Retrospective Evaluation of Cytogenetic Effects Induced by Internal Radioiodine Exposure: A 27-Year Follow-Up Study.

Radioiodine (131I) is widely used in the treatment of hyperthyroidism and as an effective ablative therapy for differentiated thyroid cancer. Radioiodine (131I) constitutes 90% of the currently used therapies in the field of nuclear medicine. Here, we report the cytogenetic findings of a long-term follow-up study of 27 years on a male patient who received two rounds of radioiodine treatment within a span of 26 months between 1992 and 1994 for his papillary thyroid cancer. A comprehensive cytogenetic follow-up study utilizing cytokinesis blocked micronucleus assay, dicentric chromosome assay, genome wide translocations and inversions was initiated on this patient since the first administration of radioiodine in 1992. Frequencies of micronuclei (0.006/cell) and dicentric chromosomes (0.008/cell) detected in the current study were grossly similar to that reported earlier in 2019. The mFISH analysis detected chromosome aberrations in 8.6% of the cells in the form of both unbalanced and balanced translocations. Additionally, a clonal translocation involving chromosomes 14p; 15q was observed in 2 of the 500 cells analyzed. Out of the 500 cells examined, one cell showed a complex translocation (involving chromosomes 9, 10, and 16) besides 5 other chromosome rearrangements. Collectively, our study indicates that the past radioiodine exposure results in long-lasting chromosome damage and that the persistence of translocations can be useful for both retrospective biodosimetry and for monitoring chromosome instability in the lymphocytes of radioiodine exposed individuals.

Evaluation of Low-dose Radiation-induced DNA Damage and Repair in 3D Printed Human Cellular Constructs.

ABSTRACT: DNA double-strand breaks (DSBs) induced by ionizing radiation (IR) are considered to be the most critical lesion that when unrepaired or misrepaired leads to genomic instability or cell death depending on the radiation exposure dose. The potential health risks associated with exposures of low-dose radiation are of concern since they are being increasingly used in diverse medical and non-medical applications. Here, we have used a novel human tissue-like 3-dimensional bioprint to evaluate low-dose radiation-induced DNA damage response. For the generation of 3-dimensional tissue-like constructs, human hTERT immortalized foreskin fibroblast BJ1 cells were extrusion printed and further enzymatically gelled in a gellan microgel-based support bath. Low-dose radiation-induced DSBs and repair were analyzed in the tissue-like bioprints by indirect immunofluorescence using a well-known DSB surrogate marker, 53BP1, at different post-irradiation times (0.5 h, 6 h, and 24 h) after treatment with various doses of γ rays (50 mGy, 100 mGy, and 200 mGy). The 53BP1 foci showed a dose dependent induction in the tissue bioprints after 30 min of radiation exposure and subsequently declined at 6 h and 24 h in a dose-dependent manner. The residual 53BP1 foci number observed at 24 h post-irradiation time for the γ-ray doses of 50 mGy, 100 mGy, and 200 mGy was not statistically different from mock treated bioprints illustrative of an efficient DNA repair response at these low-dose exposures. Similar results were obtained for yet another DSB surrogate marker, γ-H2AX (phosphorylated form of histone H2A variant) in the human tissue-like constructs. Although we have primarily used foreskin fibroblasts, our bioprinting approach-mimicking a human tissue-like microenvironment-can be extended to different organ-specific cell types for evaluating the radio-response at low-dose and dose-rates of IR.

Validation of a High-Throughput Dicentric Chromosome Assay Using Complex Radiation Exposures.

Validation of biodosimetry assays is routinely performed using primarily orthovoltage irradiators at a conventional dose rate of approximately 1 Gy/min. However, incidental/ accidental exposures caused by nuclear weapons can be more complex. The aim of this work was to simulate the DNA damage effects mimicking those caused by the detonation of a several kilotons improvised nuclear device (IND). For this, we modeled complex exposures to: 1. a mixed (photons + IND-neutrons) field and 2. different dose rates that may come from the blast, nuclear fallout, or ground deposition of radionuclides (ground shine). Additionally, we assessed whether myeloid cytokines affect the precision of radiation dose estimation by modulating the frequency of dicentric chromosomes. To mimic different exposure scenarios, several irradiation systems were used. In a mixed field study, human blood samples were exposed to a photon field enriched with neutrons (ranging from 10% to 37%) from a source that mimics Hiroshima's A-bomb's energy spectrum (0.2-9 MeV). Using statistical analysis, we assessed whether photons and neutrons act in an additive or synergistic way to form dicentrics. For the dose rates study, human blood was exposed to photons or electrons at dose rates ranging from low (where the dose was spread over 32 h) to extremely high (where the dose was delivered in a fraction of a microsecond). Potential effects of cytokine treatment on biodosimetry dose predictions were analyzed in irradiated blood subjected to Neupogen or Neulasta for 24 or 48 h at the concentration recommended to forestall manifestation of an acute radiation syndrome in bomb survivors. All measurements were performed using a robotic station, the Rapid Automated Biodosimetry Tool II, programmed to culture lymphocytes and score dicentrics in multiwell plates (the RABiT-II DCA). In agreement with classical concepts of radiation biology, the RABiT-II DCA calibration curves suggested that the frequency of dicentrics depends on the type of radiation and is modulated by changes in the dose rate. The resulting dose-response curves suggested an intermediate dicentric yields and additive effects of photons and IND-neutrons in the mixed field. At ultra-high dose rate (600 Gy/s), affected lymphocytes exhibited significantly fewer dicentrics (P < 0.004, t test). In contrast, we did not find the dose-response modification effects of radiomitigators on the yields of dicentrics (Bonferroni corrected P > 0.006, ANOVA test). This result suggests no bias in the dose predictions should be expected after emergency cytokine treatment initiated up to 48 h prior to blood collection for dicentric analysis.

Ionizing Radiation-Induced DNA Damage Response in Primary Melanocytes and Keratinocytes of Human Skin.

Currently, our knowledge of how different cell types in a tissue microenvironment respond to low and high linear energy transfer (LET) radiation is highly restricted. In this study, a comparative analysis was performed on γ-ray-induced DNA damage and repair in primary human melanocytes and keratinocytes isolated from 3 donors. Our study demonstrates a modest interindividual variability in both melanocytes and keratinocytes in terms of both spontaneous and ionizing radiation (IR)-induced 53BP1 foci formation and persistence. Melanocytes, in general, showed a slightly elevated (1.66-2.79 folds more) 53BP1 foci induction relative to keratinocytes after exposure to different doses of γ-rays (0.1-2.5 Gy) radiation. To verify the influence of ATM kinase on IR-induced 53BP1 foci formation, melanocytes and keratinocytes were treated with a specific ATM kinase inhibitor (KU55993, 10 µM) for 1 h prior to radiation. ATM kinase inhibition resulted in the reduction of both spontaneous and IR-induced 53BP1 foci by 17-42% in both melanocytes and keratinocytes of all the 3 donors. Increased persistence of IR-induced 53BP1 foci number was observed in ATM-inhibited melanocytes and keratinocytes after different post exposure times (6 h and 24 h). Taken together, our study suggests that interindividual variations exist in the induction and repair of DNA double-strand breaks (DSBs) in melanocytes and keratinocytes and that ATM is crucial for an optimal DSB repair efficiency in both human skin cell types.

A matter of delicate balance: Loss and gain of Cockayne syndrome proteins in premature aging and cancer.

DNA repair genes are critical for preserving genomic stability and it is well established that mutations in DNA repair genes give rise to progeroid diseases due to perturbations in different DNA metabolic activities. Cockayne Syndrome (CS) is an autosomal recessive inheritance caused by inactivating mutations in CSA and CSB genes. This review will primarily focus on the two Cockayne Syndrome proteins, CSA and CSB, primarily known to be involved in Transcription Coupled Repair (TCR). Curiously, dysregulated expression of CS proteins has been shown to exhibit differential health outcomes: lack of CS proteins due to gene mutations invariably leads to complex premature aging phenotypes, while excess of CS proteins is associated with carcinogenesis. Thus it appears that CS genes act as a double-edged sword whose loss or gain of expression leads to premature aging and cancer. Future mechanistic studies on cell and animal models of CS can lead to potential biological targets for interventions in both aging and cancer development processes. Some of these exciting possibilities will be discussed in this review in light of the current literature.

Analysis of ionizing radiation induced DNA damage response in human adult stem cells and differentiated neurons.

Findings of neurodegenerative features associated with human radiosensitive syndromes such as Ataxia telangiectasia suggest that DNA repair efficiency is crucial for maintaining the functional integrity of central nervous system. To gain a better understanding of ionizing radiation (IR) induced DNA damage response in undifferentiated and differentiated neural cell types and to evaluate the role of ATM in DNA double strand break (DSB) repair, an in vitro human neural cell differentiation model system was utilized in this study. As compared to adult stem cells, differentiated neurons displayed an attenuated DSB repair response (as judged by the persistence of 53BP1 foci) after IR exposure and the attenuation was even more pronounced in stem cells and neurons after suppression of ATM (Ataxia Telangiectasia Mutated) gene product suggesting the importance of ATM for an optimal DSB repair efficiency in human neural cell types. In corroboration with an attenuated DNA damage response, a sharp decline in the expression levels of several DSB repair genes was observed in neurons. Our results suggest that cellular differentiation modulates the expression of several genes thereby compromising the DSB repair fidelity in post mitotic neurons. Further studies are required to verify whether or not ATM mediated exacerbation of DNA repair deficiency in differentiated neurons leads to neurodegeneration.

A comparative validation of biodosimetry and physical dosimetry techniques for possible triage applications in emergency dosimetry.

Large-scale radiological accidents or nuclear terrorist incidents involving radiological or nuclear materials can potentially expose thousands, or hundreds of thousands, of people to unknown radiation doses, requiring prompt dose reconstruction for appropriate triage. Two types of dosimetry methods namely, biodosimetry and physical dosimetry are currently utilized for estimating absorbed radiation dose in humans. Both methods have been tested separately in several inter-laboratory comparison exercises, but a direct comparison of physical dosimetry with biological dosimetry has not been performed to evaluate their dose prediction accuracies. The current work describes the results of the direct comparison of absorbed doses estimated by physical (smartphone components) and biodosimetry (dicentric chromosome assay (DCA) performed in human peripheral blood lymphocytes) methods. For comparison, human peripheral blood samples (biodosimetry) and different components of smartphones, namely surface mount resistors (SMRs), inductors and protective glasses (physical dosimetry) were exposed to different doses of photons (0-4.4 Gy; values refer to dose to blood after correction) and the absorbed radiation doses were reconstructed by biodosimetry (DCA) and physical dosimetry (optically stimulated luminescence (OSL)) methods. Additionally, LiF:Mg,Ti (TLD-100) chips and Al2O3:C (Luxel) films were used as reference TL and OSL dosimeters, respectively. The best coincidence between biodosimetry and physical dosimetry was observed for samples of blood and SMRs exposed toγ-rays. Significant differences were observed in the reconstructed doses by the two dosimetry methods for samples exposed to x-ray photons with energy below 100 keV. The discrepancy is probably due to the energy dependence of mass energy-absorption coefficients of the samples extracted from the phones. Our results of comparative validation of the radiation doses reconstructed by luminescence dosimetry from smartphone components with biodosimetry using DCA from human blood suggest the potential use of smartphone components as an effective emergency triage tool for high photon energies.

Human RecQL4 as a Novel Molecular Target for Cancer Therapy.

Human RecQ helicases play diverse roles in the maintenance of genomic stability. Inactivating mutations in 3 of the 5 human RecQ helicases are responsible for the pathogenesis of Werner syndrome (WS), Bloom syndrome (BS), Rothmund-Thomson syndrome (RTS), RAPADILINO, and Baller-Gerold syndrome (BGS). WS, BS, and RTS patients are at increased risk for developing many age-associated diseases including cancer. Mutations in RecQL1 and RecQL5 have not yet been associated with any human diseases so far. In terms of disease outcome, RecQL4 deserves special attention because mutations in RecQL4 result in 3 autosomal recessive syndromes (RTS type II, RAPADILINO, and BGS). RecQL4, like other human RecQ helicases, has been demonstrated to play a crucial role in the maintenance of genomic stability through participation in diverse DNA metabolic activities. Increased incidence of osteosarcoma in RecQL4-mutated RTS patients and elevated expression of RecQL4 in sporadic cancers including osteosarcoma suggest that loss or gain of RecQL4 expression is linked with cancer susceptibility. In this review, current and future perspectives are discussed on the potential use of RecQL4 as a novel cancer therapeutic target.

Cytogenetic follow-up studies on humans with internal and external exposure to ionizing radiation.

Cells exposed to ionizing radiation have a wide spectrum of DNA lesions that include DNA single-strand breaks, DNA double-strand breaks (DSBs), oxidative base damage and DNA-protein crosslinks. Among them, DSB is the most critical lesion, which when mis-repaired leads to unstable and stable chromosome aberrations. Currently, chromosome aberration analysis is the preferred method for biological monitoring of radiation-exposed humans. Stable chromosome aberrations, such as inversions and balanced translocations, persist in the peripheral blood lymphocytes of radiation-exposed humans for several years and, therefore, are potentially useful tools to prognosticate the health risks of radiation exposure, particularly in the hematopoietic system. In this review, we summarize the cytogenetic follow-up studies performed by REAC/TS (Radiation Emergency Assistance Center/Training site, Oak Ridge, USA) on humans exposed to internal and external radiation. In the light of our observations as well as the data existing in the literature, this review attempts to highlight the importance of follow-up studies for predicting the extent of genomic instability and its impact on delayed health risks in radiation-exposed victims.

RENEB Inter-Laboratory comparison 2017: limits and pitfalls of ILCs.

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.

RNF8 ubiquitinates RecQL4 and promotes its dissociation from DNA double strand breaks.

Ubiquitination-dependent DNA damage response (DDR) signals play a critical role in the cellular choice of DNA damage repair pathways. Human DNA helicase RecQL4 participates in DNA replication and repair, and loss of RecQL4 is associated with autosomal recessive genetic disorders characterized by genomic instability features. In an earlier study, RecQL4 was isolated as a stable complex that contained two ubiquitin ligases of the N-end rule (UBR1 and UBR2). However, it is unknown whether or not RecQL4 ubiquitination status is critical for its DNA repair function. Here, we report that RecQL4 directly interacts with RNF8 (a RING finger ubiquitin E3 ligase), and both co-localize at DNA double-strand break (DSB) sites. Our findings indicate that RNF8 ubiquitinates RecQL4 protein mainly at the lysine sites of 876, 1048, and 1101, thereby facilitating the dissociation of RecQL4 from DSB sites. RecQL4 mutant at ubiquitination sites had a significantly prolonged retention at DSBs, which hinders the recruitment of its direct downstream DSB repair proteins (CtIP & Ku80). Interestingly, reduced DSB repair capacity observed in RecQL4 depleted cells was restored only by the reconstitution of wild-type RecQL4, but not the ubiquitination mutant. Additionally, RecQL4 directly interacts with WRAP53ß that is known to recruit RNF8 to DSBs and WRAP53ß enhances the association of RecQL4 with RNF8. WRAP53ß silencing resulted in a nearly diminished recruitment of RNF8 to DSBs and in a greatly attenuated dissociation of RecQL4 from the DSB sites. Collectively, our study demonstrates that the ubiquitination event mediated by RNF8 constitutes an essential component for RecQL4's function in DSB repair.

Retrospective cytogenetic analysis of unstable and stable chromosome aberrations in the victims of radiation accident in Bulgaria.

Five occupational workers in an industrial sterilization unit at Stamboliyski in Bulgaria were accidentally exposed to a very high specific activity of Cobalt-60 source on June 14, 2011. Initial cytogenetic analysis performed on days 2 and 7 after radiation exposure revealed the whole body absorbed radiation doses of 5.32 Gy for patient 1, 3.40 Gy for patient 2, 2.50 Gy for patient 3, 1.91 Gy for patient 4 and 1.24 Gy for patient 5 [1]. Here, a retrospective multicolor FISH analysis was performed on three patients (patients 1, 2 and 3) using the blood samples collected over a period of 4 years from 2012 through 2015. In all the three patients, cells with stable chromosome aberrations (simple and complex chromosome translocations) were 3-4 folds more than cells with unstable chromosome aberrations (dicentric, rings and excess acentric chromosome fragments). In corroboration with the results reported in the literature, we observed that the time dependent decline of dicentrics, rings and excess acentric fragments occurred much more rapidly than chromosome translocations in the blood samples of the three victims. Further, inter-individual variation in the decline of radiation induced chromosome aberrations was also noticed among the three victims. The reason for the increased persistence of balanced chromosome translocations is not entirely clear but may be attributed to certain subsets of long-lived T-lymphocytes. The retrospective cytogenetic follow up studies on radiation-exposed victims may be useful for determining the extent of genomic/chromosomal instability in the hematopoietic system.

Radiation-induced DNA damage and repair effects on 3D genome organization.

The three-dimensional structure of chromosomes plays an important role in gene expression regulation and also influences the repair of radiation-induced DNA damage. Genomic aberrations that disrupt chromosome spatial domains can lead to diseases including cancer, but how the 3D genome structure responds to DNA damage is poorly understood. Here, we investigate the impact of DNA damage response and repair on 3D genome folding using Hi-C experiments on wild type cells and ataxia telangiectasia mutated (ATM) patient cells. We irradiate fibroblasts, lymphoblasts, and ATM-deficient fibroblasts with 5 Gy X-rays and perform Hi-C at 30 minutes, 24 hours, or 5 days after irradiation. We observe that 3D genome changes after irradiation are cell type-specific, with lymphoblastoid cells generally showing more contact changes than irradiated fibroblasts. However, all tested repair-proficient cell types exhibit an increased segregation of topologically associating domains (TADs). This TAD boundary strengthening after irradiation is not observed in ATM deficient fibroblasts and may indicate the presence of a mechanism to protect 3D genome structure integrity during DNA damage repair.

The RABiT-II DCA in the Rhesus Macaque Model.

An automated platform for cytogenetic biodosimetry, the "Rapid Automated Biodosimetry Tool II (RABiT-II)," adapts the dicentric chromosome assay (DCA) for high-throughput mass-screening of the population after a large-scale radiological event. To validate this test, the U.S. Federal Drug Administration (FDA) recommends demonstrating that the high-throughput biodosimetric assay in question correctly reports the dose in an in vivo model. Here we describe the use of rhesus macaques (Macaca mulatta) to augment human studies and validate the accuracy of the high-throughput version of the DCA. To perform analysis, we developed the 17/22-mer peptide nucleic acid (PNA) probes that bind to the rhesus macaque's centromeres. To our knowledge, these are the first custom PNA probes with high specificity that can be used for chromosome analysis in M. mulatta. The accuracy of fully-automated chromosome analysis was improved by optimizing a low-temperature telomere PNA FISH staining in multiwell plates and adding the telomere detection feature to our custom chromosome detection software, FluorQuantDic V4. The dicentric frequencies estimated from in vitro irradiated rhesus macaque samples were compared to human blood samples of individuals subjected to the same ex vivo irradiation conditions. The results of the RABiT-II DCA analysis suggest that, in the lymphocyte system, the dose responses to gamma radiation in the rhesus macaques were similar to those in humans, with small but statistically significant differences between these two model systems.

USP33 deubiquitinates PRKN/parkin and antagonizes its role in mitophagy.

PRKN/parkin activation through phosphorylation of its ubiquitin and ubiquitin-like domain by PINK1 is critical in mitophagy induction for eliminating the damaged mitochondria. Deubiquitinating enzymes (DUBs) functionally reversing PRKN ubiquitination are critical in controlling the magnitude of PRKN-mediated mitophagy process. However, potential DUBs that directly target PRKN and antagonize its pro-mitophagy effect remains to be identified and characterized. Here, we demonstrated that USP33/VDU1 is localized at the outer membrane of mitochondria and serves as a PRKN DUB through their interaction. Cellular and in vitro assays illustrated that USP33 deubiquitinates PRKN in a DUB activity-dependent manner. USP33 prefers to remove K6, K11, K48 and K63-linked ubiquitin conjugates from PRKN, and deubiquitinates PRKN mainly at Lys435. Mutation of this site leads to a significantly decreased level of K63-, but not K48-linked PRKN ubiquitination. USP33 deficiency enhanced both K48- and K63-linked PRKN ubiquitination, but only K63-linked PRKN ubiquitination was significantly increased under mitochondrial depolarization. Further, USP33 knockdown increased both PRKN protein stabilization and its translocation to depolarized mitochondria leading to the enhancement of mitophagy. Moreover, USP33 silencing protects SH-SY5Y human neuroblastoma cells from the neurotoxin MPTP-induced apoptotic cell death. Our findings convincingly demonstrate that USP33 is a novel PRKN deubiquitinase antagonizing its regulatory roles in mitophagy and SH-SY5Y neuron-like cell survival. Thus, USP33 inhibition may represents an attractive new therapeutic strategy for PD patients.Abbreviations: CCCP: carbonyl cyanide 3-chlorophenylhydrazone; DUB: deubiquitinating enzymes; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; OMM: outer mitochondrial membrane; PD: Parkinson disease; PINK1: PTEN induced kinase 1; PRKN/PARK2: parkin RBR E3 ubiquitin protein ligase; ROS: reactive oxygen species; TM: transmembrane; Ub: ubiquitin; UBA1: ubiquitin like modifier activating enzyme 1; UBE2L3/UbcH7: ubiquitin conjugating enzyme E2 L3; USP33: ubiquitin specific peptidase 33; WT: wild type.

Detection of Simple, Complex, and Clonal Chromosome Translocations Induced by Internal Radioiodine Exposure: A Cytogenetic Follow-Up Case Study after 25 Years.

Here, we report the findings of a 25-year cytogenetic follow-up study on a male patient who received 2 rounds of radioiodine treatment within a span of 26 months (1.78 GBq in 1992 and 14.5 GBq in 1994). The patient was 34 years old with a body mass index of 25 at the time of the first radioiodine treatment. Multicolor FISH and multicolor banding (mBAND) techniques performed on the patient detected inter- and intrachromosomal exchanges. Although the frequency of chromosome translocations remained essentially the same as reported in our earlier study (0.09/cell), the percentage of reciprocal (balanced) translocations increased from 54.38 to 80.30% in the current study. In addition to simple chromosome translocations, complex exchanges (0.29%) involving more than 2 chromosomes were detected for the first time in this patient. Strikingly, a clonal translocation involving chromosomes 14 and 15, t(14p;15q), was found in 7 of the 677 cells examined (1.03%). The presence of complex and clonal translocations indicates the onset of chromosomal instability induced by internal radioiodine exposure. mBAND analysis using probes specific for chromosomes 1, 2, 4, 5, and 10 revealed 5 inversions in a total of 717 cells (0.69%), and this inversion frequency is several-fold higher than the baseline frequency reported in healthy individuals using the classical G-banding technique. Collectively, our study suggests that stable chromosome aberrations such as translocations and inversions can be useful not only for retrospective biodosimetry but also for long-term monitoring of chromosomal instability caused by past radioiodine exposure.

Optimization and validation of automated dicentric chromosome analysis for radiological/nuclear triage applications.

Dicentric Chromosome Assay (DCA) is the most preferred cytogenetic technique for absorbed radiation dose assessment in exposed humans. However, DCA is somewhat impractical for triage application owing to its labor intensive and time consuming nature. Although lymphocyte culture for 48 h in vitro is inevitable for DCA, manual scoring of dicentric chromosomes (DCs) requires an additional time of 24-48 h, making the overall turnaround time of 72-96 h for dose estimation. To accelerate the speed of DC analysis for dose estimation, an automated tool was optimized and validated for triage mode of scoring. Several image training files were created to improve the specificity of automated DC analysis algorithm. Accuracy and efficiency of the automated (unsupervised) DC scoring was compared with the semi-automated scoring that involved human verification and correction of DCs (elimination of false positives and inclusion of true positives). DC scoring was performed by both automated and semi-automated modes for different doses of X-rays and γ-rays (0 Gy-5 Gy). Biodoses estimated from the frequencies of DCs detected by both automated (unsupervised) and semi-automated (supervised) scoring modes were grossly similar to the actual delivered doses in the range of 0.5 to 3 Gy of low LET radiation. We suggest that the automated DC tool can be effectively used for large scale radiological/nuclear incidents where a rapid segregation is essential for prioritizing moderately or severely exposed humans to receive appropriate medical countermeasures.

Use of human lymphocyte G0 PCCs to detect intra- and inter-chromosomal aberrations for early radiation biodosimetry and retrospective assessment of radiation-induced effects.

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.

CONCEPTS OF OPERATIONS FOR A US DOSIMETRY AND BIODOSIMETRY NETWORK.

The USA must be prepared to provide a prompt, coordinated and integrated response for radiation dose and injury assessment for suspected radiation exposure, whether it involves isolated cases or mass casualties. Dose estimation for radiation accidents typically necessitates a multiple parameter diagnostics approach that includes clinical, biological and physical dosimetry to provide an early-phase radiation dose. A US Individual Dosimetry and Biodosimetry Network (US-IDBN) will increase surge capacity for civilian and military populations in a large-scale incident. The network's goal is to leverage available resources and provide an integrated biodosimetry capability, using multiple parameter diagnostics. Initial operations will be to expand an existing functional integration of two cytogenetic biodosimetry laboratories by developing Standard Operating Procedures, cross-training laboratorians, developing common calibration curves, supporting inter-comparison exercises and obtaining certification to process clinical samples. Integration with certified commercial laboratories will increase surge capacity to meet the needs of a mass-casualty incident.