RESUMO
Methods for visualizing DNA damage at the microscopic level are based on treatment of cell nuclei with saline or alkaline solutions. These procedures for achieving chromatin dispersion produce halos that surround the nuclear remnants. We improved the fast halo assay for visualizing DNA breakage in cultured cells to create a simplified method for detection and quantitative evaluation of DNA breakage. Nucleated erythrocytes from chicken blood were selected as a model test system to analyze the production of nuclear halos after treatment with X-rays or H(2)O(2). After staining with ethidium bromide or Wright's methylene blue-eosin solution, nuclear halos were easily observed by fluorescence or bright-field microscopy, respectively, which permits rapid visualization of DNA breakage in damaged cells. By using image processing and analysis with the public domain ImageJ software, X-ray dose and H(2)O(2) concentration could be correlated well with the size of nuclear halos and the halo:nucleus ratio. Our results indicate that this simplified nuclear halo assay can be used as a rapid, reliable and inexpensive procedure to detect and quantify DNA breakage induced by ionizing radiation and chemical agents. A mechanistic model to explain the differences between the formation of saline or alkaline halos also is suggested.
Assuntos
Cromatina/efeitos dos fármacos , Cromatina/efeitos da radiação , Quebras de DNA , Dano ao DNA , Animais , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/efeitos da radiação , Galinhas , Fragmentação do DNA/efeitos dos fármacos , Fragmentação do DNA/efeitos da radiação , Eritroblastos/efeitos dos fármacos , Eritroblastos/efeitos da radiação , Peróxido de Hidrogênio/toxicidade , Técnicas In Vitro , Microscopia de Fluorescência , Modelos Biológicos , Coloração e RotulagemRESUMO
Due to its link with human pathologies, including cancer, the mechanism of Nucleotide Excision Repair (NER) has been extensively studied. Most of the pathway and players have been defined using in vitro reconstitution experiments. However, in vivo, the NER machinery must deal with the presence of organized chromatin, which in some regions, such as heterochromatin, is highly condensed but still susceptible to DNA damage. A series of events involving different chromatin-remodeling factors and histone-modifying enzymes target chromatin regions that contain DNA lesions. CPDs change the structure of the nucleosome, allowing access to factors that can recognize the lesion. Next, DDB1-DDB2 protein complexes, which mono-ubiquitinate histones H2A, H3, and H4, recognize nucleosomes containing DNA lesions. The ubiquitinated nucleosome facilitates the recruitment of ATP-dependent chromatin-remodeling factors and the XPC-HR23B-Centrin 2 complex to the target region. Different ATP-dependent chromatin-remodeling factors, such as SWI/SNF and INO80, have been identified as having roles in the UV irradiation response prior to the action of the NER machinery. Subsequently, remodeling of the nucleosome allows enzymatic reactions by histone-modifying factors that may acetylate, methylate or demethylate specific histone residues. Intriguingly, some of these histone modifications are dependent on p53. These histone modifications and the remodeling of the nucleosome allow the entrance of TFIIH, XPC and other NER factors that remove the damaged strand; then, gap-filling DNA synthesis and ligation reactions are carried out after excision of the oligonucleotide with the lesion. Finally, after DNA repair, the initial chromatin structure has to be reestablished. Therefore, factors that modulate chromatin dynamics contribute to the NER mechanism, and they are significant in the future design of treatments for human pathologies related to genome instability and the appearance of drug-resistant tumors.
Assuntos
Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Cromatina/genética , Cromatina/efeitos da radiação , Dano ao DNA , Reparo do DNA , Raios Ultravioleta , Cromatina/química , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/genética , Desoxirribodipirimidina Fotoliase/metabolismo , Genes p53 , Heterocromatina/química , Heterocromatina/genética , Heterocromatina/efeitos da radiação , Histonas/química , Histonas/genética , Histonas/efeitos da radiação , Humanos , Nucleossomos/genética , Nucleossomos/efeitos da radiação , Dímeros de Pirimidina/química , Dímeros de Pirimidina/metabolismoRESUMO
OBJECTIVES: The aim of the present study was to evaluate DNA damage (micronucleus) and cellular death (pyknosis, karyolysis and karyorrhexis) in exfoliated buccal mucosa cells from adults following cone beam CT exposure. METHODS: A total of 19 healthy adults (10 men and 9 women) submitted to cone beam CT were included. RESULTS: No significant statistically differences (P > 0.05) in micronucleus frequency were seen before and after cone beam CT exposure. In contrast, the tomography was able to increase other nuclear alterations closely related to cytotoxicity such as karyorrhexis, pyknosis and karyolysis (P < 0.05). CONCLUSION: In summary, these data indicate that cone beam CT may not be a factor that induces chromosomal damage, but it is able to promote cytotoxicity.
Assuntos
Tomografia Computadorizada de Feixe Cônico , Dano ao DNA , Mucosa Bucal/efeitos da radiação , Adulto , Morte Celular/efeitos da radiação , Núcleo Celular/efeitos da radiação , Cromatina/efeitos da radiação , Tomografia Computadorizada de Feixe Cônico/efeitos adversos , Feminino , Humanos , Masculino , Micronúcleos com Defeito Cromossômico/efeitos da radiação , Mucosa Bucal/citologiaRESUMO
A common side effect of radiotherapy used in the treatment of oral cancer is the occurrence of structural and physiological alterations of the salivary glands due to exposure to ionizing radiation, as demonstrated by conditions such as decreased salivary flow. The present study evaluated ultrastructural alterations in the parotid glands of rats receiving a fractionated dose (1,500-cGy) of radiation emitted by a Cesium-137 source and rats that were not subjected to ionizing radiation. After sacrifice, the parotid glands were removed and examined by transmission electron microscopy. Damage such as cytoplasmic vacuolization, dilatation of the endoplasmic reticulum and destruction of mitochondria, as well as damage to the cellular membrane of acinar cells, were observed. These findings lead to the conclusion that ionizing radiation promotes alterations in the glandular parenchyma, and that these alterations are directly related to the dose level of absorbed radiation. Certain phenomena that appear in the cytoplasm and nuclear material indicate that ionizing radiation causes acinar cell death (apoptosis).
Assuntos
Glândula Parótida/efeitos da radiação , Animais , Apoptose , Morte Celular/efeitos da radiação , Membrana Celular/efeitos da radiação , Núcleo Celular/efeitos da radiação , Radioisótopos de Césio , Cromatina/efeitos da radiação , Citoplasma/efeitos da radiação , Desmossomos/efeitos da radiação , Fracionamento da Dose de Radiação , Retículo Endoplasmático/efeitos da radiação , Masculino , Microscopia Eletrônica de Transmissão , Mitocôndrias/efeitos da radiação , Membranas Mitocondriais/efeitos da radiação , Glândula Parótida/ultraestrutura , Ratos , Ratos Wistar , Vacúolos/efeitos da radiaçãoRESUMO
A common side effect of radiotherapy used in the treatment of oral cancer is the occurrence of structural and physiological alterations of the salivary glands due to exposure to ionizing radiation, as demonstrated by conditions such as decreased salivary flow. The present study evaluated ultrastructural alterations in the parotid glands of rats receiving a fractionated dose (1,500-cGy) of radiation emitted by a Cesium-137 source and rats that were not subjected to ionizing radiation. After sacrifice, the parotid glands were removed and examined by transmission electron microscopy. Damage such as cytoplasmic vacuolization, dilatation of the endoplasmic reticulum and destruction of mitochondria, as well as damage to the cellular membrane of acinar cells, were observed. These findings lead to the conclusion that ionizing radiation promotes alterations in the glandular parenchyma, and that these alterations are directly related to the dose level of absorbed radiation. Certain phenomena that appear in the cytoplasm and nuclear material indicate that ionizing radiation causes acinar cell death (apoptosis).
Um efeito colateral comum da radioterapia usada no tratamento de câncer na cavidade oral é a ocorrência de alterações estruturais e fisiológicas sobre as glândulas salivares por exposição à radiação ionizante, como demonstrada em situações com decréscimo do fluxo salivar. O presente estudo teve por objetivo avaliar as alterações ultra-estruturais de glândulas parótidas de ratos que receberam uma dose fracionada (1500 - cGy) de radiação emitida por uma fonte de Césio 137 e ratos que não receberam a radiação ionizante. Após o sacrifício, as glândulas parótidas foram removidas e examinadas por microscopia eletrônica de transmissão. Lesões das organelas citoplasmáticas, como dilatação do retículo endoplasmático, destruição das mitocôndrias e formação das vacuolizações citoplasmáticas, além de lesão da membrana celular das células acinares foram observadas. Portanto, a radiação ionizante promove alterações no parênquima glandular, o que está diretamente relacionado com a dose de radiação absorvida. Determinados fenômenos que surgem no citoplasma e material nuclear são indicadores de que a radiação ionizante leva a célula acinar a morte programada (apoptose).
Assuntos
Animais , Masculino , Ratos , Glândula Parótida/efeitos da radiação , Apoptose , Radioisótopos de Césio , Morte Celular/efeitos da radiação , Membrana Celular/efeitos da radiação , Núcleo Celular/efeitos da radiação , Cromatina/efeitos da radiação , Citoplasma/efeitos da radiação , Fracionamento da Dose de Radiação , Desmossomos/efeitos da radiação , Retículo Endoplasmático/efeitos da radiação , Microscopia Eletrônica de Transmissão , Mitocôndrias/efeitos da radiação , Membranas Mitocondriais/efeitos da radiação , Glândula Parótida/ultraestrutura , Ratos Wistar , Vacúolos/efeitos da radiaçãoRESUMO
Interactions between tumor cells and their substratum influence cancer progression by modulating cell proliferation and survival. We now investigated whether signaling responses to UV irradiation differ on adhesion-permissive or restrictive substrates. The latter conditions diminished spreading and proliferation of neo 6.3/C8161 melanoma in which metastasis is suppressed by introduction of neo-tagged chromosome 6, but permitted proliferation of human metastatic C8161 melanoma. Apoptosis-associated PARP cleavage and DNA fragmentation induced by UV irradiation were diminished on the restrictive substrate in C8161 melanoma. Genotoxic responses to UV irradiation like persistent increases in the phosphorylation of histone H2AX, induction of the tumor suppressor p53 protein and greater binding of this protein to its DNA consensus sequence, were all decreased on the restrictive substrate. The latter also promoted a 2 fold increase of DNA condensation in chromatin and enhanced activation of the survival - and invasion-associated MMP-9 gelatinase B, preferentially in metastatic C81261 melanoma. Our data suggest that adaptation to restrictive substrates in metastatic C8161 melanoma decreases UV-induced apoptosis, partly through attenuation of DNA damage signaling responses and changes in genomic organization.
Assuntos
Núcleo Celular/metabolismo , Dano ao DNA , DNA/metabolismo , Histonas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Sequência de Bases , Adesão Celular/efeitos da radiação , Núcleo Celular/efeitos da radiação , Proliferação de Células/efeitos da radiação , Cromatina/metabolismo , Cromatina/efeitos da radiação , Sequência Consenso , Ciclina D1/metabolismo , Fragmentação do DNA/efeitos da radiação , Indução Enzimática/efeitos da radiação , Humanos , Metaloproteinase 9 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/metabolismo , Metástase Neoplásica , Fosforilação/efeitos da radiação , Poli(ADP-Ribose) Polimerases/metabolismo , Ligação Proteica/efeitos da radiação , Processamento de Proteína Pós-Traducional/efeitos da radiação , Especificidade por Substrato/efeitos da radiação , Células Tumorais Cultivadas , Raios UltravioletaRESUMO
Gamma radiation induces apoptosis in the proliferative zone (neuroblastic layer) of the developing rat retina. We asked whether sensitivity to apoptosis might be related to distinct phases of the cell cycle. Explants of newborn rat retina or newborn pups were gamma-irradiated and apoptosis was detected by chromatin condensation, DNA fragmentation in situ and DNA electrophoresis. After 6 hours, early appearing apoptotic bodies were located mainly towards the outer tier of the neuroblastic layer. In contrast, after 24 hours, late-appearing apoptotic cells were located towards the inner margin of the neuroblastic layer, a region associated with the S phase of the cell cycle. Labeling of a cohort of cells with the nucleotide analog bromo-deoxyuridine (BrdU) at the time of irradiation, showed that these cells die in the late wave of apoptosis. BrdU given 3 hours before fixation labeled a large number of late apoptotic cells, but no early apoptotic cells. After labeling of all cycling cells with BrdU, 40% of the early apoptotic profiles were unlabeled, and thus post-mitotic. The same schedules of cell death were identified after gamma irradiation in vivo. The results show that irradiation leads to two waves of apoptosis in distinct cell populations. An early wave comprises both post-mitotic cells and proliferating cells out of the S phase. The late wave comprises cells in S phase, which pass through this phase again to die. The antioxidant pyrrolidinedithiocarbamate prevented the early but not the late wave of apoptosis following irradiation, and blocked lipid peroxidation at 6 hours after the insult, suggesting that the two waves of apoptosis are indeed mediated by distinct mechanisms.
Assuntos
Apoptose/efeitos da radiação , Raios gama , Retina/efeitos da radiação , Animais , Animais Recém-Nascidos , Ciclo Celular/fisiologia , Ciclo Celular/efeitos da radiação , Cromatina/fisiologia , Cromatina/efeitos da radiação , Cromatina/ultraestrutura , Fragmentação do DNA , Cinética , Técnicas de Cultura de Órgãos , Ratos , Retina/citologia , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/efeitos da radiaçãoRESUMO
Radiaçöes ionizantes afetam a estrutura das moléculas devido à destruiçäo das suas ligaçöes químicas. Estas alteraçöes químicas poderäo levar a uma mudança nas propriedades biológicas das mesmas, como tem sido demosntrado na literatura. A Crotamina foi obtida a partir de um "pool" do veneno de Crotalus durissus terrificus através de cromatografia de exclusäo molecular, sendo posteriormente irradiada em soluçäo numa concentraçäo de 2 mg/ml de NaCl 0,85% com radiaçäo gama produzida por uma fonte de 60Co. Foram adotadas as doses de 100 Gy, 250 Gy, 500 Gy, 1000 Gy e 2000 Gy (taxa de dose = 1,19. 10**3 Gy/h). Realizou-se os seguintes ensaios: presença de grupos SH livres, determinaçäo do número de bandas na SDS-PAGE (sugerindo a formaçäo de agregados protéicos) que foi proporcional ao aumento das doses. Pela imunodifusäo näo houve perda da atividade imunoquímica quando testadas contra o antisoro produzido pelo Instituto Butantan