RESUMEN
Biological Dosimetry is a necessary support for national radiation protection programmes and emergency response schemes. The Latin American Biological Dosimetry Network (LBDNet) was formally founded in 2007 to provide early biological dosimetry assistance in case of radiation emergencies in the Latin American Region. Here are presented the main topics considered in the foundational document of the network, which comprise: mission, partners, concept of operation, including the mechanism to request support for biological dosimetry assistance in the region, and the network capabilities. The process for network activation and the role of the coordinating laboratory during biological dosimetry emergency response is also presented. This information is preceded by historical remarks on biological dosimetry cooperation in Latin America. A summary of the main experimental and practical results already obtained by the LBDNet is also included.
Asunto(s)
Protección Radiológica/métodos , Liberación de Radiactividad Peligrosa/prevención & control , Radiometría/métodos , Planificación en Desastres/métodos , Humanos , Agencias Internacionales , Cooperación Internacional , América LatinaRESUMEN
The bottleneck in data acquisition during biological dosimetry based on a dicentric assay is the need to score dicentrics in a large number of lymphocytes. One way to increase the capacity of a given laboratory is to use the ability of skilled operators from other laboratories. This can be done using image analysis systems and distributing images all around the world. Two exercises were conducted to test the efficiency of such an approach involving 10 laboratories. During the first exercise (E1), the participant laboratories analysed the same images derived from cells exposed to 0.5 and 3 Gy; 100 images were sent to all participants for both doses. Whatever the dose, only about half of the cells were complete with well-spread metaphases suitable for analysis. A coefficient of variation (CV) on the standard deviation of â¼15 % was obtained for both doses. The trueness was better for 3 Gy (0.6 %) than for 0.5 Gy (37.8 %). The number of estimated doses classified as satisfactory according to the z-score was 3 at 0.5 Gy and 8 at 3 Gy for 10 dose estimations. In the second exercise, an emergency situation was tested, each laboratory was required to score a different set of 50 images in 2 d extracted from 500 downloaded images derived from cells exposed to 0.5 Gy. Then the remaining 450 images had to be scored within a week. Using 50 different images, the CV on the estimated doses (79.2 %) was not as good as in E1, probably associated to a lower number of cells analysed (50 vs. 100) or from the fact that laboratories analysed a different set of images. The trueness for the dose was better after scoring 500 cells (22.5 %) than after 50 cells (26.8 %). For the 10 dose estimations, the number of doses classified as satisfactory according to the z-score was 9, for both 50 and 500 cells. Overall, the results obtained support the feasibility of networking using electronically transmitted images. However, before its implementation some issues should be elucidated, such as the number and resolution of the images to be sent, and the harmonisation of the scoring criteria. Additionally, a global website able to be used for the different regional networks, like Share Points, will be desirable to facilitate worldwide communication.
Asunto(s)
Aberraciones Cromosómicas/efectos de la radiación , Cromosomas Humanos/efectos de la radiación , Rayos gamma/efectos adversos , Laboratorios/normas , Linfocitos/efectos de la radiación , Bioensayo , Relación Dosis-Respuesta en la Radiación , Humanos , RadiometríaRESUMEN
Well-defined protocols and quality management standards are indispensable for biological dosimetry laboratories. Participation in periodic proficiency testing by interlaboratory comparisons is also required. This harmonization is essential if a cooperative network is used to respond to a mass casualty event. Here we present an international intercomparison based on dicentric chromosome analysis for dose assessment performed in the framework of the IAEA Regional Latin American RLA/9/054 Project. The exercise involved 14 laboratories, 8 from Latin America and 6 from Europe. The performance of each laboratory and the reproducibility of the exercise were evaluated using robust methods described in ISO standards. The study was based on the analysis of slides from samples irradiated with 0.75 (DI) and 2.5 Gy (DII). Laboratories were required to score the frequency of dicentrics and convert them to estimated doses, using their own dose-effect curves, after the analysis of 50 or 100 cells (triage mode) and after conventional scoring of 500 cells or 100 dicentrics. In the conntional scoring, at both doses, all reported frequencies were considered as satisfactory, and two reported doses were considered as questionable. The analysis of the data dispersion among the dicentric frequencies and among doses indicated a better reproducibility for estimated doses (15.6% for DI and 8.8% for DII) than for frequencies (24.4% for DI and 11.4% for DII), expressed by the coefficient of variation. In the two triage modes, although robust analysis classified some reported frequencies or doses as unsatisfactory or questionable, all estimated doses were in agreement with the accepted error of ±0.5 Gy. However, at the DI dose and for 50 scored cells, 5 out of the 14 reported confidence intervals that included zero dose and could be interpreted as false negatives. This improved with 100 cells, where only one confidence interval included zero dose. At the DII dose, all estimations fell within ±0.5 Gy of the reference dose interval. The results obtained in this triage exercise indicated that it is better to report doses than frequencies. Overall, in both triage and conventional scoring modes, the laboratory performances were satisfactory for mutual cooperation purposes. These data reinforce the view that collaborative networking in the case of a mass casualty event can be successful.
Asunto(s)
Radiometría/métodos , Aberraciones Cromosómicas/efectos de la radiación , Urgencias Médicas , Femenino , Humanos , Agencias Internacionales , Laboratorios , Persona de Mediana Edad , Dosis de Radiación , Liberación de Radiactividad Peligrosa , TriajeRESUMEN
The mechanisms of formation of sister chromatid exchanges (SCEs) and chromosome aberrations following inhibition of poly(ADP-ribose) polymerase by 3-aminobenzamide were studied in Chinese hamster ovary cell lines deficient in different repair pathways. The results confirm earlier findings that (a) the 'spontaneous' SCEs are formed due to the incorporated BrdU in the DNA, (b) 'spontaneous' and induced SCEs originate from different mechanisms, and (c) SCEs and chromatid exchanges are formed by different pathways.
Asunto(s)
Benzamidas/farmacología , Reparación del ADN , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Intercambio de Cromátides Hermanas , Animales , Bromodesoxiuridina/metabolismo , Línea Celular , Cromátides/metabolismo , Aberraciones Cromosómicas , HumanosRESUMEN
The fact that eukaryotic DNA is packed into chromatin constitutes a physical barrier to enzymes and regulatory factors to reach the DNA molecule for replication, transcription, recombination and repair. Although most studies in this field have concentrated on how chromatin regulates transcription, there is a recent emphasis on studying the role of chromatin in the response to DNA damage. Two main chromatin-remodeling mechanisms have been identified, namely, ATP-dependent chromatin-remodeling complexes and histone post-translational modifications (PTMs). PTMs constitute reversible covalent modifications in aminoacidic residues, such as serine and threonine phosphorylation, lysine acetylation, lysine and arginine methylation and lysine ubiquitylation, among others. Moreover, nucleosome composition can be modified by the incorporation of histone variants, which are assembled into nucleosomes independently of DNA replication. The phosphorylation of the histone variant H2AX (gammaH2AX) is one of the best examples of histone PTMs in response to DNA damage induction, but many others have recently been revealed. In this review, we focus on and summarize the best-known histone PTMs observed in excision repair (base excision and nucleotide excision) and double-strand break (non-homologous end-joining and homologous recombination) repair pathways. In brief, the interplay between chromatin remodelers and DNA repair factors is discussed in relation to DNA damage response mechanisms.
Asunto(s)
Reparación del ADN , Histonas/metabolismo , Animales , Ensamble y Desensamble de Cromatina , Daño del ADN , Humanos , Procesamiento Proteico-PostraduccionalRESUMEN
In order to shed more light on the influence of DNA replication on the formation and distribution of chromosome aberrations, breakpoints (BP) produced by UV-C and AluI were assigned either to the early replicating short euchromatic arm (Xp(e)) or to the late replicating long heterochromatic arm (Xq(h)) of the Chinese hamster (CHO9) X chromosome. Early (ES) or late (LS) S-phase cells were assessed by pulse incorporating the base analogue 5-bromo-2'-deoxyuridine (BrdU) immediately after UV-C irradiation (30 J/m(2)) or AluI (20 U) poration followed by BrdU immunodetection with FITC-tagged antibodies in metaphase spreads. Short (30 s) UV-C exposures (1 J/m(2)/s) induced BP preferentially in Xq(h) in LS cells and a random distribution of BP along Xp(e) and Xq(h) in ES cells. However, BP induced by long (5 min) UV-C exposures (0.1 J/m(2)/s) clustered according to arm replication time (Xp(e) during ES and Xq(h) along LS). Giemsa-stained metaphases showed elevated frequencies of UV-C induced chromatid-type aberrations and gaps, especially in cells exposed to longer UV-C irradiation. BP induced by AluI clustered in Xp(e) in ES but distributed randomly during LS. In contrast to UV-C, AluI did not produce an increase in the yield of gaps, neither in ES nor in LS cells. Putative mechanisms underlying the observed distributions of chromosome damage in replicating CHO9 cells exposed to UV-C and AluI are discussed.
Asunto(s)
Replicación del ADN , Eucromatina/metabolismo , Heterocromatina/metabolismo , Animales , Células CHO , Puntos de Rotura del Cromosoma , Cricetinae , Cricetulus , Daño del ADN/efectos de los fármacosRESUMEN
Cells with a transcription coupled repair (TCR) deficiency are characterized by a higher sensitivity to UVC irradiation and by an increase in apoptosis and chromosomal aberration frequencies. It has been claimed that the higher frequency of chromosomal aberrations results from the transcription blockage caused by UVC-lesions located in the transcribed strands of the genome. The distribution of chromosome breakpoints in euchromatic and heterochromatic regions of the X chromosome from TCR deficient and proficient Chinese hamster cell lines was studied. Most UVC-induced breakpoints occurred in euchromatic regions of the X chromosome in both cell lines. No increase of UVC-induced breakpoints in the euchromatic region of the UV61 X chromosome was observed, indicating that TCR failure alone cannot be responsible for the increased frequency of chromosomal aberrations. Differential chromatin remodeling in the TCR defective cell line is proposed as a possible mechanism involved in the distribution of UVC-induced breakpoints along the Chinese hamster X chromosome. A similar explanation for the increase of UVC-induced chromosomal aberrations in TCR defective cells is given.
Asunto(s)
Aberraciones Cromosómicas , Transcripción Genética , Cromosoma X , Animales , Células CHO , Puntos de Rotura del Cromosoma , Cricetinae , Cricetulus , Reparación del ADN , Rayos UltravioletaRESUMEN
PURPOSE: Non-random occurrence of induced chromosome breakpoints (BP) has been repeatedly reported. DNA synthesis and chromatin remodeling may influence chromosome BP localization. The CHO9 X chromosome exhibits an early replicating short euchromatic arm (Xpe) and a late replicating long heterochromatic arm (Xqh). We investigated the role played by DNA replication and related chromatin remodeling processes on BP distribution in eu/heterochromatin using the CHO9 X chromosome as a model. MATERIALS AND METHODS: BP induced by etoposide, a topoisomerase II inhibitor, as well as by the S-dependent clastogens ultraviolet-C light (UV-C) and methyl methanesulfonate (MMS) were mapped to CHO9 X chromosome arms. The base analogue 5-bromo-2'-deoxyuridine (BrdUrd) was pulse-added immediately after UV-C irradiation or during etoposide and MMS treatments (40 min) to identify cells in early S-phase (Xpe labeled) or late S-phase (Xqh labeled) after indirect BrdUrd immunodetection in metaphase spreads using primary anti-BrdUrd and secondary fluorochrome-tagged antibodies. RESULTS: During early S-phase, BP induced by etoposide and MMS mapped preferentially to Xpe while BP produced by UV-C localized randomly. BP induced by all agents during late S-phase clustered in Xqh. CONCLUSIONS: Results obtained suggest that replication time of eu/heterochromatin as well as chromatin remodeling may determine BP localization on the CHO9 X chromosome.
Asunto(s)
Aberraciones Cromosómicas/efectos de la radiación , Daño del ADN/genética , Replicación del ADN/fisiología , Replicación del ADN/efectos de la radiación , Cromosoma X/genética , Cromosoma X/efectos de la radiación , Animales , Células CHO , Cricetinae , Cricetulus , Relación Dosis-Respuesta en la Radiación , Dosis de RadiaciónRESUMEN
Histone acetylation/deacetylation constitute the most relevant chromatin remodelling mechanism to control DNA access to nuclear machinery as well as to mutagenic agents. Thus, these epigenetics mechanisms could be involved in processing DNA lesions into chromosomal aberrations. Although radiation-induced DNA lesions are believed to occur randomly, in most cases chromosome breakpoints appear distributed in a non-random manner. In order to study the distribution of chromosome damage induced by clastogenic agents in relation to chromosome histone acetylation patterns, an experimental model based on treating Chinese hamster cells with endonucleases and ionizing radiations as well as immunolabelling metaphase chromosomes with antibodies to acetylated histone H4 was developed. The analysis of intra- and interchromosome breakpoint distribution has been carried out on G-banded chromosomes, and results obtained were correlated with chromosome acetylated histone H4 profiles. A co-localization of intrachromosomal breakpoints induced by Alu I, Barn HI and DNase I as well as by neutrons and gamma-rays was observed. Radiation- and endonuclease-induced breakpoints tend to cluster in less condensed chromosome regions (G-light bands) that show the highest levels of acetylated histone H4. The analysis of interchromosomal distribution of radiation-induced lesions showed a concentration ofbreakpoints in Chinese hamster chromosomes with particular histone acetylation patterns. The fact that chromosome break-points occur more frequently in transcriptionally competent chromosome regions suggests that chromatin conformation and nuclear architecture could play a role in the distribution of chromosome lesions.
Asunto(s)
Ensamble y Desensamble de Cromatina/fisiología , Rotura Cromosómica/fisiología , Histonas/metabolismo , Acetilación , Animales , Células CHO , Ensamble y Desensamble de Cromatina/efectos de la radiación , Bandeo Cromosómico , Cricetinae , Análisis Citogenético , Endonucleasas/metabolismo , Histonas/efectos de la radiación , Radiación IonizanteRESUMEN
SORB (selected observed residual breakpoints) induced by ionizing radiation or endonucleases are often non-randomly distributed in mammalian chromosomes. However, the role played by chromatin structure in the localization of chromosome SORB is not well understood. Anti-topoisomerase drugs such as etoposide are potent clastogens and unlike endonucleases or ionizing radiation, induce DNA double-strand breaks (DSB) by an indirect mechanism. Topoisomerase II (Topo II) is a main component of the nuclear matrix and the chromosome scaffold. Since etoposide leads to DSB by influencing the activity of Topo II, this compound may be a useful tool to study the influence of the chromatin organization on the distribution of induced SORB in mammalian chromosomes. In the present work, we compared the distribution of SORB induced during S-phase by etoposide or X-rays in the short euchromatic and long heterochromatic arms of the CHO9 X chromosome. The S-phase stage (early, mid or late) at which CHO9 cells were exposed to etoposide or X-rays was marked by incorporation of BrdU during treatments and later determined by immunolabeling of metaphase chromosomes with an anti-BrdU FITC-coupled antibody. The majority of treated cells were in late S-phase during treatment either with etoposide or X-rays. SORB induced by etoposide mapped preferentially to Xq but random localization was observed for SORB produced by X-rays. Possible explanations for the uneven distribution of etoposide-induced breakpoints along Xq are discussed.
Asunto(s)
Células CHO/efectos de los fármacos , Células CHO/efectos de la radiación , Rotura Cromosómica , Inhibidores Enzimáticos/toxicidad , Etopósido/toxicidad , Inhibidores de Topoisomerasa II , Cromosoma X/efectos de los fármacos , Cromosoma X/efectos de la radiación , Animales , Células CHO/ultraestructura , Cromátides/efectos de los fármacos , Cromátides/efectos de la radiación , Cromátides/ultraestructura , Aberraciones Cromosómicas , Mapeo Cromosómico , Cricetinae , Cricetulus , ADN/efectos de los fármacos , ADN/efectos de la radiación , Daño del ADN , Femenino , Fase S/efectos de los fármacos , Fase S/efectos de la radiación , Cromosoma X/genética , Cromosoma X/ultraestructuraRESUMEN
Chromosomal aberrations (CA) are the microscopically visible part of a wide spectrum of DNA changes generated by different repair mechanisms of DNA double strand breaks (DSB). The method of fluorescence in situ hybridisation (FISH) has uncovered unexpected complexities of CA and this will lead to changes in our thinking about the origin of CA. The inter- and intrachromosomal distribution of breakpoints is generally not random. CA breakpoints occur preferentially in active chromatin. Deviations from expected interchromosomal distributions of breakpoints may result from the arrangement of chromosomes in the interphase nucleus and/or from different sensitivities of chromosomes with respect to the formation of CA. Telomeres and interstitial telomere repeat like sequences play an important role in the formation of CA. Subtelomeric regions are hot spots for the formation of symmetrical exchanges between homologous chromatids and cryptic aberrations in these regions are associated with human congenital abnormalities.
Asunto(s)
Aberraciones Cromosómicas , Pintura Cromosómica/métodos , Animales , Rotura Cromosómica/genética , Daño del ADN , Reparación del ADN , Humanos , Telómero/genéticaRESUMEN
Previously, we have shown that breakpoints induced by the endonucleases AluI, BamHI and DNase I in CHO chromosomes are localized mainly in G-light bands. Neutrons and gamma rays produced similar breakpoint clusters to endonucleases in most CHO chromosomes. Here we compare endonuclease- and radiation-induced breakpoint maps with hyperacetylation patterns of histone H4. The H4 acetylation pattern in chromosomes is similar to the pattern of G-light, or R-bands, and breakpoints are clustered in highly acetylated chromosome regions. These findings indicate that chromosomal aberrations occur more frequently in active than in inactive chromatin.
Asunto(s)
Cromosomas/efectos de los fármacos , Cromosomas/efectos de la radiación , Daño del ADN , Endonucleasas/metabolismo , Histonas/genética , Acetilación , Animales , Células CHO , Células Cultivadas , Cromatina/metabolismo , Bandeo Cromosómico , Cricetinae , Histonas/metabolismo , Procesamiento de Imagen Asistido por Computador , Cariotipificación , Transcripción GenéticaRESUMEN
It has been shown repeatedly that following irradiation of human lymphocytes in the G0 stage, more translocations are induced than dicentrics. To check the role of DNA double-strand breaks (DSB) alone for the induction of symmetrical and asymmetrical chromosome aberrations, the frequencies of induced exchange aberrations by the restriction enzyme AluI were analyzed. The enzyme was introduced into cells using the pellet pipetting technique. Frequencies of induced translocations and dicentrics were determined using a chromosome painting assay with chromosome-specific DNA libraries for chromosomes 1, 4 and X (representing 16.8% of the human genome). The number of translocations detected was approximately 3-fold higher than the number of dicentrics, indicating that the increased frequency of translocations compared with dicentrics found in irradiated human lymphocytes does not result from DNA lesions other than DSB but from differential processing of DSB.
Asunto(s)
Aberraciones Cromosómicas/genética , Pintura Cromosómica/métodos , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Translocación Genética/genética , Centrómero/efectos de los fármacos , Humanos , Hibridación Fluorescente in Situ/métodos , Linfocitos/ultraestructura , MasculinoRESUMEN
Chromosome breakpoints induced by neutrons or gamma rays in Chinese hamster ovary cells were mapped to Giemsa-light or Giemsa-dark bands or to band junctions. Radiation-induced breakpoints were found to be distributed nonrandomly according to chromosome or band length. More than 60% of the breakpoints were localized in G-light bands. A group of 13 bands which corresponded to only 7% of the total chromosome length contained 22% of the breakpoints produced by neutrons and 14% of those induced by gamma rays. Seven of these 13 bands are also preferentially damaged by AluI, BamHI and DNase I as reported previously. The results indicate that chromatin and nuclear structure may play a role in the distribution of breakpoints produced by ionizing radiation and endonucleases.
Asunto(s)
Aberraciones Cromosómicas , Rayos gamma , Neutrones , Animales , Células CHO , Bandeo Cromosómico , Cricetinae , Cricetulus , Desoxirribonucleasa BamHI/metabolismo , Desoxirribonucleasa I/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Mapeo Restrictivo , Fase SRESUMEN
Restriction endonucleases and ionizing radiations have been extensively used to study the origin of chromosomal aberrations. Although a non-random distribution of chromosome breakpoints induced by these agents has been claimed by several authors, the significance of the chromatin structure and nuclear architecture in the localization of breakpoints is still not well understood. Breakpoint patterns produced by endonucleases targeted to specific genome sequences or by ionizing radiations could provide additional evidence to clarify this point. Results obtained from the localization of breakpoints induced by AluI, BamHI or DNase I as well as by neutrons or gamma-rays in G-banded Chinese hamster ovary (CHO) chromosomes are presented. AluI and BamHI were electroporated into CHO cells either during the G1 or S-phase of the cell cycle. A co-localization of breakpoints was found with a preferential occurrence in G-light bands independent of the cell cycle stage in which aberration production took place. Since AluI and BamHI recognition sequences are partitioned in the housekeeping and tissue-specific subgenomes respectively, we postulated that nuclease sensitive sites in active chromatin could be the main targets for the induction of breakpoints by these endonucleases. This assumption is supported by the finding that DNase I-induced breakpoint patterns in CHO cells are similar to those produced by AluI and BamHI. Digestion of fixed CHO chromosomes with these endonucleases induced G-banding suggesting a higher sensitivity of G-light chromatin. For comparison purposes, CHO cells were irradiated with neutrons or gamma-rays and breakpoints localized in G-banded chromosome aberrations. A higher occurrence of breakpoints in G-light bands was also observed. We detected seven breakage-prone G-light bands that were preferentially damaged by the three endonucleases and by both types of radiation. These results emphasize the possible implication of the chromatin structure and the nuclear architecture in the localization of chromosome breakpoints induced by endonucleases, neutrons and gamma-rays.
Asunto(s)
Cromatina/química , Rotura Cromosómica/genética , Cromosomas/metabolismo , Animales , Células CHO , Ciclo Celular/fisiología , Núcleo Celular/fisiología , Bandeo Cromosómico , Cricetinae , Electroporación , Endonucleasas/metabolismo , Rayos gamma , NeutronesRESUMEN
A physical method is described to permeabilize human peripheral lymphocytes in culture for the restriction endonuclease AluI. Blood cultures are incubated for 20 h, pelleted, strongly pipetted with different types of Pasteur pipettes in the presence of AluI and recovered for up to 54 h in the presence of 5-bromodeoxyuridine. Up to 20% of aberrant first posttreatment (M1) metaphases with chromosome-type aberrations are found. The method works with whole blood cultures and is therefore easy to perform.