ABSTRACT
The purpose of this study was to evaluate the in vivo dose-response relation of chromosome aberration formation and distribution in a context of localised and fractionated radiotherapy. Cytogenetic analysis was applied to eight patients, all treated for the same tumour localisation; the same localisation was used to prevent the variability usually observed between patients treated with radiotherapy and to allow the corresponding roles of the size of irradiation field and of the dose rate to be studied. The yield of dicentrics, centric rings and fragments was measured in blood samples taken before treatment, during the course of radiotherapy and up to 6 months after. After the first fraction of radiotherapy, we observed that the whole-body dose estimated from the yield of dicentrics and rings was higher (0.35+/-0.2 Gy) than the calculated equivalent whole-body dose (0.07+/-0.04 Gy). By contrast, the partial-body dose derived from the Qdr (quotient of dicentrics and rings) model was estimated to be 2.2+/-0.3 Gy, which agreed quite well with the dose delivered to the tumour (2.1+/-0.1 Gy). We also found a correlation between the yield of induced chromosome aberrations and the target field size (p = 0.014). U-value analysis showed that the distribution of dicentrics and rings was overdispersed, despite the fractionation of the exposure, and a positive correlation between the U-value and the dose rate was observed (p = 0.017). Overall, these results suggest that the proportion of undamaged lymphocytes could increase with the dose rate.
Subject(s)
Chromosome Aberrations , Head and Neck Neoplasms/radiotherapy , Lymphocytes/radiation effects , Aged , Cytogenetic Analysis/methods , Dose Fractionation, Radiation , Dose-Response Relationship, Radiation , Female , Head and Neck Neoplasms/genetics , Humans , Male , Middle AgedABSTRACT
PURPOSE: To compare translocation rate using either M-FISH or FISH-3 in two patients treated for head and neck cancer, with a view to retrospective dosimetry. MATERIALS AND METHODS: Translocation analysis was performed on peripheral blood lymphocyte cultures from blood samples taken at different times during the radiotherapy (0 Gy, 12 Gy and 50 Gy) and a few months after the end of the treatment (follow-up). RESULTS: Estimated translocation yield varied according to the FISH technique used. At 50 Gy and follow-up points, the translocation yields were higher with FISH-3 than with M-FISH. This difference can be attributed to three events. First, an increase in complex aberrations was observed for 50 Gy and follow-up points compared with 0 Gy and 12 Gy points. Second, at the end of treatment for patient A, involvement of chromosomes 2, 4, 12 in translocations was less than expected according to the Lucas formula. Third, a clone bearing a translocation involving a FISH-3 painted chromosome was detected. CONCLUSIONS: More translocations were detected with M-FISH than with FISH-3, and so M-FISH is expected to improve the accuracy of chromosome aberration analyses in some situations.
Subject(s)
Chromosome Painting/methods , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/radiotherapy , In Situ Hybridization, Fluorescence/methods , Microscopy, Fluorescence, Multiphoton/methods , Translocation, Genetic/genetics , Translocation, Genetic/radiation effects , Aged , Humans , Male , Middle Aged , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
PURPOSE: The purpose of this study was to examine a new approach to retrospective biological dosimetry, by using a long-term animal model to determine the stability of translocation frequency after in vivo irradiation. While the frequency of dicentrics is known to decrease over time, the persistence of more stable chromosomal aberrations such as translocations could be useful if their stability were definitively proved. MATERIALS AND METHODS: Four monkeys (Macaca fascicularis) were exposed to two different doses of ionizing radiation: 2 Gy whole body irradiation for two and 4 Gy for two others. Blood samples were obtained at various times after irradiation. Both total and two-way translocations were detected by fluorescence in situ hybridization. Translocations were scored in stable cells, that is, those without dicentrics, rings or fragments. The course of translocation frequency was analysed at four time-points: one hour (H1), 2 months (M2), 10 months (M10) and 31 months (M31) after irradiation. RESULTS: We observed two separate trends in translocation frequency: Total translocation frequency decreased slightly in animals irradiated with a dose of 2 Gy, while two-way translocation frequency was relatively stable in all irradiated animals. CONCLUSIONS: We confirmed the long-term stability of translocations and found that it seems to depend on the type of the translocation recorded. Overall translocations were stable for up to 31 months regardless of dose, but two-way translocations were more stable than those that were non-reciprocal, especially in stable cells.
Subject(s)
Chromosome Aberrations/radiation effects , Chromosomes/genetics , Chromosomes/radiation effects , Gamma Rays , Lymphocytes/radiation effects , Radiation Monitoring/methods , Risk Assessment/methods , Animals , Chromosome Painting , Dose-Response Relationship, Radiation , Follow-Up Studies , Humans , Macaca fascicularis , Male , Radiation Dosage , Risk FactorsABSTRACT
PURPOSE: To compare the efficiency of different cytogenetic tools in estimating the doses received by four people involved in the Lilo accident and to monitor the dose estimate over 4.5 years. MATERIALS AND METHODS: Several young Georgian frontier guards handled at least one of the 12 Caesium sources found in a former Russian military camp. Overexposure lasted from July 1996 to May 1997. The Institute for Radiological Protection and Nuclear Safety (IRSN) obtained blood samples taken at several intervals post-exposure from the four most highly-exposed people. Dose estimation was performed using dicentric and translocation scoring. RESULTS: The first dose estimations performed by dicentric scoring gave whole-body doses ranging from 0.4 to 1.3 Gy. Overexposure was complex and several mathematical models were used to take this complexity into account. This could provide information concerning the circumstances of overexposure. Concerning follow-up, the yield of dicentrics decreased by about 50% in the first 4 months following the end of overexposure whereas translocations were stable over the period of analysis. CONCLUSION: It has been useful to compare cytogenetic results with clinical results. The results presented here reveal good stability of translocations. However the first dose estimation was not attempted until 6 months after the last exposure.
Subject(s)
Chromosome Aberrations , Radiation, Ionizing , Radioactive Hazard Release , Radiometry , Humans , Translocation, GeneticABSTRACT
We propose a new method of biodosimetry that could be applied in cases of localized irradiation. The approach is based on excess chromosome segments determination by the PCC-FISH technique in fibroblasts isolated from skin biopsy. Typically, 0 to 10 Gy ex vivo gamma-irradiated human skin biopsies were dissociated and fibroblasts were isolated and grown for several days. Cells next underwent PCC-FISH painting of whole chromosome 4, and the number of excess chromosome segments per metaphase was determined. An ex vivo reference curve correlating the number of excess chromosome segments per metaphase to the radiation dose was established and used to assess the dose delivered to the skin of one of the victims of the radiological accident that occurred at Lia in Georgia in December 2001. Specifically, the victim suffering from moist desquamation underwent skin excision in Hospital Percy (France). Measurement of excess chromosome segments per metaphase was done in fibroblasts isolated and grown from removed wounded skin and subsequent conversion to radiation doses was performed. The radiation dose map obtained was shown to be in accordance with clinical data and physical dosimetry as well as with conventional biodosimetry. These results demonstrated that PCC-FISH painting applied to skin fibroblasts may be a suitable technique for dose estimation. To assess its worth, this approach needs to be extended to future accidents involving localized radiation exposure.
Subject(s)
Fibroblasts/ultrastructure , In Situ Hybridization, Fluorescence/methods , Radioactive Hazard Release , Radiometry , Apoptosis , Biopsy , Cell Division , Cell Survival , Cells, Cultured , Chromosome Aberrations , Chromosome Painting , Chromosomes/radiation effects , Chromosomes/ultrastructure , Chromosomes, Human, Pair 4/radiation effects , Dose-Response Relationship, Radiation , Fibroblasts/metabolism , Fibroblasts/radiation effects , Gamma Rays , Georgia (Republic) , Humans , Immunohistochemistry , Ki-67 Antigen/biosynthesis , Metaphase , Mitosis , Radiation Dosage , Radiation Injuries , Skin/radiation effects , Time FactorsABSTRACT
The Institute of Radiation Protection and Nuclear Safety (IRSN) organized a biological dosimetry international intercomparison with the purpose of comparing (i) dicentrics yield produced in human lymphocytes; (ii) the gamma and neutron dose estimate according to the corresponding laboratory calibration curve. The experimental reactor SILENE was used with different configurations: bare source 4 Gy, lead shield 1 and 2 Gy and a 60Co source 2 Gy. An increasing variation of dicentric yield per cell was observed between participants when there were more damages in the samples. Doses were derived from the observed dicentric rates according to the dose-effect relationship provided by each laboratory. Differences in dicentric rate values are more important than those in the corresponding dose values. The doses obtained by the participants were found to be in agreement with the given physical dose within 20%. The evaluation of the respective gamma and neutron dose was achieved only by four laboratories, with some small variations among them.
Subject(s)
Chromosome Aberrations , Chromosomes/radiation effects , Leukocytes, Mononuclear/radiation effects , Radiation Protection/methods , Radioactive Hazard Release , Radiometry/methods , Risk Assessment/methods , Dose-Response Relationship, Radiation , France , Gamma Rays , Humans , Internationality , Leukocytes, Mononuclear/pathology , Neutrons , Nuclear Reactors , Observer Variation , Quality Assurance, Health Care/methods , Radiation Dosage , Radiation Protection/standards , Radiometry/standards , Reference Standards , Relative Biological Effectiveness , Reproducibility of Results , Risk Assessment/standards , Risk Factors , Safety Management/methods , Sensitivity and SpecificityABSTRACT
The purpose of this paper is to investigate how well various assays on blood can detect radiation dose to people exposed many years previously and, if possible, to estimate that dose. The assays were applied to persons resident close to Chernobyl in 1986. Blood samples were taken 13-15 years after the reactor accident. The assays used were the frequencies of lymphocyte chromosomal translocations, micronuclei, HPRT mutations and apoptotic cells. Translocation yields in the exposed groups were marginally higher than in their respective controls, leading to dose estimates of about 0.2 Gy but with large uncertainties. All other assays showed inconsistency from person to person or other variations apparently not related to dose. The measurement of translocations, it is concluded, is the biological method of choice for retrospective dosimetry.
Subject(s)
Chernobyl Nuclear Accident , Micronucleus Tests/methods , Power Plants , Radiation Protection/methods , Radioactive Hazard Release , Radioisotopes/blood , Radiometry/methods , Risk Assessment/methods , Adult , Body Burden , Chromosomes/radiation effects , Female , Humans , Male , Radiation Dosage , Radioisotopes/adverse effects , Relative Biological Effectiveness , Reproducibility of Results , Republic of Belarus/epidemiology , Risk Factors , Sensitivity and Specificity , Ukraine/epidemiologyABSTRACT
Because of the large number of cells to be analyzed in cases of overexposure to ionizing radiation, an automated imaging system is desirable for scoring both translocations and dicentrics. This system should include three essential steps: automatic metaphase finding, automatic image capture at high magnification, and, finally, optimized data analysis for aberration interpretation. We evaluated a new image analysis system (CYTOGEN, IMSTAR, France) and found that its metaphase finder saved time, as much as quadrupling the speed of scoring chromosomal aberrations. Automatic metaphase selection did not appear to induce bias. We confirmed the equivalence of observing aberrations on a screen after automatic image capture and direct observation under a microscope. This work validated all of the steps necessary for obtaining images for automatic chromosomal aberration detection. The protocols for the detection of translocations may now be applied for biological dosimetry. This step will be validated in a future study.
Subject(s)
Image Processing, Computer-Assisted , In Situ Hybridization, Fluorescence , Metaphase/physiology , Radiometry/methodsABSTRACT
When accidental exposure to ionizing radiations is suspected, optimal choice of a treatment strategy requires, in addition to information about the clinical signs and physical dosimetry, a determination by biological parameters of the dose received. The scoring of unstable chromosomal aberrations in peripheral blood lymphocytes is the current reference method. Preparation of these samples depends on the goal sought--an exact assessment of several irradiations or rapid triage in the case of a large-scale accident. Moreover, some adaptation may be necessary if the irradiation is either heterogenous or not recent. Despite the robustness and adaptability of this procedure, conventional cytogenetics remains a tedious and time-consuming technique, and it requires specialized staff. Scoring micronuclei in binucleated lymphocytes may be an easier, simpler altemative to a dicentric assay. This paper, which is based on the experience acquired by the IPSN in recent years in expert assessment of suspected radiations, has as its goal to provide a succinct technical guideline of these different approaches, as they are adapted to suspected recent irradiation and triage.
Subject(s)
Chromosome Aberrations/radiation effects , Radiation Injuries/diagnosis , Radiation Injuries/genetics , Radioactive Hazard Release , Adult , Calibration , Cell Cycle/radiation effects , Child , Dose-Response Relationship, Radiation , Female , Georgia (Republic) , Humans , Lymphocytes/pathology , Lymphocytes/radiation effects , Male , Micronuclei, Chromosome-Defective/radiation effects , Micronucleus Tests , Mitosis/radiation effects , Radiation Injuries/complications , Radiation Injuries/pathology , Radiation, Ionizing , Radiometry , Time Factors , Triage/methodsABSTRACT
Organotypic cultures established from the third thoracic ganglion of locust embryo have been used to investigate dopamine receptors. In this in vitro system, neurites emerge directly from the explants and form a dense network around the explants, presenting cell surface freely exposed for experimental labelling. Polyclonal anti-idiotypic antibodies raised in rabbits to antibodies against dopamine conjugate, and previously found to bind to dopamine receptors, have been used to investigate putative dopamine receptors in these neurites. Immunocytochemical detection by light microscopy employing immunofluorescence labelling, was correlated with electron microscopy, using peroxidase staining. In addition to a location for dopamine receptors on the neurite surface, intracellular binding sites were also found in neurites. This internal labelling might represent an intracellular pool of dopamine receptor precursors. The labelling was specific in that it was not present when the anti-idiotypic dopamine antibodies were replaced with non-immune serum or when preincubation with conjugated dopamine preceded incubation with anti-idiotypic dopamine antibodies.
Subject(s)
Central Nervous System/metabolism , Dopamine/immunology , Grasshoppers/physiology , Neurites/metabolism , Receptors, Dopamine/metabolism , Animals , Central Nervous System/ultrastructure , Fluorescent Antibody Technique , Ganglia, Invertebrate/cytology , Ganglia, Invertebrate/physiology , Ganglia, Invertebrate/ultrastructure , Immunoglobulin Allotypes/biosynthesis , Immunoglobulin Allotypes/immunology , Immunohistochemistry , In Vitro Techniques , Microscopy, Electron , Neurites/immunology , Neurites/ultrastructure , Receptors, Dopamine/immunology , Receptors, Dopamine/ultrastructureABSTRACT
Neurosecretory brain cells from embryonic locusts cultured in serum-free medium failed to show any visible signs of growth. In contrast, the same neurons co-cultured with CNS explants (brain-retrocerebral complexes and thoracic ganglia) show excellent axonal growth: sprouting occurs after one day of co-culture and increases within the first week. These results indicate the production of an active neurite outgrowth stimulating factor by co-cultured CNS explants. The similarity of the stimulating effects by the two explants on neurite outgrowth rule out brain neurohormones as probable candidates for the stimulating factor. In addition, neither insulin nor neuroparsin added to the culture medium to test their trophic effect improves the growth of the cells. Conditioned medium derived from cultures of brain-retrocerebral complexes produced no neurite outgrowth, suggesting that the active factor released in the medium by brain explants does not remain free in solution but binds to the substratum. Finally, neurons co-cultured with CNS explants attached to the bottom of the culture dish develop neurites only when in close proximity to the explants. This observation strongly suggests the binding of an active neurite outgrowth stimulating factor to the substratum in the vicinity of the explants. As a control for CNS explants, the action of non-nervous tissue was tested: a similar, but less extensive neurotrophic effect, was observed with esophagus segments co-cultured with neurosecretory brain cells. These results demonstrate that locust neurosecretory neurons isolated in cell culture require combined explants for elaborating processes and suggest that the neurite promoting effect is mediated by a substrate-associated molecule(s).(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Brain/embryology , Grasshoppers/physiology , Neurosecretory Systems/embryology , Animals , Axons/physiology , Brain/cytology , Brain/ultrastructure , Cells, Cultured , Culture Media, Conditioned , Embryo, Nonmammalian , Esophagus/cytology , Esophagus/embryology , Ganglia/cytology , Ganglia/embryology , Grasshoppers/embryology , Microscopy, Electron , Neurons/physiology , Neurons/ultrastructure , Neurosecretory Systems/cytology , Neurosecretory Systems/ultrastructure , Neurotransmitter Agents/pharmacology , Neurotransmitter Agents/physiologyABSTRACT
Up to the present it has not been possible to obtain viable glial cells from dissociated insect nervous system cultures. We report here that the use of explant culture of locust embryo central nervous system (CNS) has been successful in allowing the proliferation of glial cells derived from glial precursors located at the periphery of the embryonic CNS. In such cultures, maintained for 3 months under specific conditions, 4 cell types at intermediate stages of differentiation can be distinguished around the explants after 2 weeks in vitro. They have been identified by scanning and transmission electron microscopy. The first type (stage 1) consists of flat epithelioid cells with an abundant and lucent cytoplasm containing little granular endoplasmic reticulum. These earliest cells subsequently develop flat ameboid prolongations forming an intermediate cell type (stage 2) which then differentiates into protuberant bipolar cells (stage 3) in which appear well-organized cisterns of granular endoplasmic reticulum. The last stage of differentiation (stage 4) is composed of multipolar cells with an electron-dense cytoplasm and well-defined processes characterized by the presence of ribosomes and granular endoplasmic reticulum. These (stage 4) differentiated cells resemble mature glial cells. In the same in vitro system, neurites, growing from neurons originating in the explants, form a network lying upon the glial cells and in close relationship with them. Neurites present growth cones and lack ribosomes and granular endoplasmic reticulum. We conclude that this model is a potentially useful system for use in in vitro studies of insect glia and neurite-glia interactions.
Subject(s)
Central Nervous System/embryology , Grasshoppers/embryology , Neuroglia/cytology , Animals , Axons/ultrastructure , Cell Differentiation , Cells, Cultured , Central Nervous System/ultrastructure , Culture Media , Microscopy, Electron , Microscopy, Electron, Scanning , Neuroglia/ultrastructureABSTRACT
The presence of a large amount of glycoconjugates on the anuran amphibian germ cells was demonstrated using fluorescein isothiocyanate lectins binding specifically to D-galactose and at a lower level, by other lectins binding specifically to N-acetyl-galactosamine. Glycoconjugates including D-galactose were found near the pseudopodial expansions and in the extracellular space, between germ cells and follicular cells. They were also disseminated in the cytoplasm. The injection of PNA lectin (from Arachis hypogea) into the endoderm inhibited the migration of 90% of the germ cells. This inhibition was lectin-concentration dependent. Ultrastructural study of germ cells, the migration of which was inhibited, showed that they were degenerating. These results suggest that glycoconjugates are related to the migratory activity of germ cells.
Subject(s)
Germ Cells/physiology , Glycosides/physiology , Lectins/pharmacology , Acetylgalactosamine/metabolism , Animals , Bufo bufo , Cell Count , Cell Movement/drug effects , Galactose/metabolism , Germ Cells/metabolism , Germ Cells/ultrastructure , Microscopy, Electron , Ranidae , Xenopus laevisABSTRACT
Freeze-fracture was used to study Anura Amphibia primordial germ cells (PGCs) from the time when they have invaded genital ridges until the time when sexual differentiation has begun. We observed tight junctions with a variety of configurations including linear, macular, and extensive occluding cross-linking complexes. True gap junctions were not observed. Rod-shaped particles were found disseminated among particles on the P fracture faces of the germ cells.
Subject(s)
Germ Cells/ultrastructure , Gonads/embryology , Intercellular Junctions/ultrastructure , Rana pipiens/embryology , Animals , Cell Nucleus/ultrastructure , Freeze Fracturing , Gonads/ultrastructure , Microscopy, Electron , Mitochondria/ultrastructure , Organoids/ultrastructure , Sex DifferentiationABSTRACT
Effect of injection of Arachis hypogea (PNA) and Soybean (SBA) lectins on the migration of Xenopus primordial germ cells (PGCs) were investigated. PNA inhibited almost entirely the PGCs migration, more than SBA. Our results indicated that the inhibition of the migration could be a consequence of the lectin-induced alterations of cell surface properties.
Subject(s)
Germ Cells/drug effects , Lectins/pharmacology , Plant Lectins , Soybean Proteins , Xenopus laevis/embryology , Animals , Cell Membrane/drug effects , Cell Movement/drug effects , Galactose/physiology , Glycoproteins/physiology , Gonads/embryology , Peanut AgglutininABSTRACT
Primordial germ cells (PGCs) from the dorsal side of midgut endoderm and from within the dorsal mesentery were examined by transmission and scanning electron microscopy. During migration of these cells, lamellipodia and filopodia, develop in a polarized pattern. Large amounts of extracellular material accumulate around the lamellipodia at the leading end of a migrating cell. It is suggested that the polarized pseudopodia function in conjunction with extracellular matrix as the means by which PGCs move en route to gonadal ridges.
Subject(s)
Bufo bufo/embryology , Endoderm/physiology , Germ Cells/physiology , Mesentery/physiology , Ranidae/embryology , Animals , Cell Movement , Germ Cells/ultrastructure , Microscopy, Electron , Microscopy, Electron, ScanningABSTRACT
A study of primordial germ cells (PGC) of Amphibia Anura was carried out after treatment of sections by different fluorescein isothiocyanate conjugated lectins (FITC-lectins). Specific labelling on the PGC is obtained with lectins, the activity of which is inhibited by D-galactose or N-acetyl-galactosamine. These osidic groups appear to be located more specifically on the PGC. The same labelling pattern is not obtained with lectins possessing major affinity for mannose, glucose, fucose and N-acetyl-glucosamine. Furthermore, changes in labelling pattern are observed during migration of PGC. It is suggested that D-galactose and N-acetyl-galactosamine might be related to membrane activity of PGC during migration. Ultrastructural study of the visualization of cell surface carbohydrates supplies some information on the localisation of these lectins receptors.
Subject(s)
Embryo, Nonmammalian/cytology , Germ Cells/cytology , Lectins , Animals , Bufonidae , Embryo, Nonmammalian/ultrastructure , Fluorescein-5-isothiocyanate , Fluoresceins , Fluorescent Dyes , Microscopy, Electron , Microscopy, Fluorescence , Ranidae , Thiocyanates , XenopusABSTRACT
Transmission electron microscopy was used to study Anura Amphibia primordial germ cells (PGCs). Our results demonstrate the evidence of an extracellular matrix and many various cellular junctions between the somatic cells and the PGCs. Furthermore, from cytochemical investigation, it appears that there is abundant cytoplasmic glycogen and also membrane adenyl-cyclase activity. At last, the results are analysed, discussed and related to the hypothesis of the guidance of the migration of the Anura PGCs by cAMP diffusing from the chordo-mesoderm.
Subject(s)
Anura/embryology , Adenylyl Cyclases/metabolism , Animals , Cell Membrane/metabolism , Cytoplasm/metabolism , Germ Cells/ultrastructure , Glycogen/metabolism , Histocytochemistry , Intercellular Junctions/ultrastructure , Microscopy, ElectronABSTRACT
Chordo-mesoderm of Anura Amphibia embryos was investigated by electron microscopy and a cytochemical method was used to study the localization of adenyl-cyclase activity, in this part of embryo. It seem that, in Anura, at a given stage of development, there is some adenyl-cyclase activity, and, consequently, some liberation of AMPc. Pleurodeles waltlii (Urodela Amphibia) appears to be devoid of this adenyl-cyclase activity. AMPc could be the substance provided by chordo-mesoderm attracting germinal cells.