vitro. Multiplex fluorescence in situ hybridisation (M-FISH) analysis of naïve T-cell populations showed the occurrence of stable (clonal) complex chromosome aberrations consistent with those that are characteristically induced in spherical cells by the traversal of a single α-particle track. Additionally, complex chromosome exchanges were observed in the progeny of irradiated mature T-cell populations. In addition to this, newly arising de novo chromosome aberrations were detected in cells which possessed clonal markers of α-particle exposure and also in cells which did not show any evidence of previous exposure, suggesting ongoing genomic instability in these populations. Our findings support the usefulness and reliability of employing complex chromosome exchanges as indicators of past or ongoing exposure to high-LET radiation and demonstrate the potential applicability to evaluate health risks associated with α-particle exposure.

Micronucleation of chromosomal DNA is an effective indicator of DNA damage and micronucleus (MN) analysis is a valuable tool for radiation biodosimetry studies. To gain a comprehensive knowledge of micronucleation process after ionising radiation (IR) exposure, whole genome-wide chromosome analysis is desirable. With this objective, multicolour fluorescence in situ hybridization (M-FISH) technique was utilised in the present study to characterise the chromosome content of spontaneous and IR-induced micronuclei in three human donors. M-FISH analysis revealed a radiation dose-dependant increase in the number of micronuclei with multi-chromosome material above 2 Gy and as many as 3-6 multicolour signals were detected in micronuclei after high γ-rays radiation doses (5-10 Gy). Involvement of each human chromosome material was more frequently detected in multicoloured micronuclei than in single-coloured micronuclei at high radiation doses (>2 Gy). Observation of dose-dependant increase in the MN frequency with multi-chromosome material may be due to misrepair of DNA double-strand breaks involving multiple chromosomes leading to asymmetric dicentric or ring chromosomes and acentric fragments. Chromosomes belonging to groups A (1, 2 and 3) and B (4 and 5) were frequently detected in 35-45% of the total micronuclei either as single entities or in combination with other chromosomes. Among the A and B groups, chromosome 1 material was consistently detected at high MN frequencies after radiation exposure in all the donors. Additionally, chromosomes 13 and 19 were more frequently observed in micronuclei than the expected frequency based on DNA content. Our whole genome approach utilising the M-FISH technique revealed that MN formation at high radiation doses might be complex involving multiple chromosome fragments. Understanding the fate and biological consequences of these multi-chromosome-containing micronuclei may provide key molecular insights for some aspects of IR-induced genomic instability and cancer development processes., (© The Author 2014. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The combination of automatic image acquisition and automatic image analysis of premature chromosome condensation (PCC) spreads was tested as a rapid biodosimeter protocol. Human peripheral lymphocytes were irradiated with (60)Co gamma rays in a single dose of between 1 and 20 Gy, stimulated with phytohaemaglutinin and incubated for 48 h, division blocked with Colcemid, and PCC-induced by Calyculin A. Images of chromosome spreads were captured and analysed automatically by combining the Metafer 4 and CellProfiler platforms. Automatic measurement of chromosome lengths allows the calculation of the length ratio (LR) of the longest and the shortest piece that can be used for dose estimation since this ratio is correlated with ionizing radiation dose. The LR of the longest and the shortest chromosome pieces showed the best goodness-of-fit to a linear model in the dose interval tested. The application of the automatic analysis increases the potential use of the PCC method for triage in the event of massive radiation causalities., (© The Author 2014. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.)

Down syndrome (DS) is caused by trisomy of chromosome 21 (Hsa21) and presents a complex phenotype that arises from abnormal dosage of genes on this chromosome. However, the individual dosage-sensitive genes underlying each phenotype remain largely unknown. To help dissect genotype--phenotype correlations in this complex syndrome, the first fully transchromosomic mouse model, the Tc1 mouse, which carries a copy of human chromosome 21 was produced in 2005. The Tc1 strain is trisomic for the majority of genes that cause phenotypes associated with DS, and this freely available mouse strain has become used widely to study DS, the effects of gene dosage abnormalities, and the effect on the basic biology of cells when a mouse carries a freely segregating human chromosome. Tc1 mice were created by a process that included irradiation microcell-mediated chromosome transfer of Hsa21 into recipient mouse embryonic stem cells. Here, the combination of next generation sequencing, array-CGH and fluorescence in situ hybridization technologies has enabled us to identify unsuspected rearrangements of Hsa21 in this mouse model; revealing one deletion, six duplications and more than 25 de novo structural rearrangements. Our study is not only essential for informing functional studies of the Tc1 mouse but also (1) presents for the first time a detailed sequence analysis of the effects of gamma radiation on an entire human chromosome, which gives some mechanistic insight into the effects of radiation damage on DNA, and (2) overcomes specific technical difficulties of assaying a human chromosome on a mouse background where highly conserved sequences may confound the analysis. Sequence data generated in this study is deposited in the ENA database, Study Accession number: ERP000439.

Dicentric assay is the international gold standard for cytogenetic biodosimetry after radiation exposure, despite being very labour-intensive, time-consuming, and highly expertise-dependent. It involves the identification of centromeres and structure of solid-stained chromosomes and the enumeration of dicentric chromosomes in a large number of first-division metaphases of cultured T lymphocytes. The dicentric yield is used to estimate the radiation exposure dosage according to a statistically derived and predetermined dose-response curve. It can be used for population triage after large-scale accidental over-exposure to ionising radiation or with a view to making clinical decisions for individual patients receiving substantial radiation. In this report, we describe our experience in the establishment of a cytogenetic biodosimetry laboratory in Queen Elizabeth Hospital, Hong Kong. This was part of the contingency plan for emergency measures against radiation accidents at nuclear power stations.

Intra-individual variation in G(2) chromosomal radiosensitivity was examined by repeatedly taking blood samples from two individuals. Two healthy female volunteers provided a total of 44 blood samples, Donor 1 gave 28 samples in four time periods between 2001 and 2006 and Donor 2 gave 16 samples in two of the same time periods. Lymphocytes were cultured for 72 h prior to irradiation with 0.5 Gy, 300 kV X-rays. Colcemid was added 30 min post-irradiation. Cultures were harvested 90 min post-irradiation and analysed for chromatid gaps and breaks. Donor 1 exhibited significant intra-individual variation in G(2) chromosomal radiosensitivity for two of the four time periods. Variation was not significant for Period 1 (13 samples, P = 0.111) and Period 2 (six samples, P = 0.311) but was significant for Period 3 (two samples, P = 0.030) and Period 4 (seven samples, P = 0.005). Significant intra-individual variation was observed for both time periods involving Donor 2, these being Period 2 (nine samples, P = 0.002) and Period 4 (seven samples, P < 0.001). The combined data from all time periods exhibited a significant intra-individual variation for Donor 1 (P < 0.001) and Donor 2 (P < 0.001). These findings led to the conclusion that too much reliance should not be placed on the result from a single sample when assessing individual radiosensitivity status.

Chromosome translocations are the hallmark of exposure to ionizing radiation, but they also occur spontaneously, and their frequencies increase dramatically with age. This complicates dosimetry unless a pre-exposure sample is available for each putatively exposed individual. Here we use published values for translocations in unexposed subjects from a wide range of ages, together with data from an in vitro (137)Cs dose-response curve, to estimate the minimum dose of whole-body radiation that is detectable by translocation analyses in individuals of a given age. For subjects aged 20 to 69 years, we show that the minimum detectable acute dose increases linearly with age at a rate of 0.179, 0.218 and 0.256 cGy per year for significance levels of P  =  0.05, P  =  0.01 and P  =  0.001, respectively. For chronic exposures, the corresponding minimum detectable doses are 1.591, 2.270 and 3.055 cGy per year. For newborns, the 95th and 99th percentiles of translocation frequencies are 0.20 and 0.31 per 100 cell equivalents, respectively, indicating that values greater than these are consistent with exposures at P  =  0.05 and P  =  0.01, respectively. These results improve our understanding of the requirements and limitations for performing biological dosimetry when only the age of the exposed individual is known.

Background: Radiotherapists are highly interested in optimizing doses especially for patients who tend to suffer from side effects of radiotherapy (RT). It seems to be helpful to identify radiosensitive individuals before RT. Thus we examined aberrations in FISH painted chromosomes in in vitro irradiated blood samples of a group of patients suffering from breast cancer. In parallel, a follow-up of side effects in these patients was registered and compared to detected chromosome aberrations., Methods: Blood samples (taken before radiotherapy) were irradiated in vitro with 3 Gy X-rays and analysed by FISH-painting to obtain aberration frequencies of first cycle metaphases for each patient. Aberration frequencies were analysed statistically to identify individuals with an elevated or reduced radiation response. Clinical data of patients have been recorded in parallel to gain knowledge on acute side effects of radiotherapy., Results: Eight patients with a significantly elevated or reduced aberration yield were identified by use of a t-test criterion. A comparison with clinical side effects revealed that among patients with elevated aberration yields one exhibited a higher degree of acute toxicity and two patients a premature onset of skin reaction already after a cumulative dose of only 10 Gy. A significant relationship existed between translocations in vitro and the time dependent occurrence of side effects of the skin during the therapy period., Conclusions: The results suggest that translocations can be used as a test to identify individuals with a potentially elevated radiosensitivity.

Biological dosimetry is an essential tool for estimating radiation dose. The dicentric chromosome assay (DCA) is currently the tool of choice. Because the assay is labor-intensive and time-consuming, strategies are needed to increase throughput for use in radiation mass casualty incidents. One such strategy is to truncate metaphase spread analysis for triage dose estimates by scoring 50 or fewer metaphases, compared to a routine analysis of 500 to 1000 metaphases, and to increase throughput using a large group of scorers in a biodosimetry network. Previously, the National Institutes for Allergies and Infectious Diseases (NIAID) and the Armed Forces Radiobiology Research Institute (AFRRI) sponsored a double-blinded interlaboratory comparison among five established international cytogenetic biodosimetry laboratories to determine the variability in calibration curves and in dose measurements in unknown, irradiated samples. In the present study, we further analyzed the published data from this previous study to investigate how the number of metaphase spreads influences dose prediction accuracy and how this information could be of value in the triage and management of people at risk for the acute radiation syndrome (ARS). Although, as expected, accuracy decreased with lower numbers of metaphase spreads analyzed, predicted doses by the laboratories were in good agreement and were judged to be adequate to guide diagnosis and treatment of ARS. These results demonstrate that for rapid triage, a network of cytogenetic biodosimetry laboratories can accurately assess doses even with a lower number of scored metaphases.

Individual response to ionizing radiation is an important information required to apply an efficient radiotherapy treatment against tumour and to avoid any adverse effects in normal tissues. In 1981, Fertil and Malaise have demonstrated that the post-irradiation local tumor control determined in vivo is correlated with clonogenic cell survival assessed in vitro. Furthermore, these authors have reminded the relevance of the concept of intrinsic radiosensitivity that is specific to each individual organ (Fertil and Malaise, 1981) [1]. To date, since clonogenicity assays are too time-consuming and do not provide any other molecular information, a plethora of research groups have attempted to determine the molecular bases of intrinsic radiosensitivity in order to propose reliable and faster predictive assays. To this aim, several approaches have been developed. Notably, the recent revolution in genomic and proteomic technologies is providing a considerable number of data but their link with radiosensitivity still remains to be elucidated. On another hand, the systematic screening of some candidate genes potentially involved in the radiation response is highlighting the complexity of the molecular and cellular mechanisms of DNA damage sensoring and signalling and shows that an abnormal radiation response is not necessarily due to the impairment of one single protein. Finally, more modest approaches consisting in focusing some specific functions of DNA repair seem to provide more reliable clues to predict over-acute reactions caused by radiotherapy. In this review, we endeavoured to analyse the contributions of these major approaches to predict human radiosensitivity., (Copyright © 2011 Académie des sciences. Published by Elsevier SAS. All rights reserved.)

Solar radiation is one of the most common threats to the skin, with exposure eliciting a specific protective cellular response. To decrypt the underlying mechanism, we used whole genome microarrays (Agilent 44K) to study epidermis gene expression in vivo in skin exposed to simulated solar radiation (SSR). We procured epidermis samples from healthy Caucasian patients, with phototypes II or III, and used two different SSR doses (2 and 4 J/cm(2)), the lower of which corresponded to the minimal erythemal dose. Analyses were carried out five hours after irradiation to identify early gene expression events in the photoprotective response. About 1.5% of genes from the human genome showed significant changes in gene expression. The annotations of these affected genes were assessed. They indicated a strengthening of the inflammation process and up-regulation of the JAK-STAT pathway and other pathways. Parallel to the p53 pathway, the p38 stress-responsive pathway was affected, supporting and mediating p53 function. We used an ex vivo assay with a specific inhibitor of p38 (SB203580) to investigate genes the expression of which was associated with active p38 kinase. We identified new direct p38 target genes and further characterized the role of p38. Our findings provide further insight into the physiological response to UV, including cell-cell interactions and cross-talk effects.

Chromosomal copy number alterations and chromosomal rearrangements are frequent mutations in human cancer. Unlike copy number alterations, little is known about the role and occurrence of chromosomal rearrangements in breast cancer. This may be due to the fact that chromosome-based breakpoint analysis is widely restricted to cultured cells. In order to identify gene rearrangements in breast cancer, we studied the chromosomal breakpoints in radiation-transformed epithelial breast cell lines using a high-resolution array-based approach using 1 Mb bacterial artificial chromosome (BAC) arrays. The breakpoints were further narrowed down by fluorescence in situ hybridisation (FISH) with clones from the 32 k BAC library. The analysis of the cell lines B42-11 and B42-16 revealed rearrangements of chromosomes 7, 8, 10 and 12. We identified the genes Has2, Grid1, Ret, Cpm, Tbx3, Tbx5, Tuba1a, Wnt1 and Arf3 within the breakpoint regions. Quantitative RT-PCR showed a deregulated expression of all of these candidate genes except for Tbx5 and Tbx3. This is the first study demonstrating gene rearrangements and their deregulated mRNA expression in radiation-transformed breast cells. Since the gene rearrangements occurred in the transformed and tumourigenic cell lines only, it is likely that these were generated in conjunction with malignant transformation of the epithelial breast cells and therefore might reflect early molecular events in breast carcinogenesis. Initial studies indicate that these gene alterations are also found in sporadic breast cancers.

We previously described an enhanced sensitivity for cell killing and gamma-H2AX focus induction after both high-dose-rate and continuous low-dose-rate gamma irradiation in 14 primary fibroblast strains derived from hereditary-type retinoblastoma family members (both affected RB1(+/-) probands and unaffected RB1(+/+) parents). Here we present G(2)-phase chromosomal radiosensitivity assay data for primary fibroblasts derived from these RB family members and five Coriell cell bank controls (four apparently normal individuals and one bilateral RB patient). The RB family members and two normal Coriell strains had significantly higher ( approximately 1.5-fold, P < 0.05) chromatid-type aberration frequencies in the first postirradiation mitosis after doses of 50 cGy and 1 Gy of (137)Cs gamma radiation compared to the remaining Coriell strains. The induction of chromatid-type aberrations by high-dose-rate G(2)-phase gamma irradiation is significantly correlated to the proliferative ability of these cells exposed to continuous low-dose-rate gamma irradiation (reported in Wilson et al., Radiat. Res. 169, 483-494, 2008). Our results suggest that these moderately radiosensitive individuals may harbor hypomorphic genetic variants in genomic maintenance and/or DNA repair genes or may carry epigenetic changes involving genes that more broadly modulate such systems, including G(2)-phase-specific DNA damage responses.

The study was undertaken to establish a dose calibration curve for a practical PCC ring assay and to apply it in a simulated mass casualty accident. The PCC assay was validated against the conventional dicentric assay. A linear relationship was established for PCC rings after (60)Co gamma irradiation with doses up to 20 Gy. In the simulated accident experiment, 62 blood samples were analyzed with both the PCC ring assay and the conventional dicentric assay, applying a triage approach. Samples received various uniform and non-uniform (10-40% partial-body) irradiations up to doses of 13 Gy. The results indicated that both assays yielded good dose estimates for the whole-body exposure scenario, although in the lower-dose range (0-6 Gy) dicentric scoring resulted in more accurate whole-body estimates, whereas PCC rings were better in the high-dose range (>6 Gy). Neither assay was successful in identifying partial-body exposures, most likely due to the low numbers of cells scored in the triage mode. In conclusion, the study confirmed that the PCC ring assay is suitable for use as a biodosimeter after whole-body exposure to high doses of radiation. However, there are limitations for its use in the triage of people exposed to high, partial-body doses.

Superb biological effectiveness and dose conformity represent a rationale for heavy-ion therapy, which has thus far achieved good cancer controllability while sparing critical normal organs. Immediately after irradiation, heavy ions produce dense ionization along their trajectories, cause irreparable clustered DNA damage, and alter cellular ultrastructure. These ions, as a consequence, inactivate cells more effectively with less cell-cycle and oxygen dependence than conventional photons. The modes of heavy ion-induced cell death/inactivation include apoptosis, necrosis, autophagy, premature senescence, accelerated differentiation, delayed reproductive death of progeny cells, and bystander cell death. This paper briefly reviews the current knowledge of the biological aspects of heavy-ion therapy, with emphasis on the authors' recent findings. The topics include (i) repair mechanisms of heavy ion-induced DNA damage, (ii) superior effects of heavy ions on radioresistant tumor cells (intratumor quiescent cell population, TP53-mutated and BCL2-overexpressing tumors), (iii) novel capacity of heavy ions in suppressing cancer metastasis and neoangiogenesis, and (iv) potential of heavy ions to induce secondary (especially breast) cancer.

We evaluated the chromosomal aberration (CA) frequencies in the peripheral blood lymphocytes of ten female patients, age average 43.7 +/- 12.9, with thyroid cancer (TC) who had been exposed to 100-200 mCi therapeutic doses of 131I. The blood samples were obtained before-treatment and at 2 and 24 h after-treatment. Radiation was measured in the samples by means of dysprosium-activated calcium sulfate thermoluminescent dosimetry. The maximum radiation levels were detected in the samples taken 2 h after treatment. A positive correlation was found between the sample-emitted radiation values and the frequencies of CAs (r = 0.495; p < 0.01). The average baseline frequency of aberrations found in the ten studied patients was 0.009 per cell. Upon application of the 131I therapeutic dose, this frequency increased to 0.04 and 0.02 CAs/cell at 2 and 24 h after-treatment, respectively (p < 0.05). Break-type aberrations experienced a peak at 2 h after-treatment, whereas rejoined aberrations, such as dicentrics, rings, and radial figures, increased with sampling time. Seven patients with metastases had high amounts of CAs at 2 and 24 h after-treatment, in comparison to three patients without metastases who had a lower frequency of CAs at 24h aftertreatment. This difference could be due to the fact that circulating lymphocytes were exposed to a greater cancerous tissue mass, which retains 131I during the diagnostic and therapeutic processes. These results demonstrate the importance of detecting and surgically removing the largest possible amount of thyroid tissue in order to diminish the exposure of normal cells to radiation.

Background: Lymphocytes of Fanconi anemia (FA) show an increased sensitivity to the alkylating agents such as mitomycin C (MMC), but their responses to gamma-irradiation is controversial. The extent of DNA damage in leukocytes of FA patients following irradiation and MMC treatment was studied at cellular and single chromosome level., Methods: DNA damage induced by gamma-rays and MMC was measured in leukocytes of FA patients and carriers at whole genome level using the comet assay. Also, at the DNA level of specific chromosome involved in this disease using a modified comet-FISH protocol with whole chromosome painting probes (chromosomes 16 and 13), DNA damage in leukocytes of FA patients and heterozygotes were compared to healthy individuals., Results: Baseline DNA damage in leukocytes of patients and heterozygotes was higher than in controls. Net induced DNA damage by gamma-rays in leukocytes of FA cases was not significantly different from that of healthy donors and heterozygotes. Net induced DNA damage by MMC was statistically higher and significantly different (P<0.05) in patients than other groups. Hybridization of chromosome 16 reveals more signals in the tail but the number of spots in the tail was not significantly higher than the hybridization spots for chromosome 13 in both gamma-irradiated and MMC treated samples., Conclusion: Results indicate that DNA damage induced by MMC could be a better index for diagnosis of FA patients compared to gamma-rays. Results of comet-FISH showed no difference between the sensitivity of chromosome 16 and 13 to MMC and radiation. It may indicate that, although the FA-A gene is located on chromosome 16, this chromosome might have a similar sensitivity as other chromosomes.

The association between chromosomal radiosensitivity and genetic predisposition to head and neck cancer was investigated in this study. In all, 101 head and neck cancer patients and 75 healthy control individuals were included in the study. The G(2) assay was used to measure chromosomal radiosensitivity. The results demonstrated that head and neck cancer patients had a statistically higher number of radiation-induced chromatid breaks than controls, with mean values of 1.23 and 1.10 breaks per cell, respectively (P<0.001). Using the 90th percentile of the G(2) scores of the healthy individuals as a cutoff value for chromosomal radiosensitivity, 26% of the cancer patients were radiosensitive compared with 9% of the healthy controls (P=0.008). The mean number of radiation-induced chromatid breaks and the proportion of radiosensitive individuals were highest for oral cavity cancer patients (1.26 breaks per cell, 38%) and pharynx cancer patients (1.27 breaks per cell, 35%). The difference between patients and controls was most pronounced in the lower age group (

Pathology archives provide unique and abundant opportunities to investigate human carcinogenesis and identify potential targets for cancer therapy. Microwaving was introduced into various procedures used in histopathology two decades ago, although the precise mechanisms underlying its effectiveness in any of the procedures, including antigen retrieval, acceleration of fixation and nucleic acid hybridization, are not known. Since microwaving was first applied to fluorescence in situ hybridization (FISH), many pathologists and researchers have enjoyed the benefits of excellent preservation of histological structures as well as good retrieval of FISH signals by this method. Microwave-assisted fluorescence in situ hybridization (MW-FISH) has proved to be especially useful in retrospective investigations of tissues fixed and preserved for long periods of time, and the success rates in the randomly selected pathology archives have been greater (70-95%) than by the conventional protocol (

Adaptive response (AR) is a well-documented phenomenon by which cells or organisms exposed to low dose of a genotoxicant become less sensitive to subsequent high-dose exposure to the same or another genotoxicant. AR, if induced can modify the efficacy leading to drug or radio-resistance, during anti-neoplastic drug or radiation treatment. Contradictions exist in AR induction by different genotoxicants with respect to the biomarkers, time schedules, and inter-individual variability, reflecting the complexity of AR in eukaryotic cells. In order to further ascertain these factors, AR induced by anti-neoplastic agents mitomycin C (MMC), bleomycin (BLM) and chemosterilant quinacrine dihydrochloride was examined in different donors and time schedules using cytogenetic biomarkers chromosome aberrations, sister chromatid exchanges and micronuclei (MN). BLM- and hyperthermia (HT)-induced cross-resistance to gamma rays and MMC/BLM, respectively, was also studied. Difference between MMC- and BLM-induced protective effects in biomarkers examined in the same donors was noticed. Adaptation to BLM and HT showed cross-resistance to chromosome damage induction by gamma rays and BLM/MMC, respectively. Cell cycle analysis indicated that adaptation is not caused by change in the rate of cell proliferation after challenge dose. MN as a chromosomal biomarker in large-scale population studies on AR is advocated, based on similar AR induced in all donors by MMC/BLM and rapid assessment in binucleated cells. Influence of certain genotypes on chromosomal biomarkers used in AR studies and role of AR in radiation and chemotherapy need to be further deciphered.

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.

Mazor, R., Korenstein-Ilan, A., Barbul, A., Eshet, Y., Shahadi, A., Jerby, E. and Korenstein, R. Increased Levels of Numerical Chromosome Aberrations after In Vitro Exposure of Human Peripheral Blood Lymphocytes to Radiofrequency Electromagnetic Fields for 72 Hours. Radiat. Res. 169, 28-37 (2008). We investigated the effects of 72 h in vitro exposure of 10 human lymphocyte samples to radiofrequency electromagnetic fields (800 MHz, continuous wave) on genomic instability. The lymphyocytes were exposed in a specially designed waveguide resonator at specific absorption rates (SARs) of 2.9 and 4.1 W/kg in a temperature range of 36-37 degrees C. The induced aneuploidy of chromosomes 1, 10, 11 and 17 was determined by interphase FISH using semi-automated image analysis. We observed increased levels of aneuploidy depending on the chromosome studied as well as on the level of exposure. In chromosomes 1 and 10, there was increased aneuploidy at the higher SAR, while for chromosomes 11 and 17, the increases were observed only for the lower SAR. Multisomy (chromosomal gains) appeared to be the primary contributor to the increased aneuploidy. The effect of temperature on the level of aneuploidy was examined over the range of 33.5-40 degrees C for 72 h with no statistically significant difference in the level of aneuploidy compared to 37 degrees C. These findings suggest the possible existence of an athermal effect of RF radiation that causes increased levels of aneuploidy. These results contribute to the assessment of potential health risks after continuous chronic exposure to RF radiation at SARs close to the current levels set by ICNIRP guidelines.

Mean terminal restriction fragment (TRF) lengths in white blood cells (WBCs) have been previously found to be associated with breast cancer. To assess whether this marker could be used as a test for breast cancer susceptibility in women, TRF length was measured in 72 treated female breast cancer patients and 1696 unaffected female controls between the ages of 45 and 77 from the Twin Research Unit at St Thomas' Hospital, as well as 140 newly diagnosed breast cancer cases and 108 mammographically screened unaffected controls from Guy's Hospital. Mean TRF was also tested for correlation with chromosome radiosensitivity and apoptotic response in the Guy's Hospital patients. After adjusting for age, smoking and body mass index, there was no significant difference in TRF lengths between the treated breast cancer patients and unaffected controls (P=0.71). A positive correlation between age-adjusted apoptotic response and mean TRF in newly diagnosed untreated breast cancer patients (P=0.008) was identified but no significant difference in TRF lengths between breast cancer patients and unaffected controls was detected (P=0.53). This suggests that TRF lengths in WBC, is not a marker of breast cancer susceptibility and does not vary significantly between affected women before and after treatment.

Background: Previous studies have demonstrated that telomeres in somatic cells are not randomly distributed at the end of the chromosomes. We hypothesize that these chromosome arm specific differences in telomere length (the telomere length pattern) may be actively maintained. In this study we investigate the existence and maintenance of the telomere length pattern in stem cells. For this aim we studied telomere length in primary human mesenchymal stem cells (hMSC) and their telomerase-immortalised counterpart (hMSC-telo1) during extended proliferation as well as after irradiation. Telomere lengths were measured using Fluorescence In Situ Hybridization (Q-FISH)., Results: A telomere length pattern was found to exist in primary hMSC's as well as in hMSC-telo1. This pattern is similar to what was previously found in lymphocytes and fibroblasts. The cells were then exposed to a high dose of ionizing radiation. Irradiation caused profound changes in chromosome specific telomere lengths, effectively destroying the telomere length pattern. Following long term culturing after irradiation, a telomere length pattern was found to re-emerge. However, the new telomere length pattern did not resemble the telomere length pattern observed before irradiation., Conclusion: Our findings indicate that a telomere length pattern does exist in mesenchymal stem cells and that the pattern is not actively re-established after destruction by irradiation.

Ionizing radiation can induce chromosome instability that is transmitted over many generations after irradiation in the progeny of surviving cells, but it remains unclear why this instability can be transmitted to the progeny. To acquire knowledge about the transmissible nature of genomic instability, we transferred an irradiated human chromosome into unirradiated mouse recipient cells by microcell fusion and examined the stability of the transferred human chromosome in the microcell hybrids. The transferred chromosome was stable in all six microcell hybrids in which an unirradiated human chromosome had been introduced. In contrast, the transferred chromosome was unstable in four out of five microcell hybrids in which an irradiated human chromosome had been introduced. The aberrations included changes in the irradiated chromosome itself and rearrangements with recipient mouse chromosomes. Thus the present study demonstrates that genomic instability can be transmitted to the progeny of unirradiated cells by a chromosome exposed to ionizing radiation, implying that the instability is caused by the irradiated chromosome itself and also that the instability is induced by the nontargeted effect of radiation.

Genetic heterogeneity in Nijmegen breakage syndrome (NBS) is highlighted by patients showing clinical and cellular features of NBS but with no mutations in NBS1 and normal levels of nibrin. NBS is an autosomal recessive disorder, whose clinical cellular signs include growth and developmental defects, dysmorphic facies, immunodeficiency, cancer predisposition, chromosomal instability and radiosensitivity. NBS is caused by mutations in the NBS1 gene, whose product is part of the MRE11/RAD50/NBS1 complex involved in the DNA double-strand break (DSB) response pathway. Since the identification of the NBS1 gene, patients with NBS clinical signs, particularly severe congenital microcephaly, are screened for mutations in the NBS1 gene. Further analyses include X-ray-induced chromosome aberrations, telomere analysis, kinetics of DSBs repair, levels of a panel of proteins involved in the maintenance of genetic stability, radiation-induced phosphorylation of various substrates and cell cycle analysis. We describe a patient with a NBS clinical phenotype, chromosomal sensitivity to X-rays but without mutations in the whole NBS1 or in the Cernunnos gene. Enhanced response to irradiation was mediated neither by DSBs rejoining defects nor by the NBS/AT-dependent DNA-damage response pathway. Notably, we found that primary fibroblasts from this patient displayed telomere length alterations. Cross-talk between pathways controlling response to DSBs and those involved in maintaining telomeres has been shown in the present patient. Dissecting the cellular phenotype of radiosensitive NBS-like patients represents a useful tool for the research of new genes involved in the cellular response to DSBs.

Unlabelled: The aim of this study was to investigate if irradiation with X-rays in different cell cycle phases resulted in a different response as measured with the micronucleus technique. In addition, the influence of irradiation temperature was investigated., Materials and Methods: Cells from a non-transformed human fibroblast cell line, HS2429, and a human breast cancer cell line, MCF-7, were synchronized by thymidine block and irradiated at either 2 degrees C or 37 degrees C in the G1-, S- and G2/M-phases. After cytokinesis-block by cytochalasin B, the frequency of micronuclei was determined., Results: Clear dose-response relationships were found. More micronuclei were detected in fibroblast cells irradiated in G1 and S than in G2/M, while the differences were not as prominent in MCF-7 cells. The irradiation temperature had no significant influence on the formation of micronuclei in either of the cell lines., Conclusion: The formation of micronuclei varies with the cell cycle stage at the time of irradiation.

We previously identified SIRT2, an nicotinamide adenine dinucleotide (NAD)-dependent tubulin deacetylase, as a protein downregulated in gliomas and glioma cell lines, which are characterized by aneuploidy. Other studies reported SIRT2 to be involved in mitotic progression in the normal cell cycle. We herein investigated whether SIRT2 functions in the mitotic checkpoint in response to mitotic stress caused by microtubule poisons. By monitoring chromosome condensation, the exogenously expressed SIRT2 was found to block the entry to chromosome condensation and subsequent hyperploid cell formation in glioma cell lines with a persistence of the cyclin B/cdc2 activity in response to mitotic stress. SIRT2 is thus a novel mitotic checkpoint protein that functions in the early metaphase to prevent chromosomal instability (CIN), characteristics previously reported for the CHFR protein. We further found that histone deacetylation, but not the aberrant DNA methylation of SIRT2 5'untranslated region is involved in the downregulation of SIRT2. Although SIRT2 is normally exclusively located in the cytoplasm, the rapid accumulation of SIRT2 in the nucleus was observed after treatment with a nuclear export inhibitor, leptomycin B and ionizing radiation in normal human fibroblasts, suggesting that nucleo-cytoplasmic shuttling regulates the SIRT2 function. Collectively, our results suggest that the further study of SIRT2 may thus provide new insights into the relationships among CIN, epigenetic regulation and tumorigenesis.

Aim: To determine whether the number of non-rejoining G2-chromatid breaks can predict the radiosensitivity of human cell lines., Methods: Cell lines of human ovary carcinoma cells (HO8910), human hepatoma cells (HepG2) and liver cells (L02) were irradiated with a range of doses and assessed both of cell survival and non-rejoining G2-chromatid breaks at 24 h after irradiation. Cell survival was documented by a colony assay. Non-rejoining G2-chromatid breaks were measured by counting the number of non-rejoining G2 chromatid breaks at 24 h after irradiation, detected by the prematurely chromosome condensed (PCC) technique., Results: A linear-quadratic survival curve was observed in three cell lines, and HepG2 was the most sensitive to gamma-radiation. A dose-dependent linear increase was observed in radiation-induced non-rejoining G2-PCC breaks measured at 24 h after irradiation in all cell lines, and HepG2 was the most susceptible to induction of non-rejoining G2-PCC breaks. A close correlation was found between the clonogenic radiosensitivity and the radiation-induced non-rejoining G2-PCC breaks (r = 0.923). Furthermore, survival-aberration correlations for two or more than two doses lever were also significant., Conclusion: The number of non-rejoining G2 PCC breaks holds considerable promise for predicting the radiosensitivity of normal and tumor cells when two or more than two doses lever is tested.

Histone H2AX is phosphorylated and forms foci in response to exposure to ionizing radiation. It has been thought that phosphorylated histone H2AX foci reflect unrepaired DNA double-strand breaks; however, we report here the localization of phosphorylated histone H2AX foci at the site of rejoined DNA double-strand breaks. We observed that phosphorylated histone H2AX foci remained even 96 h after exposure to X rays in interphase cells. To clarify the localization of residual phosphorylated histone H2AX foci, we examined localization of focus formation on mitotic chromosomes irradiated with X rays. We found that phosphorylated histone H2AX foci were located not only on chromosomal fragments but also on intact metaphase chromosomes without fragments. In anaphase cells, chromosomal bridges, which resulted from illegitimate rejoining of DNA broken ends, had phosphorylated histone H2AX foci. These foci were detected as individual small spots 30 min after X irradiation, but foci detected 20 or 96 h after X irradiation were clustered along the chromosomal bridges. These results indicate that phosphorylated histone H2AX foci persist if DNA breaks are rejoined. It is suggested that "residual" foci indicate an aberrant chromatin structure by illegitimate rejoining but not a DNA double-strand break itself.

Checkpoint abrogation in G(2) compromises repair of DNA double-strand breaks (DSB) and confers enhanced G(2) 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 G(2) checkpoint in irradiated primary AT and normal human lymphocytes and their lymphoblastoid derivatives. Direct analysis of radiation damage in G(2) 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 G(2) 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 G(2) checkpoint without significantly affecting DSB rejoining increases metaphase chromosome damage of normal cells to the AT level but leaves unchanged interphase chromosome damage in G(2). Caffeine has no effect on any of these end points in AT cells. These observations represent the first direct evidence that the G(2) 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 G(2)-to-M phase transition.

The melanoma genome possesses numerous recurrent chromosomal rearrangements, and embedded within this complexity are clues critical to disease pathogenesis and response to therapy. High-resolution genome-wide DNA copy number approaches, in conjunction with gene-specific mutational analyses, appear poised to define keystone molecular events, provide more accurate classification schemes, and set the stage for the design of rational therapies that may finally have an impact on survival of this deadly disease.

Cells respond to genotoxic insults such as ionizing radiation by halting in the G2 phase of the cell cycle. Delayed cell death (mitotic death) can occur when the cell is released from G2, and specific spindle defects form endopolyploid cells (endoreduplication/tetraploidy). Enhanced G2 chromosomal radiosensitivity has been observed in many cancers and genomic instability syndromes, and it is manifested by radiation-induced chromatid aberrations observed in lymphocytes of patients. Here we compare the G2 chromosomal radiosensitivity in prostate patients with benign prostatic hyperplasia (BPH) or prostate cancer with disease-free controls. We also investigated whether there is a correlation between G2 chromosomal radiosensitivity and aneuploidy (tetraploidy and endoreduplication), which are indicative of mitotic cell death. The G2 assay was carried out on all human blood samples. Metaphase analysis was conducted on the harvested chromosomes by counting the number of aberrations and the mitotic errors (endoreduplication/tetraploidy) separately per 100 metaphases. A total of 1/14 of the controls were radiosensitive in G2 compared to 6/15 of the BPH patients and 15/17 of the prostate cancer patients. Radiation-induced mitotic inhibition was assessed to determine the efficacy of G2 checkpoint control in the prostate patients. There was no significant correlation of G2 radiosensitivity scores and mitotic inhibition in BPH patients (P = 0.057), in contrast to prostate cancer patients, who showed a small but significant positive correlation (P = 0.029). Furthermore, there was no significant correlation between G2 radiosensitivity scores of BPH patients and endoreduplication/ tetraploidy (P = 0.136), which contrasted with an extremely significant correlation observed in prostate cancer patients (P < 0.0001). In conclusion, cells from prostate cancer patients show increased sensitivity to the induction of G2 aberrations from ionizing radiation exposure but paradoxically show reduced mitotic indices and aneuploidy as a function of aberration frequency.

In order to investigate the relationship between chromosomal radiosensitivity and early-onset cancer, the G(2) chromosomal radiosensitivity assay was undertaken on a group of 23 Danish survivors of childhood and adolescent cancer, a control group comprising their partners and a group of 38 of their offspring. In addition, the previously reported in-house control group from Westlakes Research Institute (WRI) was extended to 27 individuals. When using the 90th percentile cutoff for the WRI control group, the proportion of individuals with elevated radiosensitivity was 11, 35, 52 and 53% for the WRI control, partner control, cancer survivor and the offspring groups, respectively, with significant differences between the WRI control group and the cancer survivor group (P=0.002) and the offspring group (P<0.001). However, while the comparisons with the WRI control group support an association of chromosomal radiosensitivity with cancer predisposition, when the partner control group was used to define the radiosensitivity cutoff point, no significant differences in radiosensitivity profiles were found between the partner control group and either the cancer survivor group or the offspring group. The failure to distinguish between the G(2) aberration profiles of the apparently normal group of partners and the cancer survivor group suggests that any association with cancer should be viewed with caution, but also raises questions as to the suitability of the partners of cancer survivors to act as an appropriate control group. Heritability of the radiosensitive phenotype was examined by segregation analysis of the Danish families and suggested that 67.3% of the phenotypic variance of G(2) chromosomal radiosensitivity is attributable to a putative major gene locus with dominant effect.

Purpose: Despite its prominent contribution to cancer cure and palliation, around 1% to 5% of cancer patients suffer serious side effects from radiotherapy. A cardinal goal in the fields of radiobiology and oncology is to predict normal tissue radiosensitivity of a cancer patient before radiotherapy. Higher tumor control rates are likely if radiotherapy individualization could be achieved by applying predictive approaches., Experimental Design: Here, we make use of the cytokinesis block micronucleus assay to assess radiosensitivity in cell lines derived from two different cell lineages obtained from clinically radiosensitive patients. We determined the micronucleus frequency after graded doses of ionizing radiation to primary fibroblasts and lymphoblast cell lines derived from 36 highly radiosensitive cancer patients., Results: Many cell lines, following exposure to ionizing radiation, from patients with severe clinical reactions to radiotherapy showed statistically significantly higher frequencies of micronuclei than those from patients who had normal reactions to radiotherapy. One individual revealed significantly higher micronucleus frequencies in both cell lineages. Interestingly, lymphoblast cell lines from one patient showed micronucleus frequencies similar to ataxia telangiectasia mutated-deficient cells., Conclusions: These results indicate that the micronucleus assay may have use for identifying predisposition to clinical radiosensitivity, at least in a subset of patients as a component of a pretreatment radiosensitivity assay for use in the clinic.

Cytokinesis-blocked micronucleus assay (MNT) was applied in the peripheral blood lymphocytes of patients undergoing radioiodine-131 ((131)I) therapy for differentiated thyroid carcinoma (DTC) after thyroidectomy to assess the genotoxic risk of this therapy. The amount of administered (131)I activity varied from 3,330 to 4,030 MBq. Blood samples were taken immediately before (131)I administration and one month later, along with routine blood tests. Twenty-six patients underwent radioiodine ablation (RIA) or radioiodine therapy (RIT) after radical thyroidectomy. The aim of this therapy was to ablate residual thyroid tissue, to treat lymph node metastases and/or distant lung metastases. The amount of orally administered activity of (131)I ranged between 3,330 and 4,030 MBq according to the kind of therapy (RIA or RIT). In five patients the cytogenetic analysis was performed immediately before and one month after the second therapy which was given to them 6 months to 1 year after the first. Three patients were male and 23 female. The age of the patients ranged between 23 and 76 years (mean age: 48.6 years). Results show that after radioiodine therapy there is a significant increase in the frequency of micronuclei. Comparing the average frequency of micronuclei in the patients studied before and after (131)I, a more than doubling increase was found. Mean values +/- SD of the patients before and after (131)I therapy were 10.72 per thousand +/- 5.84 per thousandand 25.28 per thousand +/- 12.6 per thousand respectively. These findings indicate a genotoxic activity of (131)I therapy estimated after a period of one month.

In the present paper the relationship between cell cycle delays induced by Fe-ions of differing LET and the aberration yield observable in human lymphocytes at mitosis was examined. Cells of the same donor were irradiated with 990 MeV/n Fe-ions (LET=155 keV/micrometers), 200 MeV/n Fe-ions (LET=440 keV/micrometers) and X-rays and aberrations were measured in first cycle mitoses harvested at different times after 48-84 h in culture and in prematurely condensed G2-cells (PCCs) collected at 48 h using calyculin A. Analysis of the time-course of chromosomal damage in first cycle metaphases revealed that the aberration frequency was similar after X-ray irradiation, but increased two and seven fold after exposure to 990 and 200 MeV/n Fe-ions, respectively. Consequently, RBEs derived from late sampling times were significantly higher than those obtained at early times. The PCC-data suggest that the delayed entry of heavily damaged cells into mitosis results especially from a prolonged arrest in G2. Preliminary data obtained for 4.1 MeV/n Cr-ions (LET=3160 keV/micrometers) revealed, that these delays are even more pronounced for low energy Fe-like particles. Additionally, for the different radiation qualities, BrdU-labeling indices and apoptotic indices were determined at several time-points. Only the exposure to low energy Fe-like particles affected the entry of lymphocytes into S-phase and generated a significant apoptotic response indicating that under this particular exposure condition a large proportion of heavily damaged cells is rapidly eliminated from the cell population. The significance of this observation for the estimation of the health risk associated with space radiation remains to be elucidated., (c2004 COSPAR. Published by Elsevier Ltd. All rights reserved.)

Enhanced chromosomal radiosensitivity in breast cancer patients has been demonstrated in several studies. To investigate the chromosomal radiosensitivity of lymphocytes in breast cancer patients the G2 and micronucleus (MN) assays are often used. In these assays blood samples are exposed to ionizing radiation and the number of radiation-induced micronuclei or chromatid breaks are scored. In most studies investigating the in vitro chromosomal radiosensitivity of breast cancer patients the G2 and MN assays were performed on freshly drawn blood. The disadvantage of working with fresh blood samples is that in most cases only one blood sample can be obtained and that the assay cannot be easily repeated without further blood sampling. To allow repeated testing we propose the use of long-term cultures of T lymphocytes (IL-2 cultures). In this study we therefore investigated whether the radiation-induced MN response in IL-2 cultures was the same as in concordant whole blood cultures. For this study the MN assay (2 Gy) was performed on IL-2 cultures of 11 sensitive breast cancer patients and 20 healthy women. The results demonstrate that the enhanced chromosomal radiosensitivity observed in whole blood cultures of breast cancer patients is not present in IL-2 cultures derived from the same blood samples. Therefore, care has to be taken when IL-2 cultures are used to assess chromosomal radiosensitivity in breast cancer patients.

A multicolor banding (mBAND) fluorescence in situ hybridization technique was used to investigate the presence inhuman populations of a stable biomarker-intrachromosomal chromosome aberrations-of past exposure to high-LET radiation. Peripheral blood lymphocytes were taken from healthy Russian nuclear workers occupationally exposed from 1949 onward to either plutonium, gamma rays or both. Metaphase spreads were produced and chromosomes 1 and 2 were hybridized with mBAND FISH probes and scored for intra-chromosomal aberrations. A large yield of intrachromosomal aberrations was observed in both chromosomes of the individuals exposed to high doses of plutonium, whereas there was no significant increase over the (low) background control rate in the population who were exposed to high doses of gamma rays. Interchromosome aberration yields were similar in both the high plutonium and the high gamma-ray groups. These results for chromosome 1 and 2 confirm and extend data published previously for chromosome 5. Intrachromosomal aberrations thus represent a potential biomarker for past exposure to high-LET radiations such as alpha particles and neutrons and could possibly be used as a biodosimeter to estimate both the dose and type of radiation exposure in previously exposed populations.

Chromosome analysis using fluorescence in situ hybridization was undertaken on 294 retired workers from the British Nuclear Fuels plc facility at Sellafield, 95 with external occupational exposure <50 mSv, 108 with 50-499 mSv, and 91 with >500 mSv. In univariate analyses, external dose (P <10-s) and age (P = 0.0075) were significantly associated with translocation frequency, but no effect was found for smoking status. In a multivariate analysis with age and external dose as continuous variables, the slopes were 0.017 +/- 0.0075 x 10(-2) translocations per cell per year for age (P = 0.024) and 1.11+/- 0.190 x 10(-2) translocations per cell per sievert for external dose (P < 10(-5)). The dose response for translocation induction for occupational workers is similar to the linear component of in vitro dose-response curves, thus supporting the use of translocation frequency for retrospective biological dosimetry in situations of chronic low-dose exposure occurring over many years. The dose response obtained in this study is lower than the linear component of the dose response for stable chromosome aberrations obtained for the Japanese atomic bomb survivors. Thus, if chromosome aberration levels are indicative of cancer risk, this would suggest that low-dose risks derived from the Japanese atomic bomb survivor data will overestimate the risks associated with the occupational exposure encountered by the men in this study.

In the present study, we examined the potential contribution of irradiated medium to the bystander effect using custom-made double-Mylar stainless steel rings. Exponentially growing human-hamster hybrid (A(L)) cells were plated on either one or both sides of double-Mylar dishes 2-4 days before irradiation. One side (with or without cells) was irradiated with alpha particles using the track segment mode of a 4 MeV Van de Graaff accelerator at the Radiological Research Accelerator Facility of Columbia University. Since alpha particles can traverse only a very limited distance (around 23 microm in water), cells plated on the other side of a medium-filled Mylar dish will not be irradiated by the alpha particles. The results of the cytogenetic assay of unirradiated target cells that were attached to the top Mylar layer indicate that the number of chromatid-type aberrations was higher when there was a bottom layer of cells in the medium-filled chambers than with just medium alone. Furthermore, when the medium was transferred from these cell-irradiated dishes to fresh A(L) cell cultures, chromatid-type aberrations were produced in the unirradiated fresh cells. In contrast, medium irradiated in the absence of cells had no effect on chromatid aberrations. These results suggest that certain unidentified modulating factors secreted from the irradiated cells on the bottom Mylar layer into the medium induce chromatin damage in the unirradiated bystander cells.

To investigate the chromosomal radiosensitivity of lymphocytes in cancer patients the micronucleus (MN) assay is often used and performed on freshly drawn peripheral blood lymphocytes. The use of Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines may have a lot of advantages (e.g. large pool of cells) compared with fresh blood samples. In this study we have investigated whether the response of EBV-transformed lymphoblastoid cell lines to irradiation in the G1/S/G2 phases of the cell cycle is the same as in concordant whole blood cultures where primary lymphocytes were irradiated in the G0 phase of the cell cycle. For this study the MN assay (2 Gy) was performed on EBV-transformed cell lines of breast cancer patients and a group of healthy women. Those breast cancer patients were selected who showed an elevated chromosomal radiosensitivity in fresh blood samples in a previous study. The results demonstrated that the enhanced chromosomal radiosensitivity observed in fresh blood cultures of breast cancer patients is not present in EBV-transformed cell lines derived from the same blood samples. Therefore, care must be taken when EBV cell lines are used to assess chromosomal radiosensitivity in breast cancer patients.

The purpose of this study is to investigate the normal tissue damage caused by carbon-ion therapy. We measured chromosomal aberrations in peripheral blood lymphocytes before, during, and after radiotherapy, using the techniques of fluorescence in situ hybridization (FISH) and chemically induced premature chromosome condensation (PCC). Twenty-two lung cancer patients treated at HIMAC (Heavy Ion Medical Accelerator in Chiba) entered the study and signed an informed consent. Frequencies of lymphocytes with chromosomal aberrations at the end of carbon-ion therapy varied among the patients. This frequency was significantly correlated to the radiation field size and weakly correlated to the counts of white blood cells and lymphocytes during the treatment. As a result, we have found that chromosomal aberrations in peripheral blood lymphocytes from patients treated for lung cancer by carbon-ions were dependent on target volume, possibly reflecting the increased involvement of lymph nodes in the target field.

The inactivation of TP53 by transfection of a dominant- negative mutated TP53 (MP53.13 cells) was compared with inactivation of TP53 by transfection with the HPV E6 gene (RC10.1 cells) with respect to PLD repair, G(1)-phase arrest, and induction of color junctions. Functional G(1) arrest was demonstrated in parental (RKO) cells with wild-type TP53, while in RC10.1 cells the G(1) arrest was eliminated. In MP53.13 cells an intermediate G(1) arrest was found. Functionality of endogenous TP53 was confirmed in RKO and MP53.13 cells by accumulation of TP53 protein and its downstream target CDKN1A (p21). Radiation survival of MP53.13 cells was higher than that of RKO cells, and PLD repair was found in RKO cells and MP53.13 cells but not in RC10.1 cells. Both with and without irradiation, the number of color junctions was 50 to 80% higher in MP53.13 cells than in RKO and RC10.1 cells. In the MP53.13 cells, the genetic instability appears to lead to more aberrations and to radioresistance. In spite of the presence of an excess of mutated TP53, wild- type TP53 functions appear to be affected only partly or not at all.

Schmid et al. recently reported on the maximum low-dose RBE for mammography X rays (29 kV) for the induction of dicentrics in human lymphocytes. To obtain additional information on the RBE for this radiation quality, experiments with monochromatized synchrotron radiation were performed. Monochromatic 17.4 keV X rays were chosen for comparison with the diagnostic mammography X-ray spectrum to evaluate the spectral influence, while monochromatic 40 keV X rays represent a higher-energy reference radiation, within the experiment. The induction of dicentric chromosomes in human lymphocytes from one blood donor irradiated in vitro with 17.4 keV and 40 keV monochromatic X rays resulted in alpha coefficients of (3.44 +/- 0.87) x 10(-2) Gy(-1) and (2.37 +/- 0.93) x 10(-2) Gy(-1), respectively. These biological effects are only about half of the alpha coefficients reported earlier for exposure of blood from the same donor with the broad energy spectra of 29 kV X rays (mean energy of 17.4 keV) and 60 kV X rays (mean energy of 48 keV). A similar behavior is evident in terms of RBEM. Relative to weakly filtered 220 kV X rays, the RBEM for 17.4 and 40 keV monochromatic X rays is 0.86 +/- 0.23 and 0.59 +/- 0.24, respectively, which is in contrast to the RBEM of 1.64 +/- 0.27 for 29 kV X rays and 1.10 +/- 0.19 for 60 kV X rays. It is evident that the monochromatic radiations are less effective in inducing dicentric chromosomes than broad-spectrum X rays with the corresponding mean energy value. Therefore, it can be assumed that, for these X-ray qualities with broad energy spectra, a large fraction of the effects should be attributed predominantly to photons with energies well below the mean energy.

Confluent human fibroblast cells (AG1522) were irradiated with gamma rays, 490 MeV/nucleon silicon ions, or iron ions at either 200 or 500 MeV/nucleon. The cells were allowed to repair at 37 degrees C for 24 h after exposure, and a chemically induced premature chromosome condensation (PCC) technique was used to condense chromosomes in the G2 phase of the cell cycle. Incomplete and complex exchanges were analyzed in the irradiated samples. To verify that chromosomal breaks were truly unrejoined, chromosome aberrations were analyzed using a combination of whole-chromosome specific probes and probes specific for the telomere region of the chromosome. Results showed that the frequency of unrejoined chromosome breaks was higher after irradiation with the heavy ions of high LET, and consequently the ratio of incomplete to complete exchanges increased steadily with LET up to 440 keV/microm, the highest LET included in the present study. For samples exposed to 200 MeV/nucleon iron ions, chromosome aberrations were analyzed using the multicolor FISH (mFISH) technique, which allows identification of both complex and truly incomplete exchanges. Results of the mFISH study showed that 0.7 and 3 Gy iron ions produced similar ratios of complex to simple exchanges and incomplete to complete exchanges; these ratios were higher than those obtained after exposure to 6 Gy gamma rays. After 0.7 Gy of iron ions, most complex aberrations were found to involve three or four chromosomes, which is a likely indication of the maximum number of chromosome domains traversed by a single iron-ion track.