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1.
Toxicol Lett ; 327: 58-68, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32247831

RESUMO

The in vivo comet assay is an established genotoxicity test, with an OECD test guideline, but in its standard form it measures only DNA strand breaks. Including in the assay an additional step, in which the DNA is incubated with a lesion-specific enzyme, can provide important information about the nature of the DNA damage. Formamidopyrimidine DNA glycosylase, 8-oxoguanine DNA glycosylase or endonuclease III are commonly used in the in vitro genotoxicity test and in human biomonitoring to detect oxidised bases, but in vivo applications are rarer. A systematic literature search has identified a total of 60 papers that report such in vivo experiments, testing a variety of agents. In many cases, strand breaks were not seen, but significant levels of enzyme-sensitive sites were induced - indicating a mechanism of action involving oxidative stress. Compounds such as methyl methanesulfonate (MMS) or ethyl methanesulfonate (EMS) could be used as positive controls in both the standard and the enzyme-modified in vivo comet assays.


Assuntos
Monitoramento Biológico/métodos , Ensaio Cometa/métodos , Animais , DNA , Dano ao DNA , Humanos , Testes de Mutagenicidade/métodos , Estresse Oxidativo
2.
J Vis Exp ; (157)2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32281969

RESUMO

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other environmental stressors. Use of the comet assay in regulatory testing for genotoxic potential in rodents has been driven by adoption of an Organisation for Economic Co-operation and Development (OECD) test guideline in 2014. Comet assay slides are typically prepared from fresh tissue at the time of necropsy; however, freezing tissue samples can avoid logistical challenges associated with simultaneous preparation of slides from multiple organs per animal and from many animals per study. Freezing also enables shipping samples from the exposure facility to a different laboratory for analysis, and storage of frozen tissue facilitates deferring a decision to generate DNA damage data for a given organ. The alkaline comet assay is useful for detecting exposure-related DNA double- and single-strand breaks, alkali-labile lesions, and strand breaks associated with incomplete DNA excision repair. However, DNA damage can also result from mechanical shearing or improper sample processing procedures, confounding the results of the assay. Reproducibility in collection and processing of tissue samples during necropsies may be difficult to control due to fluctuating laboratory personnel with varying levels of experience in harvesting tissues for the comet assay. Enhancing consistency through refresher training or deployment of mobile units staffed with experienced laboratory personnel is costly and may not always be feasible. To optimize consistent generation of high quality samples for comet assay analysis, a method for homogenizing flash frozen cubes of tissue using a customized tissue mincing device was evaluated. Samples prepared for the comet assay by this method compared favorably in quality to fresh and frozen tissue samples prepared by mincing during necropsy. Moreover, low baseline DNA damage was measured in cells from frozen cubes of tissue following prolonged storage.


Assuntos
Ensaio Cometa/métodos , Dano ao DNA/genética , Humanos , Reprodutibilidade dos Testes
3.
Mutat Res ; 783: 108288, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32192646

RESUMO

The comet assay is a well-accepted biomonitoring tool to examine the effect of dietary, lifestyle, environmental and occupational exposure on levels of DNA damage in human cells. With such a wide range of determinants for DNA damage levels, it becomes challenging to deal with confounding and certain factors are inter-related (e.g. poor nutritional intake may correlate with smoking status). This review describes the effect of intrinsic (i.e. sex, age, tobacco smoking, occupational exposure and obesity) and extrinsic (season, environmental exposures, diet, physical activity and alcohol consumption) factors on the level of DNA damage measured by the standard or enzyme-modified comet assay. Although each factor influences at least one comet assay endpoint, the collective evidence does not indicate single factors have a large impact. Thus, controlling for confounding may be necessary in a biomonitoring study, but none of the factors is strong enough to be regarded a priori as a confounder. Controlling for confounding in the comet assay requires a case-by-case approach. Inter-laboratory variation in levels of DNA damage and to some extent also reproducibility in biomonitoring studies are issues that have haunted the users of the comet assay for years. Procedures to collect specimens, and their storage, are not standardized. Likewise, statistical issues related to both sample-size calculation (before sampling of specimens) and statistical analysis of the results vary between studies. This review gives guidance to statistical analysis of the typically complex exposure, co-variate, and effect relationships in human biomonitoring studies.


Assuntos
Monitoramento Biológico/métodos , Ensaio Cometa/métodos , Dano ao DNA , Estresse Oxidativo , Adulto , Fatores Etários , DNA-Formamidopirimidina Glicosilase , Exposição Ambiental , Proteínas de Escherichia coli , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Obesidade/genética , Obesidade/metabolismo , Fatores de Risco , Estações do Ano , Fatores Sexuais , Fumar Tabaco
5.
Chemosphere ; 247: 125748, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31954338

RESUMO

INTRODUCTION: Radon-induced biological effects have been studied mainly through epidemiological investigations, and well-controlled in vitro and in vivo experiments. To provide data explaining radon exposure-induced harmful effects in natural environment, exposure assessment under these conditions is needed. The objective of the study was to examine the level of genetic damage assessed with biomarkers of DNA single- and double-strand breaks (SSBs and DSBs) in peripheral blood mononuclear cells obtained from individuals continuously exposed to Rn in homes. Naturally elevated Rn concentrations in homes can be found in the South of Poland, in Kowary city. METHODS: Measurements of expression of phosphorylated histone γH2AX was used as a marker of DNA double strand breaks. To detect DNA single and double-strand breaks and alkali labile sites, the alkaline comet assay was used. Oxidative damage of DNA was evaluated by formamidopyrimidyne (FPG)-modified comet assay. The blood was collected from 94 volunteers living in Kowary. Subjects were grouped according to their status of living in radon concentration ≥100 Bq/m3 (n = 67), and <100 Bq/m3 (n = 27). RESULTS: The statistically significant differences in levels of DNA damage in peripheral lymphocytes assessed with comet assay were found to be associated with levels of radon exposure in indoor air (p = 0.034). DNA damage in the comet assay was significantly correlated with DNA damage assessed with γH2AX staining. CONCLUSIONS: Results of the present study indicate the suitability of alkaline comet assay for the detection of DNA damage in peripheral blood lymphocytes of people environmentally exposed to radon.


Assuntos
Poluentes Radioativos do Ar/toxicidade , Ensaio Cometa/normas , Exposição Ambiental , Linfócitos/efeitos da radiação , Radônio/análise , Ensaio Cometa/métodos , Dano ao DNA/efeitos da radiação , Histonas/análise , Histonas/genética , Humanos , Polônia , Radônio/farmacologia
6.
Bull Environ Contam Toxicol ; 104(2): 215-221, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31932906

RESUMO

Silicon nanoparticles gained a great interest due to its use in biomedical research. It is considered as safe and has been used in nanomedicine. But literature still states its toxicity depending upon the size and dose of silicon nanoparticles. So, current study was aimed to evaluate the cytotoxicity and genotoxicity of silicon dioxide nanoparticles (SiO2NPs) by Allium anaphase-telophase and Comet tests. Characterization of SiO2NPs showed the particle size as 16.12 ± 3.07 nm. The mean diameter of SiO2NPs was having range of 404.66 ± 93.39 nm in solution. Highest total anomalies (18.80 ± 0.45) were observed at 100 µg/mL, whereas least (11.2 ± 0.84) were observed by the 12.5 µg/mL concentration. There was concentration-response association in increased CAs and DNA damage. The highest concentration (100 µg/mL) of SiO2NPs induced the significant DNA damage (149.67 ± 1.15), whereas the least was observed by the negative control (2.67 ± 0.58). The current study revealed the cytotoxic and genotoxic effects of SiO2NPs on the root meristem cells of A. cepa.


Assuntos
Nanopartículas/toxicidade , Cebolas/efeitos dos fármacos , Dióxido de Silício/toxicidade , Allium , Ensaio Cometa/métodos , Dano ao DNA , Meristema/citologia , Meristema/efeitos dos fármacos , Meristema/genética , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Testes de Mutagenicidade/métodos , Cebolas/citologia , Cebolas/genética , Tamanho da Partícula , Raízes de Plantas/citologia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética
7.
Nucleic Acids Res ; 48(3): e13, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-31822921

RESUMO

Genotoxicity testing is critical for predicting adverse effects of pharmaceutical, industrial, and environmental chemicals. The alkaline comet assay is an established method for detecting DNA strand breaks, however, the assay does not detect potentially carcinogenic bulky adducts that can arise when metabolic enzymes convert pro-carcinogens into a highly DNA reactive products. To overcome this, we use DNA synthesis inhibitors (hydroxyurea and 1-ß-d-arabinofuranosyl cytosine) to trap single strand breaks that are formed during nucleotide excision repair, which primarily removes bulky lesions. In this way, comet-undetectable bulky lesions are converted into comet-detectable single strand breaks. Moreover, we use HepaRG™ cells to recapitulate in vivo metabolic capacity, and leverage the CometChip platform (a higher throughput more sensitive comet assay) to create the 'HepaCometChip', enabling the detection of bulky genotoxic lesions that are missed by current genotoxicity screens. The HepaCometChip thus provides a broadly effective approach for detection of bulky DNA adducts.


Assuntos
Ensaio Cometa/métodos , Adutos de DNA/análise , Carcinogênese , Linhagem Celular , Quebras de DNA de Cadeia Simples , Reparo do DNA , Humanos , Análise em Microsséries/métodos , Sensibilidade e Especificidade
8.
Toxicol Lett ; 319: 58-65, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31730884

RESUMO

This study proposes the application of the comet assay for the evaluation of DNA damage from frozen human whole blood samples that could be readily used in human biomonitoring and epidemiological studies. It was done on simply frozen whole blood samples collected from male volunteers (N = 60) aliquoted in small volumes and stored at -80 °C without the addition of cryopreservatives for a period of 5 years. To test the applicability of the alkaline comet assay for the evaluation of DNA damage in frozen whole blood, samples were quickly thawed at 37 °C and immediately embedded in an agarose matrix followed by an alkaline comet assay procedure. We concluded that the whole blood freezing and prolonged storage do not severely affect comet assay values, although background values were higher compared to our historical control data from the fresh whole blood. Even the influence of the variables tested, such as age, body mass index, smoking habit and alcohol consumption were in agreement with our previous data using fresh blood. The obtained results suggest that the comet assay could be applied to frozen blood samples, if properly stored, even for decades, which would certainly facilitate large-scale human biomonitoring and long-term epidemiological studies.


Assuntos
Preservação de Sangue/efeitos adversos , Sangue , Ensaio Cometa/métodos , Dano ao DNA , Adolescente , Adulto , Fatores Etários , Idoso , Índice de Massa Corporal , Criopreservação , Congelamento , Humanos , Estilo de Vida , Masculino , Pessoa de Meia-Idade , Monitorização Fisiológica , Adulto Jovem
9.
Biomed Pharmacother ; 121: 109600, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31707352

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: UVB is a high energy source that causes the major risk factor for sunburn and skin tumor. However, photochemical interactions lead to beneficial effects such as synthesis of vitamin D and corticosteroids. Therefore, a reasonable therapeutic regime is advocated to reduce UVB injuries but makes use of synthesizing sunlight metabolite. Many natural compounds improving plant cells resistant to oxidative stress by the harnessing of solar energy may be also used to protect human cells. Although many nature plants have shown photoprotective effects on skin, the mechanisms underlying of the effects are still ambiguous. AIM OF THE STUDY: This study evaluates the protective effects of cultivated Cordyceps against UVB-induced damage in human keratinocytes and identifies the photoprotective mechanisms using a transcriptomic network approach. MATERIALS AND METHODS: Cordyceps extract compositions were investigated by HPLC analysis. Cell survival, reactive oxygen species (ROS) generation, H2O2 content, aquaporin 3 (AQP3) level and DNA damage were determined upon UVB irradiation in the presence of Cordyceps extract. In addition, next-generation sequencing was used to profile transcriptomic alteration of 20 mJ/cm2 UVB and non-UV. Finally, a network pharmacology method was applied to study Cordyceps extract-related natural compounds and their UVB-induced differentially change targets using the Cytoscape 3.7.1 software. RESULTS: Adenosine and mannitol were the major contents in Cordyceps extract. Cordyceps caused a significant diminished in intracellular UVB-induced oxidative stress, including ROS production and intracellular H2O2 content. Besides, AQP3 which mediated intracellular signal transmission and transported H2O2 into cells was significantly increased in the presence of Cordyceps extract against UVB irradiation. In addition, DNA repair effect of Cordyceps extract after UV irradiation was proven to be effective by comet assay. Moreover, KEGG analysis showed steroid hormone biosynthesis, ovarian steroidogenesis, fat digestion and absorption were enriched in top 3 between 20 mJ/cm2 UVB and non-UV. Gene ontology (Go) analysis showed that steroid metabolic process, sterol metabolic process, and cholesterol metabolic process were enriched in top3 biology process. By using network analysis, 125 potential bioactive ingredients in Cordyceps and 201 targets were identified. Finally, signal pathway analyses suggested that the protective effects of Cordyceps compounds against low dose UVB­induced changes might target PPAR signaling pathway, cholesterol metabolism, and ovarian steroidogenesis. CONCLUSION: Cordyceps extract may be an ideal product for external use of skin which could not only avoid UVB-induced adverse effects, but also could application of metabolite products by UVB such us steroid hormone and vitamin D3.


Assuntos
Cordyceps , Queratinócitos/efeitos dos fármacos , Queratinócitos/efeitos da radiação , Protetores contra Radiação/farmacologia , Raios Ultravioleta/efeitos adversos , Administração Tópica , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Ensaio Cometa/métodos , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Relação Dose-Resposta a Droga , Previsões , Humanos , Queratinócitos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/efeitos da radiação , Protetores contra Radiação/isolamento & purificação , Reprodutibilidade dos Testes
10.
Int J Mol Sci ; 20(23)2019 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-31810189

RESUMO

Single cell gel electrophoresis, also known as the comet assay, has become a widespread DNA damage assessment tool due to its sensitivity, adaptability, low cost, ease of use, and reliability. Despite these benefits, this assay has shortcomings, such as long assay running time, the manipulation of multiple slides, individually, through numerous process steps, the challenge of working in a darkened environment, and reportedly considerable inter- and intra-laboratory variation. All researchers typically perform the comet assay based upon a common core approach; however, it appears that some steps in this core have little proven basis, and may exist, partly, out of convenience, or dogma. The aim of this study was to critically re-evaluate key steps in the comet assay, using our laboratory's protocol as a model, firstly to understand the scientific basis for why certain steps in the protocol are performed in a particular manner, and secondly to simplify the assay, and decrease the cost and run time. Here, the shelf life of the lysis and neutralization buffers, the effect of temperature and incubation period during the lysis step, the necessity for drying the slides between the electrophoresis and staining step, and the need to perform the sample workup and electrophoresis steps under subdued light were all evaluated.


Assuntos
Ensaio Cometa/métodos , Monitoramento Ambiental/métodos , Análise de Célula Única/métodos , Dano ao DNA/genética , Humanos , Laboratórios/normas , Temperatura
11.
An Acad Bras Cienc ; 91(3): e20180655, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31576914

RESUMO

This study evaluated 24 patients with lung cancer (CA) and 23 individuals with no smoking history or cancer in the family and without respiratory disease in childhood (CO). Peripheral blood lymphocytes was used to perform alkaline comet assay and to assess DNA damage as well as to evaluate methyl methane sulfonate (MMS) DNA repair after one hour and three hours at 37 ºC. The percentage of residual damage (RD) after three hours of MMS treatment, for each patient was assessed. The majority of patients were in the CA group, male patients, former smokers, with a history of smoking for 15 years and without associated comorbidities. Alkaline and residual damages were higher in the CA group when compared to controls (alkaline damage P = 0.015 and RD P = 0.05). After one hour of MMS treatment the DNA damage of the CA increased indicating failure to repair it, compared to the controls, and after three hours DNA repair was observed in both groups. Patients with lung cancer are mostly men, former smokers and with more than 15 years of tobacco consumption, undergoing chemotherapy, have high rates of DNA damage and deficiency in their ability to repair against induced damage when compared to controls.


Assuntos
Dano ao DNA , Reparo do DNA , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Idoso , Antineoplásicos Alquilantes/farmacologia , Estudos de Casos e Controles , Ensaio Cometa/métodos , Estudos Transversais , Feminino , Humanos , Linfócitos/efeitos dos fármacos , Masculino , Metanossulfonato de Metila/farmacologia , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Fumar/efeitos adversos , Fatores de Tempo
12.
Methods Mol Biol ; 2031: 237-257, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31473963

RESUMO

Anthropogenic activities, indiscriminate and rapid industrialization as well as pursuance of a better life has led to an increase in the concentration of chemicals, like pesticides, automobile exhausts, and new chemical entities, in the environment, which have an adverse effect on all living organisms including humans. Sensitive and robust test systems are thus required for accurate hazard identification and risk assessment. The Comet assay has been used widely as a simple, rapid, and sensitive tool for assessment of DNA damage in single cell from both in vitro and in vivo sources as well as in humans. The advantages of the in vivo Comet assay are its ability to detect DNA damage in any tissues, despite having non-proliferating cells, and its sensitivity to detect genotoxicity. The recommendations from the international workshops held for the Comet assay have resulted in establishment of guidelines, and the OECD has adopted a guideline for the in vivo Comet assay as a test for assessing DNA damage in animals. The in vitro Comet assay conducted in cultured cells can be used for screening large number of compounds and at very low concentrations. The in vitro assay has also been automated to provide a high throughput screening method for new chemical entities, as well as in environmental samples. This chapter details the in vitro Comet assay using the 96-well plate and in vivo Comet assay in multiple organs of the mouse.


Assuntos
Ensaio Cometa/métodos , Dano ao DNA/efeitos dos fármacos , Animais , Células CHO , Técnicas de Cultura de Células/métodos , Cricetulus , Camundongos , Mutagênicos/toxicidade
13.
Methods Mol Biol ; 2031: 259-274, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31473964

RESUMO

Human biomonitoring studies aim to identify potential exposures to environmental, occupational, or lifestyle toxicants in human populations and are commonly used by public health decision makers to predict disease risk. The Comet assay measures changes in genomic stability and is one of the most reliable biomarkers to indicate early biological effects and therefore accepted by various governmental regulatory agencies. The appeal of the Comet assay lies in its relative simplicity, rapidity, sensitivity, and economic efficiency. Furthermore, the assay is known for its broad versatility, as it can be applied to virtually any human cell and easily adapted in order to detect particular biomarkers of interest, such as DNA repair capacity or single and double-strand breaks. In a standard experiment, isolated single cells are first embedded in agarose, and then lysed in high-salt solutions in order to remove all cellular contents except the DNA attached to a nuclear scaffold. Subsequent electrophoresis results in accumulation of undamaged DNA sequences at the proximity of the nuclear scaffold, while damaged sequences migrate toward the anode. When visualized with fluorochromes, these migrated DNA fragments resemble a Comet tail and can be quantified for their intensity and shape according to internationally drafted guidelines.


Assuntos
Monitoramento Biológico/métodos , Ensaio Cometa/métodos , Dano ao DNA/efeitos dos fármacos , Monitoramento Biológico/instrumentação , Células Cultivadas , Ensaio Cometa/instrumentação , Desenho de Equipamento , Humanos , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Masculino , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Mutagênicos/toxicidade , Análise de Célula Única/instrumentação , Análise de Célula Única/métodos , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismo , Coloração e Rotulagem/instrumentação , Coloração e Rotulagem/métodos
14.
Methods Mol Biol ; 2031: 275-286, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31473965

RESUMO

Comet assay is a quick and versatile technique for assessing DNA damage in individual cells. It allows for the detection of DNA single- and double-strand breaks, as well as the presence of alkali labile sites and cross-links. Here we describe protocols for the single-cell gel electrophoresis (Comet assay) in its alkaline (pH > 13), mild alkaline (pH = 12.1) and neutral (pH = 8) versions when applied in marine animals.


Assuntos
Bivalves/genética , Ensaio Cometa/métodos , Análise de Célula Única/métodos , Animais , Bivalves/citologia , Bivalves/efeitos dos fármacos , Células Cultivadas , Criopreservação/métodos , Dano ao DNA/efeitos dos fármacos , Hemócitos/efeitos dos fármacos , Hemócitos/metabolismo , Concentração de Íons de Hidrogênio , Mutagênicos/toxicidade
15.
Methods Mol Biol ; 2031: 337-348, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31473970

RESUMO

In spite of its pioneer use in detecting mutational processes, Drosophila still plays an important role in those studies aiming to detect and quantify the induction of DNA damage. Here we describe two assays, one detecting primary damage (the Comet assay) and the other detecting somatic mutation and recombination effects (wing-spot test). It is important to emphasize that somatic recombination is a key event in cancer development and no assays exist at present to detect and quantify somatic recombination processes, other than the spot tests developed in Drosophila.


Assuntos
Drosophila melanogaster/genética , Testes de Mutagenicidade/métodos , Animais , Ensaio Cometa/métodos , Dano ao DNA/efeitos dos fármacos , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/ultraestrutura , Hemócitos/efeitos dos fármacos , Hemócitos/metabolismo , Mutagênicos/toxicidade , Recombinação Genética/efeitos dos fármacos , Asas de Animais/efeitos dos fármacos , Asas de Animais/metabolismo , Asas de Animais/ultraestrutura
16.
Mutat Res ; 842: 146-157, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31471003

RESUMO

The genotoxicity of nanoparticles is a major concern for nano-safety appraisal in the bryophytes as they are the primary colonizers of bare land, indicators of atmospheric pollution and excellent accumulators of trace metals. The present study for the first time evince the in planta genotoxicity of MnONP in Physcomitrella patens a model plant system utilized for evolutionary developmental genetics. The induction of DNA strand breaks was confirmed by comet assay at all tested concentrations corroborated with the enhanced generation of ROS, increase in Mn dissolution, uptake and internalization. Genotoxicity is often coupled with epigenetic alterations. In the present study, global DNA methylation pattern at the level of single cells was studied by the methylation sensitive comet assay using the isochizomeric restriction endonucleases HpaII (digests unmethylated and hemimethylated DNA) and MspI (digests methylated DNA at 5'-CmCGG-3'). MnONP incited DNA hypomethylation in P. patens gametophores treated with the highest concentration of MnONP (20 µg/mL). The DNA hypomethylation incurred upon MnONP exposure was comparable with that of the DNA methylation blocker 5-azacytidine. This can be ascribed to its clastogenic potential mediated by the formation of H2O2, OH and O2¯. There are no reports on the epigenotoxicity of nanomaterials in plants utilizing the detection of DNA damage and DNA methylation. This can open up new avenues of research on the assessment of the epigenotoxic impact of environmentally relevant nanoparticles using bryophytes as model indicator plant system.


Assuntos
Bryopsida/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Mutagênicos/toxicidade , Nanopartículas/toxicidade , Óxidos/toxicidade , Ensaio Cometa/métodos , Peróxido de Hidrogênio/toxicidade , Compostos de Manganês
17.
Environ Mol Mutagen ; 60(9): 837-844, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31490579

RESUMO

Caffeic acid is found in variety of fruits and vegetables. It is considered as possible human carcinogen (Group 2B). It is negative in Ames and mouse micronucleus (MN), but positive in mouse lymphoma and chromosomal aberration assays. The objective of this study was to evaluate the in vivo genotoxicity of caffeic acid using three different endpoints: in vivo MN, Pig-a, and comet assay. Two sets of six rats per group were administered vehicle (0.5% hydroxypropyl methylcellulose), 500, 1,000, or 2,000 mg/kg/day of caffeic acid for three consecutive days via oral gavage. One set of animals was used for the Pig-a and MN assay and the other set was used for the comet assay. N-Ethyl N-Nitrosourea was used as positive control for the Pig-a and MN assay, and ethyl methanesulfonate for the comet assay. From one set of animals, peripheral blood was collected on Days -1, 14, and 30 for the Pig-a assay and on Day 4 for the MN assay. The other set of animals was euthanized 3 hr after the last dose; liver and blood were collected for the comet assay. A statistically significant increase in the MN frequency was observed at 2,000 mg/kg/day. No increase in the red blood cells (RBCCD59- ) or reticulocytes (RETCD59- ) Pig-a mutant frequencies was observed on Days 14 or 30. No increase in DNA strand breaks was observed in the peripheral blood or liver in the comet assay. Environ. Mol. Mutagen. 2019. © 2019 Wiley Periodicals, Inc.


Assuntos
Ácidos Cafeicos/efeitos adversos , Animais , Antígenos CD59/metabolismo , Aberrações Cromossômicas/efeitos dos fármacos , Ensaio Cometa/métodos , Quebras de DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Eritrócitos/efeitos dos fármacos , Metanossulfonato de Etila/efeitos adversos , Etilnitrosoureia/efeitos adversos , Masculino , Testes para Micronúcleos/métodos , Testes de Mutagenicidade/métodos , Mutagênicos/efeitos adversos , Ratos , Ratos Sprague-Dawley , Reticulócitos/efeitos dos fármacos
18.
Mutat Res ; 845: 402995, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31561885

RESUMO

One of the problems of in vitro genotoxicity testing is the inadequate representation of drug metabolizing enzymes in indicator cells which are currently used. An alternative are human derived liver cell lines which retained the activities of enzymes that catalyze the activation and detoxification of genotoxins. Several cell lines were identified which were used in comet experiments. The most frequently employed line is HepG2, i.e. more than 400 individual compounds have been tested; furthermore, it was also used for the detection of combined effects in mixtures as drug metabolizing and antioxidant enzymes are represented in inducible form. One of the shortcomings of these cells are the strong inter-laboratory variation of the results. Recently it was postulated that HepaRG cells are an ideal model for human liver studies, but comet experiments were only partly successful and failed to detect genotoxins such as cadmium chloride, styrene and etoposide, as well as compounds that require activation via N-actetyltransferases (IQ, 2,4-DAT, 2-AAF). Furthermore, these cells are relatively insensitive towards ROS. Hep3B cells were used in a few studies but failed to detect representatives of important genotoxic carcinogens (AFB1, B(a)P, NDMA, IQ, PhiP), the line HCC1.1 was sensitive towards these chemicals but possesses an instable karyotype and a mutated p53. A more promising line is Huh6, but further validation of the usefulness for routine testing is needed. Recent developments which may lead to a better metabolic capacity of liver cells include improvement of the growth conditions (e.g. increase of serum levels, use of differentiated cells and of 3D-cultures), use of differentiated stem cells with hepatocyte like characteristics or of transformed proliferating hepatocytes.


Assuntos
Ensaio Cometa/métodos , Dano ao DNA , Hepatócitos/efeitos dos fármacos , Biotransformação , Linhagem Celular , Linhagem Celular Tumoral , Aberrações Cromossômicas , Resistência a Medicamentos , Feminino , Genes p53 , Células Hep G2 , Hepatócitos/química , Hepatócitos/enzimologia , Humanos , Neoplasias Hepáticas/patologia , Masculino , Mutagênicos/metabolismo , Mutagênicos/toxicidade , Ploidias , Análise de Célula Única , Xenobióticos/metabolismo , Xenobióticos/toxicidade
19.
Mutat Res ; 845: 402994, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31561887

RESUMO

The comet assay is a commonly used method for in vitro and in vivo genotoxicity assessment. This versatile assay can be performed in a wide range of tissues and cell types. Although most of the studies use samples immediately processed after collection, frozen biological samples can also be used. The present study aimed to optimize a collection and freezing protocol to minimize the DNA damage associated with these procedures in human cell line samples for comet assay analysis. This study was conducted in glial A172 and lung alveolar epithelial A549 cells. Two cell detachment methods (mechanical vs enzymatic) and two cryoprotective media [FBS + 10% DMSO vs Cell Culture Media (CCM) + 10% DMSO] were tested, and DNA damage assessed at four time points following storage at -80 °C (one, two, four and eight weeks). In both cell lines, no differences in % tail intensity were detected between fresh and frozen cells up to eight weeks, irrespective of the harvesting method and freezing medium used. However, freshly isolated A172 cells exhibited a significant lower DNA damage when resuspended in CCM + 10% DMSO, while for A549 fresh cells the preferable harvesting method was the enzymatic one since it induced less DNA damage. Although both harvesting methods and cryoprotective media tested were found suitable, our data indicate that enzymatic harvesting and cryopreservation in CCM + 10% DMSO is a preferable method for DNA integrity preservation of human cell line samples for comet assay analysis. Our data also suggest that CCM is a preferable and cost-effective alternative to FBS in cryopreservation media. This optimized protocol allows the analysis of in vitro cell samples collected and frozen at different locations, with minimal interference on the basal DNA strand break levels in samples kept frozen up to eight weeks.


Assuntos
Células Epiteliais Alveolares , Ensaio Cometa/métodos , Criopreservação/métodos , Dano ao DNA , Neuroglia , Manejo de Espécimes/métodos , Células A549 , Células Epiteliais Alveolares/efeitos dos fármacos , Animais , Bovinos , Linhagem Celular , Separação Celular/métodos , Crioprotetores/farmacologia , Meios de Cultura/farmacologia , Quebras de DNA , Dimetil Sulfóxido/farmacologia , Sangue Fetal , Humanos , Concentração de Íons de Hidrogênio , Neuroglia/efeitos dos fármacos , Soluções/farmacologia , Fatores de Tempo
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