Femtosecond laser-induced blastomere fusion results in embryo tetraploidy by common metaphase plate formation.

The cell fusion is a widespread process, which takes place in many systems in vivo and in vitro. Fusion of cells is frequently related to tetraploidy, which can be found within natural physiological conditions, e.g., placentation, and in pathophysiological conditions, such as cancer and early pregnancy failure in humans. Here we investigate the mechanism of tetraploidization with help of femtosecond laser-induced mouse blastomere fusion by the means of Hoechst staining, GFP, BODIPY dyes and fluorescent species generated intracellularly by a femtosecond laser. We establish diffusive mixing of cytosol, whereas the large components of a cytoplasm (organelles, cytoskeleton) are poorly diffusible and are not completely mixed after cell fusion and a subsequent division. We show that mechanisms which are responsible for the formation of a common metaphase plate triggered tetraploidization in fused mouse embryos and could be a significant factor in polyploidy formation in vivo. Thus, our results suggest that microtubules play a critical role in tetraploidization.

The Sister Chromatid Exchange (SCE) Assay.

A fully optimized staining method for detecting sister chromatid exchanges in cultured cells is presented. The method gives reproducibly robust quantitative results. Sister chromatid exchange is a classic toxicology assay for genotoxicity and for detecting alterations to the biochemistry underlying cellular homologous recombination. Growth of cells in the presence of 5'-bromo-deoxyuridine for two rounds of DNA replication followed by collecting metaphase spreads on glass slides, treatment with the UV-sensitive dye Hoechst 33258, long-wave UV light exposure, and Giemsa staining gives a permanent record of the exchanges.

The BioDoseNet image repository used as a training tool for the dicentric assay.

Purpose: This paper describes how the BioDoseNet image repository was used as a training tool for the dicentric assay.Materials and methods: The training was implemented in three phases: introduction to dicentric scoring, dose response curve elaboration and dose assessment exercise. Four labs without previous experience in the dicentric assay participated and four modules of the repository were used.Results: The labs become familiar with aberrations induced by ionizing radiation. The labs were able to generate data for the elaboration of a dose response curve and then successfully estimated doses and irradiated fractions in six blind samples.Conclusions: The performance of these laboratories during the exercise demonstrates the efficacy of the BioDoseNet image repository as a training tool and the utility of web based scoring for the dicentric assay community.

Human Lymphocyte Metaphase Chromosome Preparation for Radiation-Induced Chromosome Aberration Analysis.

Radiation-induced chromosomal aberration analysis for metaphase chromosomes is well established and the golden standard for human biodosimetry. This method can estimate doses of human radiation exposure after nuclear accident and unwanted radiation exposure from their lymphocytes. The natural background frequency of dicentric chromosome for human lymphocytes is less than 1% and any increase in dicentric and centric ring chromosomes may be highly associated with radiation exposure. With the appropriate number of metaphase cells, one can detect the exposure of more than 0.1 Gy by observing dicentric and centric ring chromosomes. Dicentric chromosome analysis is relying on morphological changes and may be difficult for researchers without appropriate training. This method is time consuming and labor intensive, but still currently the most reliable technique and analysis needs only light microscopes with high magnification objectives and trained personnel. Recent research enables us to visualize dicentric chromosomes clearly with fluorescent markers for easy detection of dicentric and centric ring chromosomes. This chapter will introduce classical dicentric analysis of human lymphocyte cells with Giemsa staining.

PNA Telomere and Centromere FISH Staining for Accurate Analysis of Radiation-Induced Chromosomal Aberrations.

Dicentric and centric ring chromosomes are used for radiation-induced damage analysis and biodosimetry after radiation exposure. However, Giemsa stain-based cytogenetic analysis is labor-intense and time-consuming. Moreover, the disadvantage of Giemsa based chromosome analysis is a potential poor reproducibility when researchers are not fully trained for analysis. These problems come from analysis of morphological abnormality of chromosomal aberrations. Locus-specific FISH probes were used to overcome this problem. Centromere probes can visualize centromere locations and help identify dicentric chromosomes and centric rings. Telomere probes help to identify terminal deletion and telomere fusions. Probes were originally designed with a DNA probe but Peptide nucleic acid (PNA) probes took the place of DNA probes. This chapter introduces PNA telomere and centromere FISH staining and accurate analysis of chromosomal aberrations.

A method for the cell-cycle-specific analysis of radiation-induced chromosome aberrations and breaks.

The classical G2-assay is widely used to assess cell-radiosensitivity and cancer phenotype: Cells are exposed to low doses of ionizing-radiation (IR) and collected for cytogenetic- analysis ˜1.5 h later. In this way, chromosome-damage is measured in cells irradiated in G2-phase, without retrieving information regarding kinetics of chromosome-break-repair. Modification of the assay to include analysis at multiple time-points after IR, has enabled kinetic-analysis of chromatid-break-repair and assessment of damage in a larger proportion of G2-phase cells. This modification, however, increases the probability that at later time points not only cells irradiated in G2-phase, but also cells irradiated in S-phase will reach metaphase. However, the response of cells irradiated in G2-phase can be mechanistically different from that of cells irradiated in S-phase. Therefore, indiscriminate analysis may confound the interpretation of experiments designed to elucidate mechanisms of chromosome-break-repair and the contributions of the different DSB-repair-pathways in this response. Here we report an EdU based modification of the assay that enables S- and G2-phase specific analysis of chromatid break repair. Our results show that the majority of metaphases captured during the first 2 h after IR originate from cells irradiated in G2-phase (EdU- metaphases) in both rodent and human cells. Metaphases originating from cells irradiated in S-phase (EdU+ metaphases) start appearing at 2 h and 4 h after IR in rodent and human cells, respectively. The kinetics of chromatid-break-repair are similar in cells irradiated in G2- and S-phase of the cell-cycle, both in rodent and human cells. The protocol is applicable to classical-cytogenetic experiments and allows the cell-cycle specific analysis of chromosomal-aberrations. Finally, the protocol can be applied to the kinetic analysis of chromosome-breaks in prematurely-condensed-chromosomes of G2-phase cells. In summary, the developed protocol provides means to enhance the analysis of IR-induced-cytogenetic-damage by providing information on the cell-cycle phase where DNA damage is inflicted.

Direct and bystander effects in human blood lymphocytes exposed to 241Am alpha particles and the relative biological effectiveness using chromosomal aberration and micronucleus assay.

Purpose: It is important to understand the significance of alpha (α) radiation-induced bystander effects (RIBE) and its relative biological effectiveness (RBE); this is because the phenomenon is not universal and the mechanism is unclear and because the RBE is widely varying and projected to be very high. Materials and methods: Isolated lymphocytes from healthy volunteers (n = 10) were exposed to either low fluence α-particles (241Am), γ-rays (60Co), or X-rays (225 kVp and 6 MV). Co-culture methodology was employed to investigate bystander effects (BEs). Chromosomal aberrations (CA) and micronucleus (MN) formation were used to study the BE and calculated RBE. Results: Lymphocytes directly exposed to the types of radiation used showed a dose-dependent increase in the frequency of CA and MN; dose independent increases in the frequency of these chromosomal damages in co-cultured bystander cells, implies that all three types of radiation-induced a BE. The calculated RBE at the level of 5% induced aberrations varied between 9 and 20. Conclusion: The magnitude of low fluence α-particle induced RIBE is higher than in low LET (linear energy transfer) radiation. The RBE also varies depending upon the endpoints used and adds up to targeted effects. Since the endpoint of CA is considered as an important and early marker of risk prediction, the RIBE and RBE using CA as a marker are relevant for radiation protection purposes.

THE PROJECT OF ANOTHER LOW-COST METAPHASE FINDER (SECOND REPORT-APPLICATION OF ARTIFICIAL INTELLIGENCE).

Biological dosimetry is used to estimate individual absorbed radiation dose by quantifying an appropriate biological marker. The most popular gold-standard marker is the appearance of dicentric chromosomes in metaphase. The metaphase finder is a tool for biological dosimetry that finds metaphase cells on glass slides. The author and a software company have designed a new system and are now preparing to produce the system commercially. The metaphase finder consists of an automated microscope, a camera, and a computer. To enhance the accuracy of the system, an artificial intelligence (AI) with deep learning was tested. A total of 1709 images of the metaphase finder detected as 'metaphases' were read into a nine-layer artificial neural network to detect true metaphases. A total of 456 images were used for training, and the rest of the images were used for validation. The accuracy of AI was 0.89 for metaphases and 0.90 for non-metaphases.

RADIATION DOSE ESTIMATION BY COMPLETELY AUTOMATED INTERPRETATION OF THE DICENTRIC CHROMOSOME ASSAY.

Accuracy of the automated dicentric chromosome (DC) assay relies on metaphase image selection. This study validates a software framework to find the best image selection models that mitigate inter-sample variability. Evaluation methods to determine model quality include the Poisson goodness-of-fit of DC distributions for each sample, residuals after calibration curve fitting and leave-one-out dose estimation errors. The process iteratively searches a pool of selection model candidates by modifying statistical and filter cut-offs to rank the best candidates according to their respective evaluation scores. Evaluation scores minimize the sum of squared errors relative to the actual radiation dose of the calibration samples. For one laboratory, the minimum score for the curve fit residual method was 0.0475 Gy2, compared to 1.1975 Gy2 without image selection. Application of optimal selection models using samples of unknown exposure produced estimated doses within 0.5 Gy of physical dose. Model optimization standardizes image selection among samples and provides relief from manual DC scoring, improving accuracy and consistency of dose estimation.

Comparative analysis the frequency and spectrum of chromosome aberrations in irradiated in vitro peripheral blood lymphocytes of the elderly and centenarians from Kyiv. / PORIVNIaL'NYY̆ ANALIZ RIVNIa TA SPEKTRU ABERATsIY̆ KhROMOSOM V OPROMINENYKh IN VITRO LIMFOTsYTAKh PERYFERYChNOÏ KROVI OSIB LITN'OGO VIKU TA DOVGOZhYTELIV m. KYIeVA.

OBJECTIVE: To establish and compare the frequency and spectrum of chromosome aberrations under X radiation exposure in vitro in dose 0.25 Gy peripheral blood lymphocytes of the elderly and centenarians. MATERIAL AND METHODS: Material of cytogenetic research were peripheral blood lymphocytes from 11 elderly and 10 centenarians, which were irradiated in vitro in dose 0.25 Gy and cultured by generally accepted semi micromethod; slides of metaphase chromosomes were GTG stained and analyzed under the microscope with magnification x 1000. RESULTS: Under irradiation of blood in vitro the mean group frequencies of chromosome aberrations exceeded such without irradiation (р < 0.001) and were 11.60 ± 0.95 аnd 6.82 ± 0.63 per 100 cells in the elderly and the centenar ians, accordingly. Radiation induced increase in the frequency of chromosomal injuries occurred due to chromo some type aberrations which are markers of radiation exposure. In the elderly the elevated frequency of chromatid type aberrations also was registered what is considered a sign of chromosome instability. CONCLUSIONS: The results indicate increased sensitivity the blood lymphocytes from the elderly to radiation expo sure in low doses and allow to assume the advantage of persons with hereditary determined chromosomal stability in achieving longevity.

Cytogenetic abnormalities of the descendants of permanent residents of heavily contaminated East Kazakhstan.

More than 400 nuclear explosion tests were conducted at the Semipalatinsk Nuclear Test Site (SNTS) and significant radioactive substances were released. The long-term consequences of the activities at the SNTS and the appearance of any hereditary effects remain insufficiently studied about 25 years after the test site was closed. The population living in villages near the SNTS are considered to have been heavily exposed to external and internal radiation. This study aims to perform an assessment and comprehensive cytogenetic analysis of the inhabitants living near the SNTS, and their first-(F1) and second-(F2) generation children. Residents of the East Kazakhstan region living in the area covered by the former SNTS were included in the study. To evaluate the hereditary effects of nuclear testing, comprehensive chromosome analyses were performed in lymphocytes using conventional Giemsa and fluorescent in situ hybridization methods in 115 F1 and F2 descendants in the villages of Dolon and Sarzhal, which were heavily contaminated. The parents of the subjects had permanently lived in the villages. A higher number of stable-type chromosome aberrations such as translocations was found in these residents than in 80 residents of the control area, Kokpecty, which indicates the possibility that radiation had biological effects on the exposed subjects.

Peripheral Blood Lymphocytes as In Vitro Model to Evaluate Genomic Instability Caused by Low Dose Radiation.

Diagnostic and therapeutic radiation fields are planned so as to reduce side-effects while maximising the dose to site but effects on healthy tissues are inevitable. Radiation causes strand breaks in DNA of exposed cells which can lead to chromosomal aberrations and cause malfunction and cell death. Several researchers have highlighted the damaging effects of high dose radiation but still there is a lacuna in identifying damage due to low dose radiation used for diagnostic purposes. Blood is an easy resource to study genotoxicity and to estimate the effects of radiation. The micronucleus assay and chromosomal aberration can indicate genetic damage and our present aim was to establish these with lymphocytes in an in vitro model to predict the immediate effects low dose radiation. Blood was collected from healthy individuals and divided into 6 groups with increasing radiation dose i.e., 0Gy, 0.10Gy, 0.25Gy, 0.50Gy, 1Gy and 2Gy. The samples were irradiated in duplicates using a LINAC in the radiation oncology department. Standard protocols were applied for chromosomal aberration and micronucleus assays. Metaphases were stained in Giemsa and 200 were scored per sample for the detection of dicentric or acentric forms. For micronuclei detection, 200 metaphases. Giemsa stained binucleate cells per sample were analysed for any abnormality. The micronuclei (MN) frequency was increased in cells exposed to the entire range of doses (0.1- 2Gy) delivered. Controls showed minimal MN formation (2.0%±0.05) with triple MN (5.6%±2.0) frequency at the lowest dose. MN formation increased exponentially with the radiation dose thereafter with a maximum at 2Gy. Significantly elevated numbers of dicentric chromosomes were also observed, even at doses of 0.1- 0.5Gy, compared to controls, and acentric chromosomes were apparent at 2Gy. In conclusion we can state that lymphocytes can be effectively used to study direct effect of low dose radiation.

Identification of Novel Chromosomal Aberrations Induced by (60)Co-γ-Irradiation in Wheat-Dasypyrum villosum Lines.

Mutations induced by radiation are widely used for developing new varieties of plants. To better understand the frequency and pattern of irradiation-induced chromosomal rearrangements, we irradiated the dry seeds of Chinese Spring (CS)-Dasypyrum villosum nullisomic-tetrasomic (6A/6D) addition (6V) line (2n = 44), WD14, with (60)Co-γ-rays at dosages of 100, 200, and 300 Gy. The M0 and M1 generations were analyzed using Feulgen staining and non-denaturing fluorescence in situ hybridization (ND-FISH) by using oligonucleotide probes. Abnormal mitotic behavior and chromosomes with structural changes were observed in the M0 plants. In all, 39 M1 plants had structurally changed chromosomes, with the B genome showing the highest frequency of aberrations and tendency to recombine with chromosomes of the D genome. In addition, 19 M1 plants showed a variation in chromosome number. The frequency of chromosome loss was considerably higher for 6D than for the alien chromosome 6V, indicating that 6D is less stable after irradiation. Our findings suggested that the newly obtained γ-induced genetic materials might be beneficial for future wheat breeding programs and functional gene analyses.

Increase in dicentric chromosome formation after a single CT scan in adults.

Excess risk of leukemia and brain tumors after CT scans in children has been reported. We performed dicentric chromosome assay (DCAs) before and after CT scan to assess effects of low-dose ionizing radiation on chromosomes. Peripheral blood (PB) lymphocytes were collected from 10 patients before and after a CT scan. DCA was performed by analyzing either 1,000 or 2,000 metaphases using both Giemsa staining and centromere-fluorescence in situ hybridization (Centromere-FISH). The increment of DIC formation was compared with effective radiation dose calculated using the computational dosimetry system, WAZA-ARI and dose length product (DLP) in a CT scan. Dicentric chromosome (DIC) formation increased significantly after a single CT scan, and increased DIC formation was found in all patients. A good correlation between the increment of DIC formation determined by analysis of 2,000 metaphases using Giemsa staining and those by 2,000 metaphases using Centromere-FISH was observed. However, no correlation was observed between the increment of DIC formation and the effective radiation dose. Therefore, these results suggest that chromosome cleavage may be induced by one CT scan, and we recommend 2,000 or more metaphases be analyzed in Giemsa staining or Centromere-FISH for DCAs in cases of low-dose radiation exposure.

Chromosomal Aberrations in Wild Mice Captured in Areas Differentially Contaminated by the Fukushima Dai-Ichi Nuclear Power Plant Accident.

Following the Fukushima Dai-ichi Nuclear Power Plant accident, radiation effects on nonhuman biota in the contaminated areas have been a great concern. The induction of chromosomal aberrations in splenic lymphocytes of small Japanese field mice (Apodemus argenteus) and house mice (Mus musculus) inhabiting Fukushima Prefecture was investigated. In mice inhabiting the slightly contaminated area, the average frequency of dicentric chromosomes was similar to that seen in mice inhabiting a noncontaminated control area. In contrast, mice inhabiting the moderately and heavily contaminated areas showed a significant increase in the average frequencies of dicentric chromosomes. Total absorbed dose rate was estimated to be approximately 1 mGy d(-1) and 3 mGy d(-1) in the moderately and heavily contaminated areas, respectively. Chromosomal aberrations tended to roughly increase with dose rate. Although theoretically, the frequency of chromosomal aberrations was considered proportional to the absorbed dose, chromosomal aberrations in old mice (estimated median age 300 days) did not increase with radiation dose at the same rate as that observed in young mice (estimated median age 105 days).

Multicolour FISH analysis of ionising radiation induced micronucleus formation in human lymphocytes.

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.

Next generation platforms for high-throughput biodosimetry.

Here the general concept of the combined use of plates and tubes in racks compatible with the American National Standards Institute/the Society for Laboratory Automation and Screening microplate formats as the next generation platforms for increasing the throughput of biodosimetry assays was described. These platforms can be used at different stages of biodosimetry assays starting from blood collection into microtubes organised in standardised racks and ending with the cytogenetic analysis of samples in standardised multiwell and multichannel plates. Robotically friendly platforms can be used for different biodosimetry assays in minimally equipped laboratories and on cost-effective automated universal biotech systems.

The Internet's role in a biodosimetric response to a radiation mass casualty event.

Response to a large-scale radiological incident could require timely medical interventions to minimize radiation casualties. Proper medical care requires knowing the victim's radiation dose. When physical dosimetry is absent, radiation-specific chromosome aberration analysis can serve to estimate the absorbed dose in order to assist physicians in the medical management of radiation injuries. A mock exercise scenario was presented to six participating biodosimetry laboratories as one individual acutely exposed to Co under conditions suggesting whole-body exposure. The individual was not wearing a dosimeter and within 2-3 h of the incident began vomiting. The individual also had other medical symptoms indicating likelihood of a significant dose. Physicians managing the patient requested a dose estimate in order to develop a treatment plan. Participating laboratories in North and South America, Europe, and Asia were asked to evaluate more than 800 electronic images of metaphase cells from the patient to determine the dicentric yield and calculate a dose estimate with 95% confidence limits. All participants were blind to the physical dose until after submitting their estimates based on the dicentric chromosome assay (DCA). The exercise was successful since the mean biological dose estimate was 1.89 Gy whereas the actual physical dose was 2 Gy. This is well within the requirements for guidance of medical management. The exercise demonstrated that the most labor-intensive step in the entire process (visual evaluation of images) can be accelerated by taking advantage of world-wide expertise available on the Internet.

Suitability of scoring PCC rings and fragments for dose assessment after high-dose exposures to ionizing radiation.

Assessment of radiation doses through measurement of dicentric chromosomes may be difficult due to the inability of damaged cells to reach mitosis. After high-dose exposures, premature chromosome condensation (PCC) has become an important method in biodosimetry. PCC can be induced upon fusion with mitotic cells, or by treatment with chemicals such as calyculin A or okadaic acid. Several different cytogenetic endpoints have been measured with chemically induced PCC, e.g., via scoring of extra chromosome pieces or ring chromosomes. The dose-effect curves published with chemically induced PCC show differences in their coefficients and in the distribution of rings among cells. Here we present a study with calyculin A to induce PCC in peripheral blood lymphocytes irradiated at nine different doses of γ-rays up to 20Gy. Colcemid was also added in order to observe metaphase cells. During microscopical analysis the chromosome aberrations observed in the different cell-cycle phases (G2/M-PCC, M/A-PCC and M cells) were recorded. The proportion of G2/M-PCC cells was predominant from 3 to 20Gy, M cells decreased above 1Gy and M/A-PCC cells remained constant at all doses and showed the highest frequencies of PCC rings. Depending on the cell-cycle phase there was a difference in the linear coefficients in the dose-effect curves of extra fragments and rings. Poisson distribution among PCC rings was observed after calyculin A+colcemid treatment, facilitating the use of this methodology also for partial body exposures to high doses. This has been tested with two simulated partial exposures to 6 and 12Gy. The estimated doses in the irradiated fraction were very close to the real dose, indicating the usefulness of this methodology.

Relative proximity of chromosome territories influences chromosome exchange partners in radiation-induced chromosome rearrangements in primary human bronchial epithelial cells.

It is well established that chromosomes exist in discrete territories (CTs) in interphase and are positioned in a cell-type specific probabilistic manner. The relative localisation of individual CTs within cell nuclei remains poorly understood, yet many cancers are associated with specific chromosome rearrangements and there is good evidence that relative territorial position influences their frequency of exchange. To examine this further, we characterised the complexity of radiation-induced chromosome exchanges in normal human bronchial epithelial (NHBE) cells by M-FISH analysis of PCC spreads and correlated the exchanges induced with their preferred interphase position, as determined by 1/2-colour 2D-FISH analysis, at the time of irradiation. We found that the frequency and complexity of aberrations induced were reduced in ellipsoid NHBE cells in comparison to previous observations in spherical cells, consistent with aberration complexity being dependent upon the number and proximity of damaged CTs, i.e. lesion proximity. To ask if particular chromosome neighbourhoods could be identified we analysed all radiation-induced pair-wise exchanges using SCHIP (statistics for chromosome interphase positioning) and found that exchanges between chromosomes (1;13), (9;17), (9;18), (12;18) and (16;21) all occurred more often than expected assuming randomness. All of these pairs were also found to be either sharing similar preferred positions in interphase and/or sharing neighbouring territory boundaries. We also analysed a human small cell lung cancer cell line, DMS53, by M-FISH observing the genome to be highly rearranged, yet possessing rearrangements also involving chromosomes (1;13) and (9;17). Our findings show evidence for the occurrence of non-random exchanges that may reflect the territorial organisation of chromosomes in interphase at time of damage and highlight the importance of cellular geometry for the induction of aberrations of varying complexity after exposure to both low and high-LET radiation.