Desenvolvimento e validação de técnica quantitativa de análise de imagem para avaliação do teste do cometa corado pela prata

Brianezi, Gabrielli; Camargo, João Lauro Viana de; Miot, Hélio Amante

doi:10.1590/s1676-24442009000400010

Cited by 17 publications

(13 citation statements)

References 33 publications

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“…After upper agarose solidification, the slides were transferred to a lysis solution (2.5 M NaCl; 10 mM Tris; 100 mM EDTA; 10% DMSO; 1% lauryl sarcosinate; 1% triton X-100; pH 10) at 4°C for 12 h and kept on electrophoresis alkaline buffer (0.3 M NaOH; 1 mM EDTA; pH 13) for 20 minutes before running electrophoresis at 25 V/300 mA for 20 minutes. After electrophoresis, DNA was silver stained according to [ 32 , 33 ]. A total of 100 cells were analyzed per slide and visually classified into four categories according to the degree of DNA damage, where 0 corresponds to no damage and 4 to a highly damaged DNA.…”

Section: Methodsmentioning

confidence: 99%

MiR-34a is up-regulated in response to low dose, low energy X-ray induced DNA damage in breast cells

et al. 2013

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BackgroundMicroRNAs are non-coding RNAs involved in the regulation of gene expression including DNA damage responses. Low doses of low energy X-ray radiation, similar to those used in mammographic exams, has been described to be genotoxic. In the present work we investigated the expression of miR-34a; a well described p53-regulated miRNA implicated in cell responses to X-ray irradiation at low doses.MethodsNon-cancerous breast cell line MCF-10A and cancerous T-47D and MCF-7 cell lines were submitted to a low-energy X-ray irradiation (ranging from 28–30 Kv) using a dose of 5 Gy. The expression level of miR-34a, let-7a and miR-21 was assessed by qRT-PCR at 4 and 24 hours post-irradiation. DNA damage was then measured by comet assay and micronuclei estimation in MCF-10A and MCF-7 cell lines, where an increase of miR-34a levels could be observed after irradiation. The rate of apoptotic cells was estimated by nuclear staining and fluorescence microscopy. These experiments were also performed at low doses (3; 12 and 48 mGy) in MCF-10A and MCF-7 cell lines.ResultsWe have observed an increase in miR-34a expression 4 hours post-irradiation at 5 Gy in MCF-10A and MCF-7 cell lines while its level did not change in T-47D, a breast cancer cell line bearing non-functional p53. At low doses, miR-34a was up-regulated in non-tumoral MCF-10A to a higher extent as compared to MCF-7. MiR-34a levels decreased 24 hours post-irradiation. We have also observed DNA damage and apoptosis at low-energy X-ray irradiation at low doses and the high dose in MCF-10A and MCF-7 4 and 24 hours post-irradiation relative to the mock control.ConclusionLow energy X-ray is able to promote DNA strand breaks and miR-34a might be involved in cell responses to low energy X-ray DNA damage. MiR-34a expression correlates with X-ray dose, time after irradiation and cell type. The present study reinforces the need of investigating consequences of low dose X-ray irradiation of breast cells.

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Section: Methodsmentioning

confidence: 99%

MiR-34a is up-regulated in response to low dose, low energy X-ray induced DNA damage in breast cells

et al. 2013

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“…The comet assay, also known as single-cell gel electrophoresis, is very useful and widely employed in the evaluation of DNA damage and repair in individual cells. 58 The primary goal of this test is to evaluate the damage that is caused by oxidative stress, through the interaction of DNA with reactive oxygen species. 59 Compound 1 exhibited a significant difference compared to the negative control after 2 h of exposure at concentrations 2 and 3; after 24 h, only the highest concentration produced a difference in both parameters evaluated in the comet assay.…”

Section: Cell Viability and Comet Assaymentioning

confidence: 99%

Synthesis, DNA Interaction and Genotoxic Evaluation of a Photoactive Benzothiadiazole with Potential Application in Photovoltaic Paint

Damiani¹,

Rohr²,

Westrup³

et al. 2017

J. Braz. Chem. Soc.

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A new fluorophore containing 2,1,3-benzothiadiazole unit with alkylated tetrazole terminal groups as electron donors connected through a π-conjugated system of the type (D-π-A-π-D) was synthesized using the Sonogashira coupling reaction with good yields. The genotoxicity of the two intermediate compounds and the final product was evaluated in vitro in consideration of the potential application of the new compound in the chemical production of photovoltaic paints. The new fluorophore did not show significant biological effects in the cell viability test and the comet assay when compared with the intermediate compounds that were obtained synthetically despite the ability to interact with deoxyribonucleic acid (DNA) as demonstrated in circular dichroism assays. Thus, the compound does not show toxic or genotoxic activity, and therefore, it can be applied as a pigment in photovoltaic paint.

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“…Após definido as características desejáveis e a aquisição de imagens por meio do teste do cometa, procedeu-se o pré-processamento, que foi executado de forma a eliminar ruídos [27], devido a necessidade de uma delimitação precisa dos cometas [18] [16] [14] [32]. Para este fim, foram usados os filtros morfológicos (erosão e dilatação), apesar de serem usualmente utilizados em imagens binárias [18] [26], como elemento de segmentação.…”

Section: Extrair O Diâmetro Da Cabeça Do Cometa Do Conjunto Total;unclassified

“…Após o pré-processamento,é utilizada uma binarização dinâmica [14] [32]. Esta consiste em utilizar intensidades de pixels, calculados através da magnitude do histograma.…”

Section: Extrair O Diâmetro Da Cabeça Do Cometa Do Conjunto Total;unclassified

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Smart Comet: Uma Abordagem Automatizada Para Detecção E Classificação De Dano Celular Dispondo Do Ensaio Cometa

Silva¹,

Silva²,

Carvalho³

et al. 2016

L&NLM

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A detecção e classificação de danos nas imagens do ensaio cometaé um importante indicativo de alguns problemas de saúde, como por exemplo o câncer. No entanto, normalmente, a detecção e classificação de danos por meio deste tipo de ensaio são realizadas manualmente, decorrendo em erros e lentidão. Embora existam algumas abordagens automatizadas para esse problema, tais abordagens detectam as células e classificam seus danos com níveis de eficiência não tão satisfatório. Neste artigo,é proposto o SMART COMET, uma solução de detecção baseada em filtros morfológicos, detecção de borda e classificação usando um sistema especialista. Experimentos mostram resultados mais eficientes que outras abordagens presentes na literatura em imagens com borda de objetiva nos protocolos do ensaio cometa de nitrato de prata e GelRed. Adicionalmente, dispondo de imagens sem borda da objetiva utilizando os protocolos de nitrato de prata e GelRed, o SMART COMET mostrou-se, também mais eficiente que outras abordagens propostas na literatura.

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Desenvolvimento e validação de técnica quantitativa de análise de imagem para avaliação do teste do cometa corado pela prata

Cited by 17 publications

References 33 publications

MiR-34a is up-regulated in response to low dose, low energy X-ray induced DNA damage in breast cells

MiR-34a is up-regulated in response to low dose, low energy X-ray induced DNA damage in breast cells

Synthesis, DNA Interaction and Genotoxic Evaluation of a Photoactive Benzothiadiazole with Potential Application in Photovoltaic Paint

Smart Comet: Uma Abordagem Automatizada Para Detecção E Classificação De Dano Celular Dispondo Do Ensaio Cometa

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