Genotoxicity of Gamma Radiation Against Lymphocytes of Radiation Workers: The Cytokinesis-Block Micronucleus Assay.

<b>Background and Objective:</b> Gamma irradiation induces genotoxicity, characterized by the formation of extra-nuclear bodies and left behind during the anaphase stage of cell division, often referred to as a micronucleus (MN). The present work aims to monitor exposure to ionizing radiation as a genotoxic agent in the lymphocytes of workers at radiation energy centers. <b>Materials and Methods:</b> The lymphocyte cytokinesis block micronucleus assay used and analyzed the correlation between the Nuclear Division Index (NDI), age, blood type and the number of micronuclei (MN). Blood samples were collected from 20 volunteers in heparin tubes, exposed to 2 Gy gamma rays and cultured <i>in vitro</i>. <b>Results:</b> A significant difference in the number of micronuclei between blood group A and blood groups A, B and AB. The Nuclear Division Index (NDI) value for lymphocytes of radiation energy center workers after gamma radiation was significant (1.74±0.1) but still within the normal range. Neither MN frequency nor NDI values correlated with age, but MN frequency showed a correlation with blood type. <b>Conclusion:</b> The gamma irradiation did not induce a cytostatic effect but proved genotoxic to the lymphocytes of radiation energy center workers. Notably, blood type A demonstrated higher sensitivity to gamma radiation.

Mobile phone specific radiation disturbs cytokinesis and causes cell death but not acute chromosomal damage in buccal cells: Results of a controlled human intervention study.

Several human studies indicate that mobile phone specific electromagnetic fields may cause cancer in humans but the underlying molecular mechanisms are currently not known. Studies concerning chromosomal damage (which is causally related to cancer induction) are controversial and those addressing this issue in mobile phone users are based on the use of questionnaires to assess the exposure. We realized the first human intervention trial in which chromosomal damage and acute toxic effects were studied under controlled conditions. The participants were exposed via headsets at one randomly assigned side of the head to low and high doses of a UMTS signal (n = 20, to 0.1 W/kg and n = 21 to 1.6 W/kg Specific Absorption Rate) for 2 h on 5 consecutive days. Before and three weeks after the exposure, buccal cells were collected from both cheeks and micronuclei (MN, which are formed as a consequence of structural and numerical chromosomal aberrations) and other nuclear anomalies reflecting mitotic disturbance and acute cytotoxic effects were scored. We found no evidence for induction of MN and of nuclear buds which are caused by gene amplifications, but a significant increase of binucleated cells which are formed as a consequence of disturbed cell divisions, and of karyolitic cells, which are indicative for cell death. No such effects were seen in cells from the less exposed side. Our findings indicate that mobile phone specific high frequency electromagnetic fields do not cause acute chromosomal damage in oral mucosa cells under the present experimental conditions. However, we found clear evidence for disturbance of the cell cycle and cytotoxicity. These effects may play a causal role in the induction of adverse long term health effects in humans.

Assessment of Micronuclei Frequency in the Peripheral Blood of Adult and Pediatric Patients Receiving Fractionated Total Body Irradiation.

The cytokinesis-block micronucleus (CBMN) assay is an established method for assessing chromosome damage in human peripheral blood lymphocytes resulting from exposure to genotoxic agents such as ionizing radiation. The objective of this study was to measure cytogenetic DNA damage and hematology parameters in vivo based on MN frequency in peripheral blood lymphocytes (PBLs) from adult and pediatric leukemia patients undergoing hematopoietic stem cell transplantation preceded by total body irradiation (TBI) as part of the conditioning regimen. CBMN assay cultures were prepared from fresh blood samples collected before and at 4 and 24 h after the start of TBI, corresponding to doses of 1.25 Gy and 3.75 Gy, respectively. For both age groups, there was a significant increase in MN yields with increasing dose (p < 0.05) and dose-dependent decrease in the nuclear division index (NDI; p < 0.0001). In the pre-radiotherapy samples, there was a significantly higher NDI measured in the pediatric cohort compared to the adult due to an increase in the percentage of tri- and quadri-nucleated cells scored. Complete blood counts with differential recorded before and after TBI at the 24-h time point showed a rapid increase in neutrophil (p = 0.0001) and decrease in lymphocyte (p = 0.0006) counts, resulting in a highly elevated neutrophil-to-lymphocyte ratio (NLR) of 14.45 ± 1.85 after 3.75 Gy TBI (pre-exposure = 4.62 ± 0.49), indicating a strong systemic inflammatory response. Correlation of the hematological cell subset counts with cytogenetic damage, indicated that only the lymphocyte subset survival fraction (after TBI compared with before TBI) showed a negative correlation with increasing MN frequency from 0 to 1.25 Gy (r = -0.931; p = 0.007). Further, the data presented here indicate that the combination of CBMN assay endpoints (MN frequency and NDI values) and hematology parameters could be used to assess cytogenetic damage and early hematopoietic injury in the peripheral blood of leukemia patients, 24 h after TBI exposure.

RENEB Inter-Laboratory Comparison 2021: The Cytokinesis-Block Micronucleus Assay.

The goal of the RENEB inter-laboratory comparison 2021 exercise was to simulate a large-scale radiation accident involving a network of biodosimetry labs. Labs were required to perform their analyses using different biodosimetric assays in triage mode scoring and to rapidly report estimated radiation doses to the organizing institution. This article reports the results obtained with the cytokinesis-block micronucleus assay. Three test samples were exposed to blinded doses of 0, 1.2 and 3.5 Gy X-ray doses (240 kVp, 13 mA, ∼75 keV, 1 Gy/min). These doses belong to 3 triage categories of clinical relevance: a low dose category, for no exposure or exposures inferior to 1 Gy, requiring no direct treatment of subjects; a medium dose category, with doses ranging from 1 to 2 Gy, and a high dose category, after exposure to doses higher than 2 Gy, with the two latter requiring increasing medical attention. After irradiation the test samples (no. 1, no. 2 and no. 3) were sent by the organizing laboratory to 14 centers participating in the micronucleus assay exercise. Laboratories were asked to setup micronucleus cultures and to perform the micronucleus assay in triage mode, scoring 500 binucleated cells manually, or 1,000 binucleated cells in automated/semi-automated mode. One laboratory received no blood samples, but scored pictures from another lab. Based on their calibration curves, laboratories had to provide estimates of the administered doses. The accuracy of the reported dose estimates was further analyzed by the micronucleus assay lead. The micronucleus assay allowed classification of samples in the corresponding clinical triage categories (low, medium, high dose category) in 88% of cases (manual scoring, 88%; semi-automated scoring, 100%; automated scoring, 73%). Agreement between scoring laboratories, assessed by calculating the Fleiss' kappa, was excellent (100%) for semi-automated scoring, good (83%) for manual scoring and poor (53%) for fully automated scoring. Correct classification into triage scoring dose intervals (reference dose ±0.5 Gy for doses ≤2.5 Gy, or reference dose ±1 Gy for doses >2.5 Gy), recommended for triage biodosimetry, was obtained in 79% of cases (manual scoring, 73%; semi-automated scoring, 100%; automated scoring, 67%). The percentage of dose estimates whose 95% confidence intervals included the reference dose was 58% (manual scoring, 48%; semiautomated scoring, 72%; automated scoring, 60%). For the irradiated samples no. 2 and no. 3, a systematic shift towards higher dose estimations was observed. This was also noticed with the other cytogenetic assays in this intercomparison exercise. Accuracy of the rapid triage modality could be maintained when the number of manually scored cells was scaled down to 200 binucleated cells. In conclusion, the micronucleus assay, preferably performed in a semi-automated or manual scoring mode, is a reliable technique to perform rapid biodosimetry analysis in large-scale radiation emergencies.

THz irradiation inhibits cell division by affecting actin dynamics.

Biological phenomena induced by terahertz (THz) irradiation are described in recent reports, but underlying mechanisms, structural and dynamical change of specific molecules are still unclear. In this paper, we performed time-lapse morphological analysis of human cells and found that THz irradiation halts cell division at cytokinesis. At the end of cytokinesis, the contractile ring, which consists of filamentous actin (F-actin), needs to disappear; however, it remained for 1 hour under THz irradiation. Induction of the functional structures of F-actin was also observed in interphase cells. Similar phenomena were also observed under chemical treatment (jasplakinolide), indicating that THz irradiation assists actin polymerization. We previously reported that THz irradiation enhances the polymerization of purified actin in vitro; our current work shows that it increases cytoplasmic F-actin in vivo. Thus, we identified one of the key biomechanisms affected by THz waves.

The cytokinesis-block micronucleus assay for cryopreserved whole blood.

PURPOSE: The cytokinesis-block micronucleus (MN) assay is a widely used technique in basic radiobiology research, human biomonitoring studies and in vitro radiosensitivity testing. Fresh whole blood cultures are commonly used for these purposes, but immediate processing of fresh samples can be logistically challenging. Therefore, we aimed at establishing a protocol for the MN assay on cryopreserved whole blood, followed by a thorough evaluation of the reliability of this assay for use in radiosensitivity assessment in patients. MATERIALS AND METHODS: Whole blood samples of 20 healthy donors and 4 patients with a primary immunodeficiency disease (PID) were collected to compare the results obtained with the MN assay performed on fresh versus cryopreserved whole blood samples. MN yields were scored after irradiation with 220 kV X-rays (dose rate 3 Gy/min), with doses ranging from 0.5-2 Gy. RESULTS: The application of the MN assay on cryopreserved blood samples was successful in all analyzed samples. The radiation-induced MN and NDI scores in fresh and cryopreserved blood cultures were found to be similar. Acceptable inter-individual and intra-individual variabilities in MN yields were observed. Repeated analysis of cryopreserved blood cultures originating from the same blood sample, thawed at different time points, revealed that MN values remain stable for cryopreservation periods up to one year. Finally, radiosensitive patients were successfully identified using the MN assay on cryopreserved samples. CONCLUSIONS: To our knowledge, this study is the first report of the successful use of cryopreserved whole blood samples for application of the MN assay. The data presented here demonstrate that the MN assay performed on cryopreserved whole blood is reliable for radiosensitivity testing. Our results also support its wider use in epidemiological, biomonitoring and genotoxicity studies. The presented method of cryopreservation of blood samples might also benefit other assays.

Repair-independent functions of DNA-PKcs protect irradiated cells from mitotic slippage and accelerated senescence.

The binding of DNA-dependent protein kinase catalytic subunit (DNA-PKcs, also known as PRKDC) to Ku proteins at DNA double-strand breaks (DSBs) has long been considered essential for non-homologous end joining (NHEJ) repair, providing a rationale for use of DNA-PKcs inhibitors as cancer therapeutics. Given lagging clinical translation, we reexamined mechanisms and observed instead that DSB repair can proceed independently of DNA-PKcs. While repair of radiation-induced DSBs was blocked in cells expressing shRNAs targeting Ku proteins or other NHEJ core factors, DSBs were repaired on schedule despite targeting DNA-PKcs. Although we failed to observe a DSB repair defect, the γH2AX foci that formed at sites of DNA damage persisted indefinitely after irradiation, leading to cytokinesis failure and accumulation of binucleated cells. Following this mitotic slippage, cells with decreased DNA-PKcs underwent accelerated cellular senescence. We identified downregulation of ataxia-telangiectasia mutated kinase (ATM) as the critical role of DNA-PKcs in recovery from DNA damage, insofar as targeting ATM restored γH2AX foci resolution and cytokinesis. Considering the lack of direct impact on DSB repair and emerging links between senescence and resistance to cancer therapy, these results suggest reassessing DNA-PKcs as a target for cancer treatment.

Correlation between cytogenetic biomarkers obtained from DC and CBMN assays caused by low dose radon exposure in smokers.

Purpose: Radon is of health concern because they can cause lung cancer when inhaled over many years. Cytogenetic biomarkers proved to be excellent methods to detect and estimate radiation induced DNA damage. Two well established cytogenetic assays are available to detect the DNA damage caused by ionizing radiation namely Dicentric (DC) assay and Cytokinesis-Blocked Micro Nucleus (CBMN) assay. Chromosomal aberrations such as dicentric and acentric fragments can be analyzed in DC assay. Micronuclei and Nucleoplasmic bridges can be analyzed in CBMN assay. The present study focuses on correlation between the aberrations observed in DC and CBMN assay caused by low doses of radon in smokers. Material and methods: Blood samples were collected, after prior consent, from smokers as well as healthy non-smoking individuals and exposed in vitro to radon ranging between 0-5.2 mGy using a simple, portable irradiation assembly designed and tested at authors' laboratory. Results: A significant increase in frequencies of dicentrics and excess acentric fragments were observed with increasing dose. Interestingly in CBMN assay also, the frequencies of micronuclei and nucleoplasmic bridges were increased with dose. There was a significant correlation between nucleoplasmic bridges and dicentrics for both smokers and non-smokers. Significant correlation between acentric fragments and micronucleus was observed only for non-smokers. Conclusions: By considering the correlation between the two cytogenetic assays, the CBMN assay can be used as an alternative to DC assay for non-smokers. In case of smokers, nucleoplasmic bridges can be used to measure the radiation exposure. This study is first of its kind to correlate the cytogenetic biomarkers obtained from DC and CBMN assay for smokers with very low doses of radon.

Automated Triage Radiation Biodosimetry: Integrating Imaging Flow Cytometry with High-Throughput Robotics to Perform the Cytokinesis-Block Micronucleus Assay.

The cytokinesis-block micronucleus (CBMN) assay has become a fully-validated and standardized method for radiation biodosimetry. The assay is typically performed using microscopy, which is labor intensive, time consuming and impractical after a large-scale radiological/nuclear event. Imaging flow cytometry (IFC), which combines the statistical power of traditional flow cytometry with the sensitivity and specificity of microscopy, has been recently used to perform the CBMN assay. Since this technology is capable of automated sample acquisition and multi-file analysis, we have integrated IFC into our Rapid Automated Biodosimetry Technology (RABiT-II). Assay development and optimization studies were designed to increase the yield of binucleated cells (BNCs), and improve data acquisition and analysis templates to increase the speed and accuracy of image analysis. Human peripheral blood samples were exposed ex vivo with up to 4 Gy of c rays at a dose rate of 0.73 Gy/min. After irradiation, samples were transferred to microtubes (total volume of 1 ml including blood and media) and organized into a standard 8 × 12 plate format. Sample processing methods were modified by increasing the blood-to-media ratio, adding hypotonic solution prior to cell fixation and optimizing nuclear DRAQ5 staining, leading to an increase of 81% in BNC yield. Modification of the imaging processing algorithms within IFC software also improved BNC and MN identification, and reduced the average time of image analysis by 78%. Finally, 50 ll of irradiated whole blood was cultured with 200 ll of media in 96-well plates. All sample processing steps were performed automatically using the RABiT-II cell: :explorer robotic system adopting the optimized IFC-CBMN assay protocol. The results presented here detail a novel, high-throughput RABiT-IFC CBMN assay that possesses the potential to increase capacity for triage biodosimetry during a large-scale radiological/nuclear event.

Micronuclei formation in rainbow trout cells exposed to multiple stressors: Morpholine, heat shock, and ionizing radiation.

Discharges from industrial cooling water systems can include low levels of morpholine (a chemical pH regulator and corrosion inhibitor), as well as transiently higher temperature effluent water which present a potential source of environmental impact to aquatic biota. The effects of environmental levels of morpholine or heat shock (HS) treatment alone and in combination with a challenge high-dose of 137Cs ionizing radiation were studied using the cytokinesis block micronucleus assay in a rainbow trout cell line (RTG-2). Morpholine treatment of 10 or 100mgL-1 alone produced no significant effects, and no interaction was observed in combination with 7.75Gy radiation. A 9°C magnitude HS treatment alone significantly increased micronuclei formation. A synergistic response was observed when 9°C HS was combined with 7.75Gy radiation, with 15% more cells containing 3 or more micronuclei than the sum of each individual stressor. A synergistic increase in the average number of micronuclei was observed when morpholine and a 9°C HS were co-treated. These results indicate that morpholine at environmentally-relevant levels does not impact micronuclei formation or cell cycle progression however 9°C HS may be of potential concern both alone and in combination with other stressor treatments.

Automatic detection of micronuclei by cell microscopic image processing.

With the development and applications of ionizing radiation in medicine, the radiation effects on human health get more and more attention. Ionizing radiation can lead to various forms of cytogenetic damage, including increased frequencies of micronuclei (MNi) and chromosome abnormalities. The cytokinesis block micronucleus (CBMN) assay is widely used method for measuring MNi to determine chromosome mutations or genome instability in cultured human lymphocytes. The visual scoring of MNi is time-consuming and scorer fatigue can lead to inconsistency. In this work, we designed software for the scoring of in vitro CBMN assay for biomonitoring on Giemsa-stained slides that overcome many previous limitations. Automatic scoring proceeds in four stages as follows. First, overall segmentation of nuclei is done. Then, binucleated (BN) cells are detected. Next, the entire cell is estimated for each BN as it is assumed that there is no detectable cytoplasm. Finally, MNi are detected within each BN cell. The designed Software is even able to detect BN cells with vague cytoplasm and MNi in peripheral blood smear. Our system is tested on a self-provided dataset and is achieved high sensitivities of about 98% and 82% in recognizing BN cells and MNi, respectively. Moreover, in our study less than 1% false positives were observed that makes our system reliable for practical MNi scoring.

Adverse and beneficial effects in Chinese hamster lung fibroblast cells following radiofrequency exposure.

In this study, the effect of radiofrequency (RF) exposure to 1950 MHz, Universal Mobile Telecommunication System signal, was investigated in Chinese hamster lung fibroblast cell line (V79). Genotoxic and cytotoxic effects of 20-h exposure at specific absorption rate (SAR) values from 0.15 W/kg to 1.25 W/kg were measured by means of cytokinesis-block micronucleus (MN) assay. Exposure was carried out blinded under strictly controlled conditions of dosimetry and temperature. The effect of RF exposure alone at four SAR values was tested, that is, 0.15, 0.3, 0.6, and 1.25 W/kg. A statistically significant increase in MN frequency was found in cultures exposed to 0.15 and 0.3 W/kg (P < 0.05) compared to sham-exposed ones, in the absence of cytotoxicity. SAR values of 0.6 and 1.25 W/kg did not exert any effect. Moreover, to evaluate the ability of RF to exert protective effects with respect to a chemical mutagen, cell cultures were also pre-exposed for 20 h at 0.3 or 1.25 W/kg, and then treated with 500 ng/ml of mitomycin-C (MMC). A significant reduction in the frequency of MN was detected in cultures pre-exposed to 1.25 W/kg compared to cultures treated with MMC alone (P < 0.05), indicating induction of adaptive response. Such a decrease was not induced by pre-exposure at 0.3 W/kg SAR. Taken together, our results indicated that V79 is a sensitive cell model to evidence either adverse or beneficial effects of RF exposure, depending on experimental conditions applied. Bioelectromagnetics. 38:245-254, 2017. © 2017 Wiley Periodicals, Inc.

Evaluation of cytological radiation damage to lymphocytes after I-131 metaiodobenzylguanidine therapy by the cytokinesis-blocked micronucleus assay.

OBJECTIVE: The purpose of this study was to evaluate the degree of cytological radiation damage to lymphocytes occurring after I-131 metaiodobenzylguanidine (MIBG) therapy as determined by the cytokinesis-blocked micronucleus assay. The chromosomal damage to lymphocytes induced by I-131 in vivo should result in augmentation of the number of cells with micronuclei. METHODS: We studied 15 patients with pheochromocytoma (14/15) or ganglioneuroma (1/15), who were treated initially with 7.4 GBq of I-131-MIBG. Isolated lymphocytes collected from patients 10 days after the therapy were harvested and treated according to the cytokinesis-blocked method of Fenech and Morley. Serial blood samples were obtained periodically only from two patients for 2 years after therapy. Micronucleus number of micronuclei per 500 binucleated cells was scored by visual inspection. As controls, lymphocytes from the same patients before the therapy were also studied. In an in vitro study, lymphocytes from eight normal volunteers were exposed to doses varying from 0.5 to 2 Gy and studied with the same method. RESULTS: The mean number (mean ± SD) of micronuclei after treatment was significantly increased (p < 0.001) as compared to control subjects (49.4 ± 8.2 vs. 11.3 ± 6.4). Internal radiation absorbed doses estimated for the 15 patients were 1.6 ± 0.3 Gy in this external irradiation study. The frequency of micronuclei post-administration of I-131-MIBG gradually decreased to near baseline (i.e., pre-therapy) levels by 2 years. CONCLUSIONS: The relatively low frequency of lymphocyte micronuclei induced by I-131-MIBG in vivo and reversal of the increasing frequency of lymphocyte micronuclei after therapy suggest that the short-term non-stochastic damage induced by this therapy with 7.4 GBq of I-131-MIBG in pheochromocytoma or ganglioneuroma patients is limited and reversible.

THE EFFECT OF AN OPTIMIZED IMAGING FLOW CYTOMETRY ANALYSIS TEMPLATE ON SAMPLE THROUGHPUT IN THE REDUCED CULTURE CYTOKINESIS-BLOCK MICRONUCLEUS ASSAY.

In cases of overexposure to ionizing radiation, the cytokinesis-block micronucleus (CBMN) assay can be performed in order to estimate the dose of radiation to an exposed individual. However, in the event of a large-scale radiation accident with many potentially exposed casualties, the assay must be able to generate accurate dose estimates to within ±0.5 Gy as quickly as possible. The assay has been adapted to, validated and optimized on the ImageStreamX imaging flow cytometer. The ease of running this automated version of the CBMN assay allowed investigation into the accuracy of dose estimates after reducing the volume of whole blood cultured to 200 µl and reducing the culture time to 48 h. The data analysis template used to identify binucleated lymphocyte cells (BNCs) and micronuclei (MN) has since been optimized to improve the sensitivity and specificity of BNC and MN detection. This paper presents a re-analysis of existing data using this optimized analysis template to demonstrate that dose estimations from blinded samples can be obtained to the same level of accuracy in a shorter data collection time. Here, we show that dose estimates from blinded samples were obtained to within ±0.5 Gy of the delivered dose when data collection time was reduced by 30 min at standard culture conditions and by 15 min at reduced culture conditions. Reducing data collection time while retaining the same level of accuracy in our imaging flow cytometry-based version of the CBMN assay results in higher throughput and further increases the relevancy of the CBMN assay as a radiation biodosimeter.

Induction of Excess Centrosomes in Neural Progenitor Cells during the Development of Radiation-Induced Microcephaly.

The embryonic brain is one of the tissues most vulnerable to ionizing radiation. In this study, we showed that ionizing radiation induces apoptosis in the neural progenitors of the mouse cerebral cortex, and that the surviving progenitor cells subsequently develop a considerable amount of supernumerary centrosomes. When mouse embryos at Day 13.5 were exposed to γ-rays, brains sizes were reduced markedly in a dose-dependent manner, and these size reductions persisted until birth. Immunostaining with caspase-3 antibodies showed that apoptosis occurred in 35% and 40% of neural progenitor cells at 4 h after exposure to 1 and 2 Gy, respectively, and this was accompanied by a disruption of the apical layer in which mitotic spindles were positioned in unirradiated mice. At 24 h after 1 Gy irradiation, the apoptotic cells were completely eliminated and proliferation was restored to a level similar to that of unirradiated cells, but numerous spindles were localized outside the apical layer. Similarly, abnormal cytokinesis, which included multipolar division and centrosome clustering, was observed in 19% and 24% of the surviving neural progenitor cells at 48 h after irradiation with 1 and 2 Gy, respectively. Because these cytokinesis aberrations derived from excess centrosomes result in growth delay and mitotic catastrophe-mediated cell elimination, our findings suggest that, in addition to apoptosis at an early stage of radiation exposure, radiation-induced centrosome overduplication could contribute to the depletion of neural progenitors and thereby lead to microcephaly.

Adaption of the Cytokinesis-Block Micronucleus Cytome Assay for Improved Triage Biodosimetry.

The purpose of this work was to adapt a more advanced form of the cytokinesis-block micronucleus (CBMN) cytome assay for triage biodosimetry in the event of a mass casualty radiation incident. We modified scoring procedures for the CBMN cytome assay to optimize field deployability, dose range, accuracy, speed, economy, simplicity and stability. Peripheral blood of 20 donors was irradiated in vitro (0-6 Gy X ray, maximum photon energy 240 keV) and processed for CBMN. Initially, we assessed two manual scoring strategies for accuracy: 1. Conventional scoring, comprised of micronucleus (MN) frequency per 1,000 binucleated (BN) cells (MN/1,000 BN cells); and 2. Evaluation of 1,000, 2,000 and 3,000 cells in total and different cellular subsets based on MN formation and proliferation (e.g., BN cells with and without MN, mononucleated cells). We used linear and logistic regression models to identify the cellular subsets related closest to dose with the best discrimination ability among different doses/dose categories. We validated the most promising subsets and their combinations with 16 blind samples covering a dose range of 0-8.3 Gy. Linear dose-response relationships comparable to the conventional CBMN assay (r(2) = 0.86) were found for BN cells with MN (r(2) = 0.84) and BN cells without MN (r(2) = 0.84). Models of combined cell counts (CCC) of BN cells with and without MN (BN(+MN) and BN(-MN)) with mononucleated cells (Mono) improved this relationship (r(2) = 0.92). Conventional CBMN discriminated dose categories up to 3 Gy with a concordance between 0.96-1.0 upon scoring 1,000 total cells. In 1,000 BN cells, concordances were observed for conventional CBMN up to 4 Gy as well as BN(+MN) or BN(-MN) (about 0.85). At doses of 4-6 Gy, the concordance of conventional CBMN, BN(+MN) and BN(-MN) declined (about 0.55). We found about 20% higher concordance and more precise dose estimates of irradiated and blinded samples for CCC (Mono + BN(+MN)) after scoring 1,000 total cells. Blinded sample analysis revealed that the mean absolute difference (MAD) of dose estimates and the number of dose estimates outside the ±0.5 Gy interval based on CCC (Mono + BN(+MN)) was comparable to conventional CBMN for doses ≤4 Gy when scoring 3,000 total cells or more. At doses >4-8.3 Gy, the MAD of CCC (Mono + BN(+MN)) declined to half of the MADs observed for conventional CBMN, suggesting that the combined cell counts approach improved the discrimination ability. Conventional CBMN at 1,000 total-cell counts performed as efficiently as counting 1,000 BN cells. Discriminating and counting only BN cells with and without MN after 1,000 BN cells at ≤4 Gy revealed performances similar to conventional CBMN. After 3,000 total cells were scored, CCC (Mono + BN(+MN)) was superior to conventional CBMN at >4 Gy up to about 8 Gy. Our modification of CBMN evaluations saved time compared to the widely established semiautomated MN scoring and extended the dose range up to approximately 6 Gy for triage biodosimetry.

The Cytome Assay as a Tool to Investigate the Possible Association Between Exposure to Extremely Low Frequency Magnetic Fields and an Increased Risk for Alzheimer's Disease.

Exposure to extremely low frequency magnetic fields (ELF-MF) has been identified as one of the potential environmental risk factors for Alzheimer's disease (AD). However, this is far from being established. So far there is no experimental evidence supporting this alleged association. We have performed an in vitro cytogenetic laboratory investigation to explore the plausibility of such association. Our investigation was based on possible similarities found in cells from AD patients and in cells exposed to ELF-MF. We especially found that 50  Hz ELF-MF increase the frequency of cells with (large) micronuclei and nuclear buds indicating that fields above 50 µT may induce chromosome instabilities as those found in AD patients. It should be stressed yet that results from the few published experimental studies on ELF-MF and AD are rather reassuring. Thus, our findings certainly do not prove anything. They only suggest that further investigations might be necessary.

The cytokinesis-blocked micronucleus assay: dose-response calibration curve, background frequency in the population and dose estimation.

An in vitro study of the dose responses of human peripheral blood lymphocytes was conducted with the aim of creating calibrated dose-response curves for biodosimetry measuring up to 4 Gy (0.25-4 Gy) of gamma radiation. The cytokinesis-blocked micronucleus (CBMN) assay was employed to obtain the frequencies of micronuclei (MN) per binucleated cell in blood samples from 16 healthy donors (eight males and eight females) in two age ranges of 20-34 and 35-50 years. The data were used to construct the calibration curves for men and women in two age groups, separately. An increase in micronuclei yield with the dose in a linear-quadratic way was observed in all groups. To verify the applicability of the constructed calibration curve, MN yields were measured in peripheral blood lymphocytes of two real overexposed subjects and three irradiated samples with unknown dose, and the results were compared with dose values obtained from measuring dicentric chromosomes. The comparison of the results obtained by the two techniques indicated a good agreement between dose estimates. The average baseline frequency of MN for the 130 healthy non-exposed donors (77 men and 55 women, 20-60 years old divided into four age groups) ranged from 6 to 21 micronuclei per 1000 binucleated cells. Baseline MN frequencies were higher for women and for the older age group. The results presented in this study point out that the CBMN assay is a reliable, easier and valuable alternative method for biological dosimetry.

Validation of the Cytokinesis-block Micronucleus Assay Using Imaging Flow Cytometry for High Throughput Radiation Biodosimetry.

The cytokinesis-block micronucleus assay can be employed in triage radiation biodosimetry to determine the dose of radiation to an exposed individual by quantifying the frequency of micronuclei in binucleated lymphocyte cells. Partially automated analysis of the assay has been applied to traditional microscope-based methods, and most recently, the assay has been adapted to an automated imaging flow cytometry method. This method is able to automatically score a larger number of binucleated cells than are typically scored by microscopy. Whole blood samples were irradiated, divided into 2 mL and 200 µL aliquots, cultured for 48 h and 72 h, and processed to generate calibration curves from 0-4 Gy. To validate the method for use in radiation biodosimetry, nine separate whole blood samples were then irradiated to known doses, blinded, and processed. Results indicate that dose estimations can be determined to within ±0.5 Gy of the delivered dose after only 48 h of culture time with an initial blood volume of 200 µL. By performing the cytokinesis-block micronucleus assay using imaging flow cytometry, a significant reduction in the culture time and volume requirements is possible, which greatly increases the applicability of the assay in high throughput triage radiation biodosimetry.

Low-Dose Gamma Radiation Does Not Induce an Adaptive Response for Micronucleus Induction in Mouse Splenocytes.

Low-dose ionizing radiation is known to induce radioadaptive responses in cells in vitro as well as in mice in vivo. Low-dose radiation decreases the incidence and increases latency for spontaneous and radiation-induced tumors in mice, potentially as a result of enhanced cellular DNA repair efficiency or a reduction in genomic instability. In this study, the cytokinesis-block micronucleus (CBMN) assay was used to examine dose response and potential radioadaptive response for cytogenetic damage and cell survival in C57BL/6 and BALB/c spleen cells exposed in vitro or in vivo to low-dose 60Co gamma radiation. The effects of genetic background, radiation dose and dose rate, sampling time and cell cycle were investigated with respect to dose response and radioadaptive response. In C57BL/6 mice, a linear-quadratic dose-response relationship for the induction of micronuclei (MN) was observed for doses between 100 mGy and 2 Gy. BALB/c mice exhibited increased radiosensitivity for MN induction compared to C57BL/6 mice. A 20 mGy dose had no effect on MN frequencies in splenocytes of either mouse strain, however, increased spleen weight and a reduced number of dead cells were noted in the C57BL/6 strain only. Multiple experimental parameters were investigated in radioadaptive response studies, including dose and dose rate of the priming dose (20 mGy at 0.5 mGy/min and 100 mGy at 10 mGy/min), time interval (4 and 24 h) between priming and challenge doses, cell cycle stage (resting or proliferating) at exposure and kinetics after the challenge dose. Radioadaptive responses were not observed for MN induction for either mouse strain under any of the experimental conditions investigated. In contrast, a synergistic response for radiation-induced micronuclei in C57BL/6 spleen was detected after in vivo 20 mGy irradiation. This increase in the percentage of cells with cytogenetic damage was associated with a reduction in the number of nonviable spleen cells, suggesting that low-dose irradiation led to a reduction in the turnover of damaged cells within the spleen of C57BL/6 mice. Overall, these results indicate that long-term protective effects against tumor latency and other beneficial health outcomes observed after low-dose irradiation are not mediated by a reduction of the proportion of cells harboring radiation-induced cytogenetic damage.