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1.
Nat Commun ; 11(1): 4034, 2020 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-32788576

RESUMO

Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency with severe platelet abnormalities and complex immunodeficiency. Although clinical gene therapy approaches using lentiviral vectors have produced encouraging results, full immune and platelet reconstitution is not always achieved. Here we show that a CRISPR/Cas9-based genome editing strategy allows the precise correction of WAS mutations in up to 60% of human hematopoietic stem and progenitor cells (HSPCs), without impairing cell viability and differentiation potential. Delivery of the editing reagents to WAS HSPCs led to full rescue of WASp expression and correction of functional defects in myeloid and lymphoid cells. Primary and secondary transplantation of corrected WAS HSPCs into immunodeficient mice showed persistence of edited cells for up to 26 weeks and efficient targeting of long-term repopulating stem cells. Finally, no major genotoxicity was associated with the gene editing process, paving the way for an alternative, yet highly efficient and safe therapy.


Assuntos
Edição de Genes , Terapia Genética , Células-Tronco Hematopoéticas/metabolismo , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/terapia , Animais , Plaquetas/metabolismo , Sistemas CRISPR-Cas/genética , Linhagem da Célula , Códon/genética , Feminino , Loci Gênicos , Células HEK293 , Transplante de Células-Tronco Hematopoéticas , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Macrófagos/metabolismo , Masculino , Camundongos , Testes de Mutagenicidade , Células Mieloides/metabolismo , Linfócitos T/metabolismo , Síndrome de Wiskott-Aldrich/patologia , Proteína da Síndrome de Wiskott-Aldrich/genética
2.
Int J Nanomedicine ; 15: 5227-5237, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32801688

RESUMO

Background: Large-scale production and application of amorphous silica nanoparticles (SiNPs) have enhanced the risk of human exposure to SiNPs. However, the toxic effects and the underlying biological mechanisms of SiNPs on Caenorhabditis elegans remain largely unclear. Purpose: This study was to investigate the genome-wide transcriptional alteration of SiNPs on C. elegans. Methods and Results: In this study, a total number of 3105 differentially expressed genes were identified in C. elegans. Among them, 1398 genes were significantly upregulated and 1707 genes were notably downregulated in C. elegans. Gene ontology analysis revealed that the significant change of gene functional categories triggered by SiNPs was focused on locomotion, determination of adult lifespan, reproduction, body morphogenesis, multicellular organism development, endoplasmic reticulum unfolded protein response, oocyte development, and nematode larval development. Meanwhile, we explored the regulated effects between microRNA and genes or signaling pathways. Pathway enrichment analysis and miRNA-gene-pathway-network displayed that 23 differential expression microRNA including cel-miR-85-3p, cel-miR-793, cel-miR-241-5p, and cel-miR-5549-5p could regulate the longevity-related pathways and inflammation signaling pathways, etc. Additionally, our data confirmed that SiNPs could disrupt the locomotion behavior and reduce the longevity by activating ins-7, daf-16, ftt-2, fat-5, and rho-1 genes in C. elegans. Conclusion: Our study showed that SiNPs induced the change of the whole transcriptome in C. elegans, and triggered negative effects on longevity, development, reproduction, and body morphogenesis. These data provide abundant clues to understand the molecular mechanisms of SiNPs in C. elegans.


Assuntos
Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Nanopartículas/toxicidade , Animais , Proteínas de Caenorhabditis elegans/genética , Redes Reguladoras de Genes/efeitos dos fármacos , Genoma Helmíntico , Humanos , Longevidade/efeitos dos fármacos , MicroRNAs/genética , Testes de Mutagenicidade/métodos , Nanopartículas/química , Reprodução/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Dióxido de Silício/química , Dióxido de Silício/toxicidade , Transcriptoma , Resposta a Proteínas não Dobradas/genética
3.
Toxicol Lett ; 332: 202-212, 2020 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-32659469

RESUMO

A variety of methods have been developed for accurate and systematic evaluation of chemical genotoxicity. Ceric ammonium nitrate (CAN) and 1,3-propane sultone (1,3-PS) have been extensively applied in industrial fields. Although 1,3-PS, but not CAN, has been reported as a potent carcinogen, systematic assessment of the genotoxic properties of these chemicals has not been conducted. The purpose of this study was to establish a decision tree for evaluating genotoxicity based on the good laboratory practices (GLP) system using 1,3-PS and CAN as test chemicals. In vitro studies were performed including the bacterial reverse mutation assay, chromosomal aberration assay, and micronucleus assay. We conducted in vivo studies using a combined micronucleus and alkaline comet (MN-CMT) assay and the Pig-a gene mutation assay, which is a promising method for detecting gene mutations in vivo. CAN showed negative responses in all in vitro genotoxicity assays and the in vivo combined MN-CMT assay. Meanwhile, 1,3-PS had positive results in all in vitro and in vivo genotoxicity assays. In this study, we confirmed the genotoxicity of 1,3-PS and CAN using both in vitro and in vivo assays. We propose a decision tree for evaluating chemical-induced genotoxicity.


Assuntos
Cério/toxicidade , Árvores de Decisões , Mutagênicos/toxicidade , Tiofenos/toxicidade , Animais , Bactérias/efeitos dos fármacos , Linhagem Celular/efeitos dos fármacos , Aberrações Cromossômicas/efeitos dos fármacos , Ensaio Cometa , Cricetinae , Cricetulus , Dano ao DNA , Relação Dose-Resposta a Droga , Feminino , Humanos , Masculino , Testes para Micronúcleos , Testes de Mutagenicidade , Ratos Sprague-Dawley
4.
Nat Commun ; 11(1): 3664, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32694532

RESUMO

Ethanol is a ubiquitous environmental stressor that is toxic to all lifeforms. Here, we use the model eukaryote Saccharomyces cerevisiae to show that exposure to sublethal ethanol concentrations causes DNA replication stress and an increased mutation rate. Specifically, we find that ethanol slows down replication and affects localization of Mrc1, a conserved protein that helps stabilize the replisome. In addition, ethanol exposure also results in the recruitment of error-prone DNA polymerases to the replication fork. Interestingly, preventing this recruitment through mutagenesis of the PCNA/Pol30 polymerase clamp or deleting specific error-prone polymerases abolishes the mutagenic effect of ethanol. Taken together, this suggests that the mutagenic effect depends on a complex mechanism, where dysfunctional replication forks lead to recruitment of error-prone polymerases. Apart from providing a general mechanistic framework for the mutagenic effect of ethanol, our findings may also provide a route to better understand and prevent ethanol-associated carcinogenesis in higher eukaryotes.


Assuntos
Replicação do DNA/efeitos dos fármacos , DNA Polimerase Dirigida por DNA/metabolismo , Etanol/toxicidade , Taxa de Mutação , Saccharomyces cerevisiae/genética , Sistemas CRISPR-Cas/genética , Proteínas de Ciclo Celular/metabolismo , DNA Fúngico/genética , Mutagênese , Testes de Mutagenicidade , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
5.
Mutat Res ; 854-855: 503199, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32660827

RESUMO

The bacterial reverse mutation test (Ames test) is the most commonly used genotoxicity test; it is a primary component of the chemical safety assessment data required by regulatory agencies worldwide. Within the current accepted in vitro genotoxicity test battery, it is considered capable of revealing DNA reactivity, and identifying substances that can produce gene mutations via different mechanisms. The previously published consolidated EURL ECVAM Genotoxicity and Carcinogenicity Database, which includes substances that elicited a positive response in the Ames test, constitutes a collection of data that serves as a reference for a number of regulatory activities in the area of genotoxicity testing. Consequently, we considered it important to expand the database to include substances that fail to elicit a positive response in the Ames test, i.e., Ames negative substances. Here, we describe a curated collection of 211 Ames negative substances, with a summary of complementary data available for other genotoxicity endpoints in vitro and in vivo, plus available carcinogenicity data. A descriptive analysis of the data is presented. This includes a representation of the chemical space formed by the Ames-negative database with respect to other substances (e.g. REACH registered substances, approved drugs, pesticides, etc.) and a description of the organic functional groups found in the database. We also provide some suggestions on further analyses that could be made.


Assuntos
Testes de Carcinogenicidade/normas , Carcinógenos/toxicidade , Bases de Dados Factuais/normas , Testes de Mutagenicidade/normas , Mutagênicos/toxicidade , Resultados Negativos/normas , Animais , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Gerenciamento de Dados/normas , Humanos
7.
Mutat Res ; 854-855: 503198, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32660822

RESUMO

TiO2 particles are broadly used in daily products, including cosmetics for their UV-absorbing property, food for their white colouring property, water and air purification systems, self-cleaning surfaces and photoconversion electrical devices for their photocatalytic properties. The toxicity of TiO2 nano- and microparticles has been studied for decades, and part of this investigation has been dedicated to the identification of their potential impact on DNA, i.e., their genotoxicity. This review summarizes data retrieved from their genotoxicity testing during the past 6 years, encompassing both in vitro and in vivo studies, mostly performed on lung and intestinal models. It shows that TiO2 particles, both nano- and micro-sized, produce genotoxic damage to a variety of cell types, even at low, realistic doses.


Assuntos
Mutagênicos/toxicidade , Titânio/toxicidade , Animais , DNA/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Humanos , Testes de Mutagenicidade/métodos
8.
Mutat Res ; 854-855: 503209, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32660823

RESUMO

We have measured the toxicity and genotoxicity of 2-methylfuran, which is formed in foods during thermal processing. The agent was administered by oral gavage to male Sprague-Dawley rats, daily for 28 days, before performing general toxicology analysis and the following genotoxicity tests: comet assay (peripheral blood, liver); Pig-a gene mutation assay (peripheral blood); micronucleus test (peripheral blood, bone marrow). Liver was the primary target organ; histological changes (oval cell hyperplasia) were observed but without significant changes in serum enzyme markers. For hepatotoxicity, the no-observed-adverse-effect level was 5 mg/kg bw/d. Histopathological changes were also seen in the bone marrow. Genotoxicity assays were uniformly negative.


Assuntos
Furanos/toxicidade , Animais , Ensaio Cometa , Dano ao DNA/efeitos dos fármacos , Fígado/efeitos dos fármacos , Masculino , Testes para Micronúcleos/métodos , Testes de Mutagenicidade/métodos , Mutagênicos/toxicidade , Ratos , Ratos Sprague-Dawley
9.
Sci Total Environ ; 726: 137862, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32481210

RESUMO

The Ames test is the most commonly used mutagenicity test worldwide. It is based on a microbial system that uses histidine auxotrophic Salmonella typhimurium strains. Due to either spontaneous mutations or mutations induced by a mutagenic compound, the cells can regain their ability to grow without histidine supplementation. The degree of mutagenicity of a sample correlates with the number of cells that are able to grow in media that lack histidine. All test variants published up to now are endpoint determinations providing no information about cell growth and respiration activity during the cultivation time. This study aimed to develop an alternative type of Ames test by characterizing the respiration activity of Salmonella typhimurium over time for dynamic mutagenicity detection. It focuses on elucidating the mechanisms underlying this novel test system, and serves as a general proof of principle. Respiration activity (oxygen transfer and uptake rate) and biomass growth of Salmonella typhimurium TA 100 and TA 98 were mechanistically modeled to understand and predict the behavior of the bacteria during the Ames test. The results simulated by the model were experimentally validated by the online monitoring of respiration activity over cultivation time using a Respiration Activity MOnitoring System (RAMOS). The simulated prediction was observed to fit well to the experimental data. When a mutagenic compound was added, its mutagenicity could be detected online due to the elevated cell number and respiration of histidine prototrophic cells. Laborious manual evaluation of mutagenicity after cultivation is not necessary. Mutagenicity evaluation with the presented alternative Ames RAMOS test fitted well to results from an Ames fluctuation test. In the future, a miniaturized RAMOS device for microtiter plates should allow for a high-throughput Ames RAMOS test.


Assuntos
Mutagênicos , Salmonella typhimurium , Histidina , Testes de Mutagenicidade , Respiração
10.
J Vis Exp ; (160)2020 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-32568251

RESUMO

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (≤14 days). These spheroid models (≥500 µm in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the 'gold standard' micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.


Assuntos
Imageamento Tridimensional/métodos , Fígado/fisiopatologia , Testes de Mutagenicidade/métodos , Nanoestruturas/química , Humanos
11.
Artigo em Inglês | MEDLINE | ID: mdl-32507983

RESUMO

Ionic lead (Pb) in the environment has accumulated due to anthropogenic activities, causing a potential threat to plants and plant consumers. We conducted this study to reveal the molecular mechanism of Pb stress response in plants. The effects of Pb (5.0 and 15.0 µM) on mitosis, DNA replication, gene expression and proteins in root-tip cells of Allium cepa var. agrogarum L. were addressed. The results indicated that root growth was inhibited dramatically in Pb treatment groups. Chromosomal aberrations were observed and the mitotic index decreased during Pb treatments at different concentrations. The accumulation of reactive oxygen species (ROS) in onion roots was induced by Pb stress. Pb increased DNA damage and suppressed cell cycle progression. The above toxic effects got more serious with increasing Pb concentration and prolonging exposure time. A total of 17 proteins were expressed differentially between control and Pb exposure groups. Under Pb treatment, the decreased expression of Anx D1 indicated decreased defensive response; the decreased expression of SHMT1 indicated decreased respiration; the decreased expression of COMT2 indicated decreased response of other funtions; the increased expression of NDPK indicated increased transcription and protein synthesis; the increased expression of PR1 and CHI1 indicated increased pathogen invasion; the increased expression of ORC5 and MPK5 indicated the reduced DNA replicating activity; the decreased expression of POLD1 indicated the reduced DNA repair activity. Our results provide new insights at the proteomic level into the Pb-induced responses, defensive responses and toxic effects, and provide new molecular markers of the early events of plant responses to Pb toxicity.


Assuntos
Chumbo/efeitos adversos , Proteínas de Plantas/genética , Proteoma/efeitos dos fármacos , Cebolinha Branca/efeitos dos fármacos , Poluentes do Solo/efeitos adversos , Meristema/efeitos dos fármacos , Meristema/crescimento & desenvolvimento , Meristema/metabolismo , Testes de Mutagenicidade , Proteínas de Plantas/metabolismo , Proteoma/genética , Proteoma/metabolismo , Cebolinha Branca/genética , Cebolinha Branca/metabolismo
12.
Mutat Res ; 853: 503171, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32522346

RESUMO

The tests used and the general principles behind test strategies are now often over 30 years old. It may be time by now, given that our knowledge of genetic toxicology has improved and that we also technically are better able to investigate DNA damage making use of modern molecular biological techniques, to start thinking on a new test strategy. In the present paper, it is discussed that the time is there to consider a new approach for genotoxicity assessment of substances. A fit for all test strategy was discussed making use of the most recent technological methods and techniques. It was also indicated that in silico tools should be more accepted by regulatory institutes/bodies as supporting information to better conclude which tests should be required for each separate substance to demonstrate its genotoxic potency. Next to that there should be a good rationale for performing in vivo studies. Finally, the need for germ cell genotoxicity testing, essential when classification and labeling of substances is mandatory, was discussed. It was suggested to change the GHS for genotoxicity classification and labelling from in vivo tests in germ cells into in vivo tests in somatic cells. Quantitative genotoxicology was also discussed. It appeared that we are currently at a transition, where the science developing to justify carrying out human health risk assessments based on genetic toxicology data sets supported by mechanistic data and exposure data. However, implementation will take time, and acceptance will be supported through the development of numerous case studies. Major remaining questions are: is genetic damage a relevant endpoint in itself, or should the risk assessment be carried out on the apical endpoint of cancer and which genotoxic endpoint should be used to derive the point of departure (PoD) for the human exposure limit?


Assuntos
Testes de Mutagenicidade/métodos , Testes de Mutagenicidade/normas , Animais , Dano ao DNA/efeitos dos fármacos , Células Germinativas/efeitos dos fármacos , Humanos , Indústrias/métodos , Indústrias/normas , Mutagênicos/toxicidade , Neoplasias/induzido quimicamente , Medição de Risco/métodos , Medição de Risco/normas
13.
Chemosphere ; 250: 126416, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32380589

RESUMO

The flavonoid metal-insecticide magnesium-hesperidin complex (MgHP) has recently been considered as a novel insecticide to replace some persistent pesticides. However, it is important to evaluate its action on non-target species, mainly those living in an aquatic environment, as these ecosystems are the final receptors of most chemicals. Reactive oxygen species, antioxidant and oxidative stress biomarkers, genotoxicity as well as cell cycle was evaluated in the liver cell line from zebrafish (Danio rerio; ZF-L) exposed to 0, 0.1, 1, 10, 100 and 1000 ng mL-1 MgHP. MgHP affected cell stability by increasing reactive oxygen species (ROS) in both exposure times (24 and 96 h) at high concentrations. Catalase (CAT) activity decreased after 24 h exposure, and glutathione and metallothionein values increased, avoiding the lipid peroxidation. Genotoxicity increased as MgHP concentration increased, after 24 h exposure, exhibiting nuclear abnormalities; it was recovered after 96 h exposure, evidencing possible stimulation of DNA repair mechanisms. The alteration in the cell cycle (increasing in the Sub-G1 phase and decreasing in the S-phase) was associated with chromosomal instability. In conclusion, the responses of ROS and the antioxidant defense system depended on MgHP concentration and time exposure, while DNA exhibited some instability after 24 h exposure, which was recovered after 96 h.


Assuntos
Ciclo Celular/efeitos dos fármacos , Inseticidas/toxicidade , Fígado/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Animais , Antioxidantes/metabolismo , Linhagem Celular , Dano ao DNA , Relação Dose-Resposta a Droga , Ecotoxicologia/métodos , Biomarcadores Ambientais/efeitos dos fármacos , Glutationa/metabolismo , Hesperidina/química , Hesperidina/toxicidade , Inseticidas/administração & dosagem , Inseticidas/química , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/citologia , Fígado/metabolismo , Magnésio/química , Testes de Mutagenicidade/métodos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Poluentes Químicos da Água/química , Peixe-Zebra
14.
Sci Total Environ ; 733: 138911, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32450377

RESUMO

The objective of the study was the assessment of the mutagenicity of chemical pollutants adsorbed on suspended particulate matter with aerodynamic diameter < 2.5 µm (PM2.5) in the four seasons. Samples were collected from the urban agglomeration of Wroclaw, Poland and evaluated for mutagenicity using two Salmonella typhimurium strains TA98 and TA100 with and without metabolic activation with microsomal fraction S9. The work covered sampling of suspended dusts in four seasons: summer, spring, autumn and winter. The dust samples were collected on glass filters using air aspirator and the organic matter of PM2.5 was extracted using Soxhlet extractor. The levels of polycyclic aromatic hydrocarbon compounds (PAH), nitro-PAH and dinitro-PAH were determined in the extract. Variable degree of air pollution with mutagenic substances was determined at the selected study site. A greater, negative effect of chemical compounds on DNA was determined in dust samples collected in the autumn-winter season in comparison to samples collected in the spring-summer season. In the majority of tests, higher mutagenicity was obtained in analyses conducted on total extracts in comparison to tests conducted in the presence of PAH pollutant fractions. The obtained mutagenic ratio values pointed to the presence of chemical compounds with a character of both promutagens and direct mutagens. Samples collected in the autumn-winter season were observed to have a higher diversity of organic substances absorbed on PM2.5 dusts. Particular samples differed in the total content and percent contribution of particular PAHs, nitro-PAHs, and other organic compounds. In addition, the identified substances included compounds belonging to different chemical classes: aliphatic compounds, cycloalkanes, mono- and bicycling arenes, polycyclic arenes, compounds containing oxygen, nitrogen, and sulphur.


Assuntos
Poluentes Atmosféricos/análise , Poluentes Ambientais , Hidrocarbonetos Policíclicos Aromáticos/análise , Testes de Mutagenicidade , Mutagênicos , Material Particulado , Polônia , Estações do Ano
15.
Toxicology ; 441: 152474, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32380031

RESUMO

2-Methoxy-4-nitroaniline (MNA), an intermediate in the synthesis of azo dyes used in textiles and paints, is structurally similar to carcinogenic anilines. Human exposure occurs primarily in the occupational setting through handling of dye dust, and through use and disposal of MNA-containing products. MNA has been reported to induce contact hypersensitivity in a human, myocardial necrosis in rats, and bacterial mutagenicity. This study assessed the subacute toxicity, genotoxicity, contact hypersensitivity, and reproductive toxicity of MNA in rodents in an effort to more fully characterize its toxicological profile. B6C3F1/N mice were exposed to 0, 650, 1250, 2500, 5000, or 10,000 ppm MNA by dosed feed for 14-days to evaluate subacute toxicity and histopathological endpoints. In female mice, decreased body weight (13.5 %) and absolute kidney weight (14.8 %), compared to control, were observed at 10,000 ppm MNA; increased relative liver weight (10-12 %), compared to control, occurred at 5,000-10,000 ppm MNA. In male mice, absolute (15 %) and relative liver weights (9-13 %) were increased at 2,500-5,000 ppm and 1250-10,000 ppm MNA, compared to control, respectively. In both sexes of mice, minimal elevations of hemosiderin pigmentation (a breakdown product of erythrocytes), relative to control, were observed in the liver (10,000 ppm); minimal to moderate elevations of hemosiderin pigmentation (5,000-10,000 ppm) and minimal increases in hematopoietic cell proliferation occurred in the spleen (≥ 1250 ppm). In a reproductive toxicity study, timed-mated female Harlan Sprague Dawley rats were exposed to 0-10,000 ppm MNA by dosed feed from gestation day 6 through postnatal day (PND) 21. Decreases in mean litter weights were observed at 5000 ppm MNA, compared to control, beginning at PND1. To evaluate potential contact hypersensitivity, MNA (2.5-50 %, in dimethylformamide) was applied to the dorsa of both ears of female Balb/c mice once daily for three days. The increase observed in lymph node cell proliferation (10-50 % increase in thymidine uptake compared to control) did not reproducibly achieve the Sensitization Index (SI) 3 level, and there was no ear swelling evident following sensitization with 10-50 % MNA and challenge with 25 % MNA in the mouse ear swelling test. In bacterial mutagenicity assays, MNA (250-1000 µg/plate) induced significant increases, compared to control, in mutant colonies with and without metabolic activation enzymes in Salmonella typhimurium strains TA100 and TA98. These data indicate that MNA is genotoxic, and may induce erythrocyte damage and reactive phagocytosis by macrophages in the liver and spleen.


Assuntos
Compostos de Anilina/toxicidade , Dermatite de Contato/etiologia , Nitrocompostos/toxicidade , Reprodução/efeitos dos fármacos , Animais , Peso Corporal/efeitos dos fármacos , Ensaio Cometa , Relação Dose-Resposta a Droga , Feminino , Fígado/efeitos dos fármacos , Linfonodos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Testes para Micronúcleos , Testes de Mutagenicidade , Tamanho do Órgão/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley
16.
Mutat Res ; 783: 108298, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32386748

RESUMO

This short review explores the utility and applications of CRISPR/Cas9 systems in radiobiology. Specifically, in the context of experimentally simulating genotoxic effects of Ionizing Radiation (IR) to determine the contributions from DNA targets and 'Complex Double-Stranded Breaks' (complex DSBs) to the IR response. To elucidate this objective, this review considers applications of CRISPR/Cas9 on nuclear DNA targets to recognize the respective 'nucleocentric' response. The article also highlights contributions from mitochondrial DNA (mtDNA) - an often under-recognized target in radiobiology. This objective requires accurate experimental simulation of IR-like effects and parameters with the CRISPR/Cas9 systems. Therefore, the role of anti-CRISPR proteins in modulating enzyme activity to simulate dose rate - an important factor in radiobiology experiments is an important topic of this review. The applications of auxiliary domains on the Cas9 nuclease to simulate oxidative base damage and multiple stressor experiments are also topics of discussion. Ultimately, incorporation of CRISPR/Cas9 experiments into computational parameters in radiobiology models of IR damage and shortcomings to the technology are discussed as well. Altogether, the simulation of IR parameters and lack of damage to non-DNA targets in the CRISPR/Cas9 system lends this rapidly emerging tool as an effective model of IR induced DNA damage. Therefore, this literature review ultimately considers the relevance of complex DSBs to radiobiology with respect to using the CRISPR/Cas9 system as an effective experimental tool in models of IR induced effects.


Assuntos
Sistemas CRISPR-Cas , Quebras de DNA de Cadeia Dupla , DNA/efeitos da radiação , Modelos Genéticos , Testes de Mutagenicidade/métodos , Proteína 9 Associada à CRISPR , DNA/metabolismo , Dano ao DNA , Reparo do DNA , DNA Mitocondrial/metabolismo , DNA Mitocondrial/efeitos da radiação , Humanos , Radiação Ionizante
17.
Mutat Res ; 850-851: 503135, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32247552

RESUMO

Use of three-dimensional (3D) tissue equivalents in toxicology has been increasing over the last decade as novel preclinical test systems and as alternatives to animal testing. In the area of genetic toxicology, progress has been made with establishing robust protocols for skin, airway (lung) and liver tissue equivalents. In light of these advancements, a "Use of 3D Tissues in Genotoxicity Testing" working group (WG) met at the 7th IWGT meeting in Tokyo in November 2017 to discuss progress with these models and how they may fit into a genotoxicity testing strategy. The workshop demonstrated that skin models have reached an advanced state of validation following over 10 years of development, while liver and airway model-based genotoxicity assays show promise but are at an early stage of development. Further effort in liver and airway model-based assays is needed to address the lack of coverage of the three main endpoints of genotoxicity (mutagenicity, clastogenicity and aneugenicity), and information on metabolic competence. The IWGT WG believes that the 3D skin comet and micronucleus assays are now sufficiently validated to undergo an independent peer review of the validation study, followed by development of individual OECD Test Guidelines.


Assuntos
Dano ao DNA/efeitos dos fármacos , Metagenômica/tendências , Testes de Mutagenicidade/métodos , Mutagênicos/toxicidade , Dano ao DNA/genética , Humanos , Fígado/efeitos dos fármacos , Fígado/patologia , Testes para Micronúcleos
18.
Mutat Res ; 850-851: 503143, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32247554

RESUMO

BACKGROUND: The cytokinesis-block micronucleus (CBMN) assay is an internationally recognized method for measuring DNA damage after exposure to genotoxic agents, as well as a biomarker for DNA repair and chromosomal instability. The high baseline level of micronuclei (MN) in the healthy population has limited the sensitivity and application of the CBMN assay for the follow-up of exposed populations. We reevaluated the sensitivity of the CBNM assay using semi-automated MN scoring following telomere and centromere (TC) staining after in vitro exposure to genotoxic agents (mitomycin or radiation) or aneugenic agents (vinblastine). MATERIALS AND METHODS: Blood samples from 12 healthy donors were exposed to 137Cs at seven doses from 0.1-4 Gy and cultured for 72 h. Cytochalasin B was added at 46 h of culture. The exposure of chemical agents (mitomycin or vinblastine) was performed after 48 h of culture for 3 h. Cytochalasin B was added after treatment and slides were prepared 24 h after. MN was semi-automatically scored following TC staining. Nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) were assessed in a human cell line after TC staining. RESULTS: The introduction TC staining to the scoring of MN not only renders MN scoring more efficient and robust, but also permits discrimination between exposure to clastogenic (MN with only telomere signals) and aneugenic agents (MN with both TC signals). The resulting improvement of MN detection led to an increase in the sensitivity of the CBMN assay following low-dose radiation exposure (0.3 versus 0.1 Gy). Hyperradiosensitivity phenomenon was observed after low dose exposure. A dose-response curve was obtained for up to 4 Gy. In addition, TC staining permits assessment of the nature of NPBs and NBUDs as biomarkers for genotoxicity and chromosomal instability. CONCLUSION: These approaches can be potentially used to follow-up populations exposed to genotoxic agents and assess cancer risk.


Assuntos
Centrômero/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Testes de Mutagenicidade , Telômero/efeitos dos fármacos , Aneugênicos/farmacologia , Centrômero/genética , Citocinese/efeitos dos fármacos , Citocinese/genética , Dano ao DNA/genética , Humanos , Linfócitos/efeitos dos fármacos , Micronúcleos com Defeito Cromossômico/efeitos dos fármacos , Testes para Micronúcleos , Mutagênicos/toxicidade , Medição de Risco , Telômero/genética
19.
Mutat Res ; 850-851: 503145, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32247555

RESUMO

5-Fluorouracil (5-FU) is a widely used antineoplastic drug. In this work, a comprehensive study was performed to detect the extent of chromosomal damage and morphological sperm defects induced by 5-FU in male mice and the possible protective role of the iridoids-rich fraction of Pentas lanceolata leaves (IFPL). Six main groups were examined in micronucleus and chromosomal assays: I- control negative, II- control positive (i.p. treated with single dose of 75 mg/kg 5-FU), III- control plant (orally administrated IFPL, 300 mg/kg, 5 consecutive days), and IV-VI- treated with IFPL (100, 200 and 300 mg/kg, 5 consecutive days) plus 5-FU (i.p. treated at the last day). Samples were taken 24 h post treatment. The study of morphological sperm anomalies, single and repeated treatments were examined and samples were taken after 35 days from the 1st treatment. In bone marrow, 5-FU induced a significant increase in the micro-nucleated polychromatic erythrocytes, chromosome anomalies (CAs) and also cytotoxic effects. A significant percentage of CAs was recorded in spermatocytes after 5-FU treatment reached 22.80 ± 1.32 vs 4.20 ± 0.37 for control (mainly X-Y univalent, 90%). IFPL was recorded to be non-mutagenic in all tests examined. In addition, it alleviated the previous defects in a dose-dependent manner. A significant and dramatic increase in the percentage of morphological sperm defects was recorded after single and repeated treatments with 5-FU reached 13.24 ± 0.24, 30.42 ± 0.32 respectively vs 2.56 ± 0.14 for control. Amorphous head-sperm and sperm with coiled tail were the most pronounced types of abnormalities. Significant protection was detected with the highest tested dose of IFPL. In conclusion: 5-FU demonstrated to be a genotoxic agent. Its genotoxicity in germ cells is serious and may lead to reproductive toxicity, infertility or heritable defects. The results also demonstrated the biosafety of IFPL and its possible protective role in combined treatment with 5-FU.


Assuntos
Iridoides/farmacologia , Extratos Vegetais/farmacologia , Rubiaceae/química , Espermatozoides/efeitos dos fármacos , Animais , Aberrações Cromossômicas/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Fluoruracila/efeitos adversos , Fluoruracila/uso terapêutico , Iridoides/química , Masculino , Camundongos , Testes de Mutagenicidade , Extratos Vegetais/química , Espermatócitos/efeitos dos fármacos , Espermatócitos/patologia , Espermatozoides/patologia
20.
Mutat Res ; 850-851: 503149, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32247558

RESUMO

Cadmium oxide nanoparticles (CdO NPs) are among some of the most studied and industrially used metal oxide NPs. They have been widely used for industrial application, such as paint pigments and electronic devices, and medical therapeutics. With increasing use of CdO NPs and concerns for their potential adverse effects on the environment and public health, evaluation of the cytotoxicity and genotoxicity of CdO NPs becomes very important. To date, there is a limited understanding of the potential hazard brought by CdO NPs and a lack of information and research, particularly on the genotoxicity assessment of these NPs. In this study, 10 nm CdO core-PEG stabilized NPs were synthesized, characterized and used for evaluation of CdO NPs' cytotoxicity and genotoxicity. Release of cadmium ions (Cd+2) from the CdO NPs in cell culture medium, cellular uptake of the NPs, and the endotoxin content of the particles were measured prior to the toxicity assays. Cytotoxicity was evaluated using the MTS assay, ATP content detection assay, and LDH assay. Genotoxicity was assessed using the Ames test, Comet assay, micronucleus assay, and mouse lymphoma assay. The cytotoxicity of cadmium chloride (CdCl2) was also evaluated along with that of the CdO NPs. The results showed that endotoxin levels within the CdO NPs were below the limit of detection. CdO NPs induced concentration-dependent cytotoxicity in TK6 and HepG2 cells with the MTS, ATP and LDH assays. Although the genotoxicity of CdO NPs was negative in the Ames test, positive results were obtained with the micronucleus, Comet, and mouse lymphoma assays. The negative response of CdO NPs with the Ames test may be the result of unsuitability of the assay for measuring NPs, while the positive responses from other genotoxicity assays suggest that CdO NPs can induce chromosomal damage, single or double strand breaks in DNA, and mutations. The toxicity of the CdO NPs results from the NPs themselves and not from the released Cd+2, because the ions released from the NPs were minimal. These results demonstrate that CdO NPs are cytotoxic and genotoxic and provide new insights into risk assessment of CdO NPs for human exposure and environmental protection.


Assuntos
Compostos de Cádmio/toxicidade , Nanopartículas Metálicas/toxicidade , Testes de Mutagenicidade , Mutagênicos/toxicidade , Óxidos/toxicidade , Animais , Compostos de Cádmio/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ensaio Cometa , Dano ao DNA/efeitos dos fármacos , Humanos , Nanopartículas Metálicas/química , Camundongos , Mutagênicos/farmacologia , Óxidos/farmacologia
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