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1.
Front Public Health ; 9: 701878, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34368064

RESUMO

The effect of the reportedly low ionizing radiation doses, such as those very often delivered to patients in interventional cardiology, remains ambiguous. As interventional cardiac procedures may have a significant impact on total collective effective dose, there are radiation protection concerns for patients and physicians regarding potential late health effects. Given that very low doses (<100 mSv) are expected to be delivered during these procedures, the purpose of this study was to assess the potency and suitability of current genotoxicity biomarkers to detect and quantitate biological effects essential for risk estimation in interventional cardiology. Specifically, the biomarkers γ-H2AX foci, dicentric chromosomes, and micronuclei, which underpin radiation-induced DNA damage, were studied in blood lymphocytes of 25 adult patients before and after interventional cardiac procedures. Even though the mean values of all patients as a group for all three endpoints tested show increased yields relative to baseline following medical exposure, our results demonstrate that only the γ-H2AX biomarker enables detection of statistically significant differences at the individual level (p < 0.001) for almost all patients (91%). Furthermore, 24 h after exposure, residual γ-H2AX foci were still detectable in irradiated lymphocytes. Their decline was found to vary significantly among the individuals and the repair kinetics of γ-H2AX foci was found to range from 25 to 95.6% of their maximum values obtained.


Assuntos
Cardiologia , Lesões por Radiação , Adulto , Biomarcadores , Dano ao DNA , Relação Dose-Resposta à Radiação , Histonas/genética , Humanos
2.
Int J Radiat Biol ; 97(10): 1404-1416, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34330206

RESUMO

PURPOSE: The development of an exposure apparatus for in situ α-irradiation studies of cells. The construction of the apparatus is simple and the apparatus is maintenance free, easy to use and of low cost. This small device can be placed in an incubator, where the exposure environment is controlled. Moreover the vapor saturated incubator protects the cells from drying out, allowing long irradiation intervals. MATERIALS AND METHODS: The system includes a 234U alpha (α)-source of total activity 0.77 ± 0.03 MBq in the form of a thin disk deposited on an aluminum substrate. The α-particles emitted in the air have a mean energy of 4.9 MeV at the disk surface. Source homogeneity has been studied via Rutherford Backscattering Spectrometry. Using SRIM 2013 and Monte Carlo (MC) simulations via the MCNP6.1 code, LET and energy deposition values have been calculated for various filling gasses. Furthermore, based on these simulations, the assembly's dimensions and equivalent irradiation rate have been determined. With respect to the aforementioned dimensions, the experimental setup is constructed in a way to provide uniform irradiation of the sample. Using Sacalc3v1.4 irradiation radial homogeneity has been studied. In order to evaluate biologically our apparatus, a well-established chromosomal aberration assay has been utilized, applied in exponentially growing hamster (CHO) cells. Furthermore, immunofluorescence gamma-H2AX/53BP1 foci assay has been performed as a 'biological detector', in order to validate α-particles surface density. RESULTS: Source surface homogeneity: emission deviations do not exceed 10-15%. The optimal distance between the source and the cells for irradiation is determined to be 14.8 mm. Irradiation radial homogeneity: a deviation of 5% occurs at the first 8 mm from the center of the irradiation area, and a 10% deviation occurs after 12 mm. Chromosomal aberrations were found in good agreement with the corresponding in bibliography. CONCLUSIONS: The current technical report describes analytically the development and evaluation stages of this experimental housing; from MC simulations to the irradiation of mammalian cells and data analysis. Moreover, guidance is provided as well as a report of the variables on which critical parameters are depended, so as to make this work useful to anyone who wants to construct a similar in-house α-irradiation apparatus for radiobiological studies using mammalian cells.


Assuntos
Partículas alfa , Radiobiologia , Partículas alfa/efeitos adversos , Animais , Aberrações Cromossômicas , Cricetinae , Método de Monte Carlo
3.
Front Public Health ; 9: 675095, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34123995

RESUMO

While technological advances in radiation oncology have led to a more precise delivery of radiation dose and a decreased risk of side effects, there is still a need to better understand the mechanisms underlying DNA damage response (DDR) at the DNA and cytogenetic levels, and to overcome tumor resistance. To maintain genomic stability, cells have developed sophisticated signaling pathways enabling cell cycle arrest to facilitate DNA repair via the DDR-related kinases and their downstream targets, so that DNA damage or DNA replication stress induced by genotoxic therapies can be resolved. ATM, ATR, and Chk1 kinases are key mediators in DDR activation and crucial factors in treatment resistance. It is of importance, therefore, as an alternative to the conventional clonogenic assay, to establish a cytogenetic assay enabling reliable and time-efficient results in evaluating the potency of DDR inhibitors for radiosensitization. Toward this goal, the present study aims at the development and optimization of a chromosomal radiosensitivity assay using the DDR and G2-checkpoint inhibitors as a novel modification compared to the classical G2-assay. Also, it aims at investigating the strengths of this assay for rapid radiosensitivity assessments in cultured cells, and potentially, in tumor cells obtained from biopsies. Specifically, exponentially growing RPE and 82-6 hTERT human cells are irradiated during the G2/M-phase transition in the presence or absence of Caffeine, VE-821, and UCN-1 inhibitors of ATM/ATR, ATR, and Chk1, respectively, and the induced chromatid breaks are used to evaluate cell radiosensitivity and their potency for radiosensitization. The increased yield of chromatid breaks in the presence of DDR inhibitors, which underpins radiosensitization, is similar to that observed in cells from highly radiosensitive AT-patients, and is considered here as 100% radiosensitive internal control. The results highlight the potential of our modified G2-assay using VE-821 to evaluate cell radiosensitivity, the efficacy of DDR inhibitors in radiosensitization, and reinforce the concept that ATM, ATR, and Chk1 represent attractive anticancer drug targets in radiation oncology.


Assuntos
Cromátides , Reparo do DNA , Dano ao DNA , Pontos de Checagem da Fase G2 do Ciclo Celular , Humanos , Tolerância a Radiação
4.
Cancers (Basel) ; 12(9)2020 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-32825012

RESUMO

For precision cancer radiotherapy, high linear energy transfer (LET) particle irradiation offers a substantial advantage over photon-based irradiation. In contrast to the sparse deposition of low-density energy by χ- or γ-rays, particle irradiation causes focal DNA damage through high-density energy deposition along the particle tracks. This is characterized by the formation of multiple damage sites, comprising localized clustered patterns of DNA single- and double-strand breaks as well as base damage. These clustered DNA lesions are key determinants of the enhanced relative biological effectiveness (RBE) of energetic nuclei. However, the search for a fingerprint of particle exposure remains open, while the mechanisms underlying the induction of chromothripsis-like chromosomal rearrangements by high-LET radiation (resembling chromothripsis in tumors) await to be elucidated. In this work, we investigate the transformation of clustered DNA lesions into chromosome fragmentation, as indicated by the induction and post-irradiation repair of chromosomal damage under the dynamics of premature chromosome condensation in G0 human lymphocytes. Specifically, this study provides, for the first time, experimental evidence that particle irradiation induces localized shattering of targeted chromosome domains. Yields of chromosome fragments and shattered domains are compared with those generated by γ-rays; and the RBE values obtained are up to 28.6 for α-particles (92 keV/µm), 10.5 for C-ions (295 keV/µm), and 4.9 for protons (28.5 keV/µm). Furthermore, we test the hypothesis that particle radiation-induced persistent clustered DNA lesions and chromatin decompaction at damage sites evolve into localized chromosome shattering by subsequent chromatin condensation in a single catastrophic event-posing a critical risk for random rejoining, chromothripsis, and carcinogenesis. Consistent with this hypothesis, our results highlight the potential use of shattered chromosome domains as a fingerprint of high-LET exposure, while conforming to the new model we propose for the mechanistic origin of chromothripsis-like rearrangements.

5.
Cancers (Basel) ; 11(8)2019 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-31390832

RESUMO

The discovery of chromothripsis in cancer genomes challenges the long-standing concept of carcinogenesis as the result of progressive genetic events. Despite recent advances in describing chromothripsis, its mechanistic origin remains elusive. The prevailing conception is that it arises from a massive accumulation of fragmented DNA inside micronuclei (MN), whose defective nuclear envelope ruptures or leads to aberrant DNA replication, before main nuclei enter mitosis. An alternative hypothesis is that the premature chromosome condensation (PCC) dynamics in asynchronous micronucleated cells underlie chromosome shattering in a single catastrophic event, a hallmark of chromothripsis. Specifically, when main nuclei enter mitosis, premature chromatin condensation provokes the shattering of chromosomes entrapped inside MN, if they are still undergoing DNA replication. To test this hypothesis, the agent RO-3306, a selective ATP-competitive inhibitor of CDK1 that promotes cell cycle arrest at the G2/M boundary, was used in this study to control the degree of cell cycle asynchrony between main nuclei and MN. By delaying the entrance of main nuclei into mitosis, additional time was allowed for the completion of DNA replication and duplication of chromosomes inside MN. We performed interphase cytogenetic analysis using asynchronous micronucleated cells generated by exposure of human lymphocytes to γ-rays, and heterophasic multinucleated Chinese hamster ovary (CHO) cells generated by cell fusion procedures. Our results demonstrate that the PCC dynamics during asynchronous mitosis in micronucleated or multinucleated cells are an important determinant of chromosome shattering and may underlie the mechanistic origin of chromothripsis.

6.
PLoS One ; 14(5): e0216081, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31059552

RESUMO

A sensitive biodosimetry tool is required for rapid individualized dose estimation and risk assessment in the case of radiological or nuclear mass casualty scenarios to prioritize exposed humans for immediate medical countermeasures to reduce radiation related injuries or morbidity risks. Unlike the conventional Dicentric Chromosome Assay (DCA), which takes about 3-4 days for radiation dose estimation, cell fusion mediated Premature Chromosome Condensation (PCC) technique in G0 lymphocytes can be rapidly performed for radiation dose assessment within 6-8 hrs of sample receipt by alleviating the need for ex vivo lymphocyte proliferation for 48 hrs. Despite this advantage, the PCC technique has not yet been fully exploited for radiation biodosimetry. Realizing the advantage of G0 PCC technique that can be instantaneously applied to unstimulated lymphocytes, we evaluated the utility of G0 PCC technique in detecting ionizing radiation (IR) induced stable and unstable chromosomal aberrations for biodosimetry purposes. Our study demonstrates that PCC coupled with mFISH and mBAND techniques can efficiently detect both numerical and structural chromosome aberrations at the intra- and inter-chromosomal levels in unstimulated T- and B-lymphocytes. Collectively, we demonstrate that the G0 PCC technique has the potential for development as a biodosimetry tool for detecting unstable chromosome aberrations (chromosome fragments and dicentric chromosomes) for early radiation dose estimation and stable chromosome exchange events (translocations) for retrospective monitoring of individualized health risks in unstimulated lymphocytes.


Assuntos
Aberrações Cromossômicas/efeitos da radiação , Linfócitos/efeitos da radiação , Radiometria/métodos , Animais , Células CHO/efeitos da radiação , Fusão Celular , Centrômero/efeitos da radiação , Cricetulus , Feminino , Raios gama/efeitos adversos , Humanos , Hibridização in Situ Fluorescente , Masculino , Lesões por Radiação/diagnóstico , Lesões por Radiação/genética , Radiação Ionizante , Estudos Retrospectivos , Cariotipagem Espectral/métodos , Telômero/efeitos da radiação , Raios X/efeitos adversos
7.
Int J Radiat Oncol Biol Phys ; 103(5): 1184-1193, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30529375

RESUMO

PURPOSE: Nontargeted effects of ionizing radiation, by which unirradiated cells and tissues are also damaged, are a relatively new paradigm in radiobiology. We recently reported radiation-induced abscopal effects (RIAEs) in normal tissues; namely, DNA damage, apoptosis, and activation of the local and systemic immune responses in C57BL6/J mice after irradiation of a small region of the body. High-dose-rate, synchrotron-generated broad beam or multiplanar x-ray microbeam radiation therapy was used with various field sizes and doses. This study explores components of the immune system involved in the generation of these abscopal effects. METHODS AND MATERIALS: The following mice with various immune deficiencies were irradiated with the microbeam radiation therapy beam: (1) SCID/IL2γR-/- (NOD SCID gamma, NSG) mice, (2) wild-type C57BL6/J mice treated with an antibody-blocking macrophage colony-stimulating factor 1 receptor, which depletes and alters the function of macrophages, and (3) chemokine ligand 2/monocyte chemotactic protein 1 null mice. Complex DNA damage (ie, DNA double-strand breaks), oxidatively induced clustered DNA lesions, and apoptotic cells in tissues distant from the irradiation site were measured as RIAE endpoints and compared with those in wild-type C57BL6/J mice. RESULTS: Wild-type mice accumulated double-strand breaks, oxidatively induced clustered DNA lesions, and apoptosis, enforcing our RIAE model. However, these effects were completely or partially abrogated in mice with immune disruption, highlighting the pivotal role of the immune system in propagation of systemic genotoxic effects after localized irradiation. CONCLUSIONS: These results underline the importance of not only delineating the best strategies for tumor control but also mitigating systemic radiation toxicity.


Assuntos
Apoptose , Quebras de DNA de Cadeia Dupla , Sistema Imunitário/fisiologia , Lesões Experimentais por Radiação/imunologia , Animais , Efeito Espectador , Quimiocina CCL2/sangue , Quimiocina CCL2/genética , DNA/isolamento & purificação , Feminino , Ligantes , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Estresse Oxidativo , Doses de Radiação , Lesões Experimentais por Radiação/etiologia , Receptor de Fator Estimulador de Colônias de Macrófagos/antagonistas & inibidores , Síncrotrons , Fator de Crescimento Transformador beta1/sangue
8.
Mutat Res Genet Toxicol Environ Mutagen ; 836(Pt A): 65-71, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30389164

RESUMO

In radiation accidents and large-scale radiological emergencies, a fast and reliable triage of individuals according to their degree of exposure is important for accident management and identification of those who need medical assistance. In this work, the applicability of cell-fusion-mediated premature chromosome condensation (PCC) in G0-lymphocytes is examined for the development of a rapid, minimally invasive and automatable micro-PCC assay, which requires blood volumes of only 100 µl and can be performed in 96-well plates, towards risk assessments and categorization of individuals based on dose estimates. Chromosomal aberrations are visualized for dose-estimation analysis within two hours, without the need of blood culturing for two days, as required by conventional cytogenetics. The various steps of the standard-PCC procedure were adapted and, for the first time, lymphocytes in blood volumes of 100 µl were successfully fused with CHO-mitotics in 96-well plates of 2 ml/well. The plates are advantageous for high-throughput analysis since the various steps required are applied to all 96-wells simultaneously. Interestingly, the use of only 1.5 ml hypotonic and Carnoy's fixative per well offers high quality PCC-images, and the morphology of lymphocyte PCCs is identical to that obtained using the conventional PCC-assay, which requires much larger blood volumes and 15 ml tubes. For dose assessments, appropriate calibration curves were constructed and for PCC analysis specialized software (MetaSystems) was used. The micro-PCC assay can be combined with fluorescence in situ hybridization (FISH), using simultaneously centromeric/telomeric (C/T) peptide nucleic acid (PNA) probes. This allows dose assessments on the basis of accurate scoring of dicentric and centric ring chromosomes in G0-lymphocyte PCCs, which is particularly helpful when further evaluation into treatment-level categories of exposed individuals is needed. The micro-PCC assay has significant advantages for early triage biodosimetry when compared to other cytogenetic biodosimetry assays. It is rapid, cost-effective, and could pave the way to its subsequent automation.


Assuntos
Bioensaio/métodos , Aberrações Cromossômicas/efeitos da radiação , Cromossomos Humanos/efeitos da radiação , Linfócitos/patologia , Medição de Risco/métodos , Animais , Células CHO , Fusão Celular , Cricetulus , Emergências , Voluntários Saudáveis , Humanos , Linfócitos/efeitos da radiação , Doses de Radiação , Liberação Nociva de Radioativos
9.
Int J Radiat Biol ; 93(1): 48-57, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27813725

RESUMO

PURPOSE: Dose assessment intercomparisons within the RENEB network were performed for triage biodosimetry analyzing G0-lymphocyte PCC for harmonization, standardization and optimization of the PCC assay. MATERIALS AND METHODS: Comparative analysis among different partners for dose assessment included shipment of PCC-slides and captured images to construct dose-response curves for up to 6 Gy γ-rays. Accident simulation exercises were performed to assess the suitability of the PCC assay by detecting speed of analysis and minimum number of cells required for categorization of potentially exposed individuals. RESULTS: Calibration data based on Giemsa-stained fragments in excess of 46 PCC were obtained by different partners using galleries of PCC images for each dose-point. Mean values derived from all scores yielded a linear dose-response with approximately 4 excess-fragments/cell/Gy. To unify scoring criteria, exercises were carried out using coded PCC-slides and/or coded irradiated blood samples. Analysis of samples received 24 h post-exposure was successfully performed using Giemsa staining (1 excess-fragment/cell/Gy) or centromere/telomere FISH-staining for dicentrics. CONCLUSIONS: Dose assessments by RENEB partners using appropriate calibration curves were mostly in good agreement. The PCC assay is quick and reliable for whole- or partial-body triage biodosimetry by scoring excess-fragments or dicentrics in G0-lymphocytes. Particularly, analysis of Giemsa-stained excess PCC-fragments is simple, inexpensive and its automation could increase throughput and scoring objectivity of the PCC assay.


Assuntos
Bioensaio/métodos , Aberrações Cromossômicas/efeitos da radiação , Testes para Micronúcleos/métodos , Garantia da Qualidade dos Cuidados de Saúde , Exposição à Radiação/análise , Monitoramento de Radiação/métodos , Bioensaio/normas , Europa (Continente) , Humanos , Linfócitos/citologia , Linfócitos/efeitos da radiação , Monitoramento de Radiação/normas , Reprodutibilidade dos Testes , Fase de Repouso do Ciclo Celular/genética , Fase de Repouso do Ciclo Celular/efeitos da radiação , Sensibilidade e Especificidade
10.
Free Radic Res ; 50(sup1): S64-S78, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27593437

RESUMO

Detrimental effects of ionizing radiation (IR) are correlated to the varying efficiency of IR to induce complex DNA damage. A double strand break (DSB) can be considered the simpler form of complex DNA damage. These types of damage can consist of DSBs, single strand breaks (SSBs) and/or non-DSB lesions such as base damages and apurinic/apyrimidinic (AP; abasic) sites in different combinations. Enthralling theoretical (Monte Carlo simulations) and experimental evidence suggests an increase in the complexity of DNA damage and therefore repair resistance with linear energy transfer (LET). In this study, we have measured the induction and processing of DSB and non-DSB oxidative clusters using adaptations of immunofluorescence. Specifically, we applied foci colocalization approaches as the most current methodologies for the in situ detection of clustered DNA lesions in a variety of human normal (FEP18-11-T1) and cancerous cell lines of varying repair efficiency (MCF7, HepG2, A549, MO59K/J) and radiation qualities of increasing LET, that is γ-, X-rays 0.3-1 keV/µm, α-particles 116 keV/µm and 36Ar ions 270 keV/µm. Using γ-H2AX or 53BP1 foci staining as DSB probes, we calculated a DSB apparent rate of 5-16 DSBs/cell/Gy decreasing with LET. A similar trend was measured for non-DSB oxidized base lesions detected using antibodies against the human repair enzymes 8-oxoguanine-DNA glycosylase (OGG1) or AP endonuclease (APE1), that is damage foci as probes for oxidized purines or abasic sites, respectively. In addition, using colocalization parameters previously introduced by our groups, we detected an increasing clustering of damage for DSBs and non-DSBs. We also make correlations of damage complexity with the repair efficiency of each cell line and we discuss the biological importance of these new findings with regard to the severity of IR due to the complex nature of its DNA damage.


Assuntos
Dano ao DNA/genética , Reparo do DNA/genética , Transferência Linear de Energia/genética , Radiação Ionizante , Humanos
11.
Radiat Prot Dosimetry ; 172(1-3): 230-237, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27344061

RESUMO

High-dose assessments using the conventional dicentric assay are essentially restricted to doses up to 5 Gy and only to lymphocytes that succeed to proceed to first post-exposure mitosis. Since G2-checkpoint activation facilitates DNA damage recognition and arrest of damaged cells, caffeine is used to release G2-blocked lymphocytes overcoming the mitotic index and dicentric yield saturation problems, enabling thus dicentric analysis even at high-dose exposures. Using the fluorescence in situ hybridization technique with telomere and centromere peptide nucleic acid probes, the released lymphocytes, identified as metaphases with decondensed chromosomes following 1.5 h caffeine treatment, show increased yield of dicentrics compared to that obtained in lymphocytes that reach metaphase without G2-checkpoint abrogation by caffeine. Here, a 3-h caffeine/colcemid co-treatment before harvesting at 55 h post-exposure is used so that the dicentric analysis using Giemsa staining is based predominantly on lymphocytes released from the G2-block, increasing thus dicentric yield and enabling construction of a dose-response calibration curve with improved precision of high-dose estimates.


Assuntos
Cafeína/administração & dosagem , Aberrações Cromossômicas/efeitos da radiação , Análise Citogenética/métodos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Linfócitos/efeitos da radiação , Radiometria/métodos , Bioensaio/métodos , Células Cultivadas , Relação Dose-Resposta à Radiação , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos da radiação , Humanos , Doses de Radiação , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
12.
Artigo em Inglês | MEDLINE | ID: mdl-26520375

RESUMO

The dual role of caffeine as a chromosomal damage inducer and G2/M-checkpoint abrogator is well known but it is observed mainly at relatively high concentrations. At low concentrations, caffeine enhances the cytogenetic effects of several carcinogens and its intake during pregnancy has been recently reported to cause adverse birth outcomes. Interestingly, a threshold below which this association is not apparent was not identified. Since chromosomal abnormalities and aneuploidy are the major genetic etiologies of spontaneous abortions and adverse birth outcomes, we re-evaluate here the effects of caffeine at the cytogenetic level and propose a model for the mechanisms involved. Our hypothesis is that low caffeine concentrations affect DNA replication and cause chromosomal aberrations and asymmetric cell divisions not easily detected at metaphase since damaged cells are delayed during their G2/M-phase transition and the low caffeine concentrations cannot abrogate the G2-checkpoint. To test this hypothesis, caffeine-induced chromatid breaks and micronuclei in peripheral blood lymphocytes (PBLs) were evaluated in vitro after low caffeine concentration exposures, followed by a short treatment with 4mM of caffeine to abrogate the G2-checkpoint. The results show a statistically significant increase in chromatid breaks at caffeine concentrations ≥1mM. When caffeine was applied for G2/M-checkpoint abrogation, a statistically significant increase in chromatid breaks, compared to an active checkpoint, was only observed at 4mM of caffeine. The potential of low concentrations to induce asymmetric cell divisions was tested by applying a methodology combining the cytochalasin-B mediated cytokinesis-block micronucleus assay (CBMN) with interphase FISH (iFISH), using selected centromeric probes. Interestingly, low caffeine concentrations induce a dose dependent aneuploidy through asymmetric cell divisions, which are caused by misalignment of chromosomes through a mechanism unrelated to the formation of chromatid breaks. The cytogenetic approach used, combining CBMN with iFISH, is proposed as a valuable tool to test chemically induced asymmetric cell divisions.


Assuntos
Cafeína/farmacologia , Divisão Celular/efeitos dos fármacos , Linfócitos/citologia , Micronúcleos com Defeito Cromossômico/efeitos da radiação , Aberrações Cromossômicas , Citocalasina B/farmacologia , Replicação do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Hibridização in Situ Fluorescente , Técnicas In Vitro , Interfase/efeitos da radiação , Linfócitos/efeitos dos fármacos , Testes para Micronúcleos
13.
Artigo em Inglês | MEDLINE | ID: mdl-26520380

RESUMO

The frequency of dicentric chromosomes in human peripheral blood lymphocytes at metaphase is considered as the "gold-standard" method for biological dosimetry and, presently, it is the most widely used for dose assessment. Yet, it needs lymphocyte stimulation and a 2-day culture, failing the requirement of rapid dose estimation, which is a high priority in radiation emergency medicine and triage biodosimetry. In the present work, we assess the applicability of cell fusion mediated premature chromosome condensation (PCC) methodology, which enables the analysis of radiation-induced chromosomal aberrations directly in non-stimulated G0-lymphocytes, without the 2-day culture delay. Despite its advantages, quantification of an exposure by means of the PCC-method is not currently widely used, mainly because Giemsa-staining of interphase G0-lymphocyte chromosomes facilitates the analysis of fragments and rings, but not of dicentrics. To overcome this shortcoming, the PCC-method is combined with fluorescence in situ hybridization (FISH), using simultaneously centromeric/telomeric peptide nucleic acid (PNA)-probes. This new approach enables an accurate analysis of dicentric and centric ring chromosomes, which are formed within 8h post irradiation and will, therefore, be present in the blood sample by the time it arrives for dose estimation. For triage biodosimetry, a dose response curve for up to 10Gy was constructed and compared to that obtained using conventional metaphase analysis with Giemsa or centromeric/telomeric PNA-probes in metaphase. Since FISH is labor intensive, a simple PCC-method scoring Giemsa-stained fragments in excess of 46 was also assessed as an even more rapid approach for triage biodosimetry. First, we studied the rejoining kinetics of fragments and constructed a dose-response curve for 24h repair time. Then, its applicability was assessed for four different doses and compared with the PCC-method using centromeric/telomeric PNA-probes, through the evaluation of speed of analysis and minimum number of cells required for dose estimation and categorization of exposed individuals.


Assuntos
Centrômero/genética , Aberrações Cromossômicas , Linfócitos/efeitos da radiação , Ácidos Nucleicos Peptídicos/genética , Radiometria/métodos , Corantes Azur , Células Cultivadas , Sondas de DNA/genética , Humanos , Hibridização in Situ Fluorescente , Linfócitos/citologia , Fase de Repouso do Ciclo Celular , Telômero/genética , Triagem/métodos
14.
Artigo em Inglês | MEDLINE | ID: mdl-26520389

RESUMO

Combination of next-generation DNA sequencing, single nucleotide polymorphism array analyses and bioinformatics has revealed the striking phenomenon of chromothripsis, described as complex genomic rearrangements acquired in a single catastrophic event affecting one or a few chromosomes. Via an unproven mechanism, it is postulated that mechanical stress causes chromosome shattering into small lengths of DNA, which are then randomly reassembled by DNA repair machinery. Chromothripsis is currently examined as an alternative mechanism of oncogenesis, in contrast to the present paradigm that considers a stepwise development of cancer. While evidence for the mechanism(s) underlying chromosome shattering during cancer development remains elusive, a number of hypotheses have been proposed to explain chromothripsis, including ionizing radiation, DNA replication stress, breakage-fusion-bridge cycles, micronuclei formation and premature chromosome compaction. In the present work, we provide experimental evidence on the mechanistic basis of chromothripsis and on how chromosomes can get locally shattered in a single catastrophic event. Considering the dynamic nature of chromatin nucleoprotein complex, capable of rapid unfolding, disassembling, assembling and refolding, we first show that chromatin condensation at repairing or replicating DNA sites induces the mechanical stress needed for chromosome shattering to ensue. Premature chromosome condensation is then used to visualize the dynamic nature of interphase chromatin and demonstrate that such mechanical stress and chromosome shattering can also occur in chromosomes within micronuclei or asynchronous multinucleate cells when primary nuclei enter mitosis. Following an aberrant mitosis, chromosomes could find themselves in the wrong place at the wrong time so that they may undergo massive DNA breakage and rearrangement in a single catastrophic event. Specifically, our results support the hypothesis that premature chromosome condensation induces mechanical stress and triggers shattering and chromothripsis in chromosomes or chromosome arms still undergoing DNA replication or repair in micronuclei or asynchronous multinucleate cells, when primary nuclei enter mitosis.


Assuntos
Núcleo Celular/genética , Cromatina/genética , Citocalasina B/farmacologia , DNA/genética , Mitose , Animais , Células CHO , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/efeitos da radiação , Células Cultivadas , Cromatina/efeitos dos fármacos , Cromatina/efeitos da radiação , Aberrações Cromossômicas , Cricetulus , DNA/efeitos dos fármacos , DNA/efeitos da radiação , Humanos , Linfócitos/efeitos dos fármacos , Linfócitos/efeitos da radiação , Mitose/efeitos dos fármacos , Mitose/efeitos da radiação
15.
Cancer Lett ; 356(1): 34-42, 2015 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-24333869

RESUMO

Radiation-induced bystander effects (RIBE), demonstrate the induction of biological non-targeted effects in cells which have not directly hit by radiation or by free radicals produced by ionization events. Although RIBE have been demonstrated using a variety of biological endpoints the mechanism(s) of this phenomenon still remain unclear. The controversial results of the in vitro RIBE and the evidence of non-targeted effects in various in vivo systems are discussed. The experimental evidence on RIBE, indicate that a more analytical and mechanistic in depth approach is needed to secure an answer to one of the most intriguing questions in radiobiology.


Assuntos
Efeito Espectador/efeitos da radiação , Citocinas/metabolismo , Dano ao DNA/efeitos da radiação , DNA/efeitos da radiação , Lesões por Radiação , Animais , Apoptose/efeitos da radiação , Arabidopsis/efeitos da radiação , Cricetinae , Citocinas/biossíntese , Instabilidade Genômica/efeitos da radiação , Humanos , Camundongos , Oncorhynchus mykiss , Lesões por Radiação/genética , Lesões por Radiação/metabolismo , Lesões por Radiação/patologia , Radiobiologia/tendências , Ratos , Transdução de Sinais , Peixe-Zebra
16.
Med Hypotheses ; 80(1): 70-4, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23111201

RESUMO

Cancer development is an evolutionary process that has been highly conserved among centuries within organisms. Based on this, the interest in cancer research focuses on cells, organelles and genes that possess a genetic conservatism from yeasts to human. Towards this thought, mitochondria, the highly conserved and responsible for the cellular bioenergetic activity organelles, might play crucial role in carcinogenesis. Interestingly, tumors with low bioenergetic signature have worse prognosis and show a decreased expression of ATPase protein. Furthermore, according to the stem-cell theory of carcinogenesis, aggressive tumors are characterized by an increase number of malignant stem-like cell population and their resistance to chemotherapy has been found to be mitochondrially driven. The above considerations triggered us to hypothesize that mitochondrial bioenergetic processes in stem-like cancer cells plays a crucial role in the highly conserved process of carcinogenesis. Specifically, we support that mitochondrial and/or nuclear DNA alterations that control stem cells' ATP production drive stem cells to "immortalization" (Otto Warburg theory) that mediates cancer initiation and progression. Substantiation of our hypothesis requires evidence that: (1) alterations in mitochondria bioenergetic metabolites and enzymes encoded either from the mtDNA or the nuclear DNA are linked to human cancer and (2) mitochondrial functions are regulated by highly conserved genes involved in cancer-related cellular processes such as apoptosis, aging and autophagy. Experimental approach on how this hypothesis might be tested and promising strategies in cancer therapeutics are also discussed. In case the hypothesis of stem-cell bioenergetic malformations' related carcinogenesis proves to be correct, it would contribute to the development of new prognostic, diagnostic and even more effective therapeutic interventions against various types of cancer.


Assuntos
Trifosfato de Adenosina/metabolismo , Transformação Celular Neoplásica/metabolismo , Metabolismo Energético/fisiologia , Mitocôndrias/fisiologia , Neoplasias/fisiopatologia , Células-Tronco Neoplásicas/fisiologia , Apoptose/fisiologia , Autofagia/fisiologia , Humanos , Modelos Biológicos
17.
Toxicol Mech Methods ; 23(5): 303-7, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23215871

RESUMO

The study of carcinogenic potential of a variety of chemical agents such as food additives and drugs of abuse via the application of various in vitro methodologies constitutes the first step for the evaluation of their toxicogenomic profile. Considering the chromosomal theories of carcinogenesis, where it is stated that aneuploidy and chromosomal imbalance (instability) are among the main causes of carcinogenesis, chemicals capable to induce such changes in the cells could be considered as potential carcinogens. Chromosomal imbalance and aneuploidy directly affect the overall DNA content of the exposed cell as well as other cellular morpho- and densitometric features. These features can be measured by means of computerized DNA image analysis technologies and include DNA content (DNA Index), Proliferation Index, Ploidy Balance, Degree of Aneuploidy, Skewness and Kurtosis. Considering the enormous number of untested chemicals and drugs of abuse that follow non-genotoxic mechanisms of carcinogenesis, the establishment of a reliable technology for the estimation of chemically induced chromosomal imbalance is of particular importance in toxicogenomic studies. In the present article and based on our previously published work, we highlight the advantages of the applications of DNA image analysis technology in an easy-to-use experimental model for the evaluation of the potential risk of various chemicals. The use of this technology for the detection of chemically induced chromosomal instability will contribute to the development of safer regulatory directives concerning the use of chemicals in food and pharmaceutical industry, as well as in the clarification of mechanisms of action of drugs of abuse.


Assuntos
Instabilidade Cromossômica , DNA/genética , Toxicogenética , Animais , Humanos
18.
Acta Biol Hung ; 63(4): 483-9, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23134605

RESUMO

The toxicity, in terms of changes in the DNA content, of two food preservatives, sodium nitrate and sodium benzoate was studied on the protozoan Tetrahymena pyriformis using DNA image analysis technology. For this purpose, selected doses of both food additives were administered for 2 h to protozoa cultures and DNA image analysis of T. pyriformis nuclei was performed. The analysis was based on the measurement of the Mean Optical Density which represents the cellular DNA content. The results have shown that after exposure of the protozoan cultures to doses equivalent to ADI, a statistically significant increase in the macronuclear DNA content compared to the unexposed control samples was observed. The observed increase in the macronuclear DNA content is indicative of the stimulation of the mitotic process and the observed increase in MOD, accompanied by a stimulation of the protozoan proliferation activity is in consistence with this assumption. Since alterations at the DNA level such as DNA content and uncontrolled mitogenic stimulation have been linked with chemical carcinogenesis, the results of the present study add information on the toxicogenomic profile of the selected chemicals and may potentially lead to reconsideration of the excessive use of nitrates aiming to protect public health.


Assuntos
Replicação do DNA/efeitos dos fármacos , DNA de Protozoário/efeitos dos fármacos , Conservantes de Alimentos/toxicidade , Macronúcleo/efeitos dos fármacos , Nitratos/toxicidade , Benzoato de Sódio/toxicidade , Tetrahymena pyriformis/efeitos dos fármacos , DNA de Protozoário/biossíntese , Macronúcleo/metabolismo , Mitose/efeitos dos fármacos , Medição de Risco , Tetrahymena pyriformis/genética , Tetrahymena pyriformis/crescimento & desenvolvimento
19.
Mol Biol Rep ; 39(1): 251-7, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21556769

RESUMO

Glutaraldehyde (GA) is a high production volume chemical that is very reactive with a wide spectrum of medical, scientific and industrial applications. Concerning the genotoxic and carcinogenic effect of GA, controversial results have been reported, while in humans no studies with positive carcinogenic results for GA have been published. However, our previous study concerning the combined effects of exposure to both GA and ionising radiation (IR) in peripheral blood lymphocytes of healthy donors has shown that non-genotoxic doses of the chemical induces a statistically significant increase in chromosomal radiosensitivity. The lack of information concerning the radiosensitizing potential of GA on cancerous cells triggered us to test the radiosensitizing effect of GA on breast cancer cells (MCF7). For this purpose the G2-chromosomal radiosensitivity assay (G2-assay) was used. The assay involves G2-phase irradiation and quantitation of the chromosomal fragility in the subsequent metaphase. The experimental data show that 48 h exposure to GA, at doses that are not clastogenic to MCF7 breast cancer cells enhances G2-chromosomal radiosensitivity of this cell line. In an effort to evaluate whether the observed increase in GAs-induced G2-chromosomal radiosensitization is linked to GA-induced alterations in the cell cycle and feedback control mechanism, Mitotic Index analysis was performed. The results have shown that such a mechanism cannot be directly related to the observed GA-induced increase in G2-chromosomal radiosensitivity. Since increased G2-chromosomal radiosensitivity has been linked with cancer proneness, the radiosensitizing effect of GA at non-clastogenic doses highlights its potential carcinogenic profile.


Assuntos
Cromossomos Humanos/efeitos dos fármacos , Fase G2/efeitos dos fármacos , Glutaral/farmacologia , Radiossensibilizantes/farmacologia , Bromodesoxiuridina , Linhagem Celular Tumoral , Cromossomos Humanos/efeitos da radiação , Feminino , Fase G2/efeitos da radiação , Humanos , Cariotipagem , Índice Mitótico , Troca de Cromátide Irmã , Estatísticas não Paramétricas
20.
Radiother Oncol ; 101(1): 28-34, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22014898

RESUMO

BACKGROUND AND PURPOSE: An increased yield of chromatid breaks following G2-phase irradiation could be a marker of radiosensitivity-predisposing genes that respond to DNA damage. We have shown that the dynamic nature of chromatin-nucleoprotein complex, which is capable of rapid unfolding, disassembling, assembling and refolding, affects repair of radiation-induced DNA-lesions and causes chromatid breaks during G2-M transition in damaged DNA sites. Here, we investigate induction and repair kinetics of chromatid breaks, their potential role in radiosensitivity predisposition and a standardized G2-assay is proposed to assess individual radiosensitivity. MATERIALS AND METHODS: Lymphocytes from 125 blood donors with significant inter-individual radiosensitivity variation (healthy, cancer, AT-patients) are used to correlate G2-checkpoint efficiency with chromatid breakage and individual radiosensitivity. Experiments involve repair kinetics of chromatid breaks using colcemid-block and treatment with caffeine to abrogate G2-checkpoint, generate internal controls and standardize the G2-assay. RESULTS: Radiation-induced chromatid breaks during G2-M transition, following 4h repair, remained unchanged and a significant correlation between G2-chromosomal radiosensitivity and G2-checkpoint efficiency to prevent chromatid breakage was found. A standardized G2-assay is developed by introducing normalization to conditions reflecting lack of checkpoint and repair similar to those of AT-patients, generating a unique standard for individual radiosensitivity testing. CONCLUSIONS: The standardized G2-assay can minimize inter-laboratory and intra-experimental variations and may have straightforward application in clinical practice for individualization of radiotherapy protocols.


Assuntos
Pontos de Checagem da Fase G2 do Ciclo Celular , Linfócitos/efeitos da radiação , Neoplasias/genética , Neoplasias/radioterapia , Tolerância a Radiação/genética , Estudos de Casos e Controles , Aberrações Cromossômicas , Dano ao DNA/efeitos da radiação , Reparo do DNA/genética , Feminino , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Humanos , Masculino , Neoplasias/patologia , Valor Preditivo dos Testes , Doses de Radiação , Tolerância a Radiação/efeitos da radiação , Valores de Referência , Estudos de Amostragem
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