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1.
Methods Mol Biol ; 2519: 65-72, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36066710

RESUMO

The comet assay is an effective method for identifying DNA breaks and alkali-labile sites induced by genotoxins. Performed as a single-cell electrophoresis, this assay is especially simplistic, and the results are easily reproducible. DNA breakage can be quantitatively assessed by the induced comet tail regions, which can be measured using a variety of comet software. This protocol will finish within approximately two hours with adequate preparation, and digitized images can be taken using a confocal or standard fluorescence microscopes after staining the cell nucleus with a DNA dye.


Assuntos
Dano ao DNA , Mutagênicos , Ensaio Cometa/métodos , DNA , Coloração e Rotulagem
2.
J Toxicol Environ Health A ; 85(22): 937-951, 2022 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-36068785

RESUMO

Coumarins and chalcones are compounds widely found in plants or obtained by synthetic methods which possess several biological properties including antioxidant, anti-inflammatory, and antitumor effects. A series of coumarin-chalcone hybrids were synthesized to improve their biological actions and reduce potential adverse effects. Considering the applications of these molecules, a coumarin-chalcone hybrid [7-methoxy-3-(E)-3-(3,4,5-trimethoxyphenyl) acryloyl-2 H-chromen-2-one] (4-MET) was synthesized and the genotoxic, cytotoxic, and protective effects assessed against damage induced by different mutagens. First, in silico tools were used to predict biological activity of 4-MET which indicated a chemopreventive potential. Subsequently, the genotoxic/antigenotoxic activities of 4-MET were determined both in vitro (Ames test) and in vivo (micronucleus (MN) test and comet assay). In addition, molecular docking simulations were performed between 4-MET and glutathione reductase, an important cellular detoxifying enzyme. Our results indicated that 4-MET was not mutagenic in the Ames test; however, when co-treated with sodium azide or 4-nitroquinoline 1-oxide (4-NQO), 4-MET significantly reduced the harmful actions of these mutagens. Except for a cytotoxic effect after 120 hr treatment, 4-MET alone did not produce cytotoxicity or genotoxicity in the MN test and comet assay. Nonetheless, all treatments of 4-MET with cyclophosphamide (CPA) showed a chemoprotective effect against DNA damage induced by CPA. Further, molecular docking analysis indicated a strong interaction between 4-MET and the catalytic site of glutathione reductase. These effects may be related to (1) damage prevention, (2) interaction with detoxifying enzymes, and (3) DNA-repair induction. Therefore, data demonstrated that 4-MET presents a favorable profile to be used in chemopreventive therapies.


Assuntos
Chalcona , Chalconas , Chalconas/farmacologia , Ensaio Cometa/métodos , Cumarínicos/farmacologia , Ciclofosfamida , Dano ao DNA , Reparo do DNA , Glutationa Redutase , Testes para Micronúcleos , Simulação de Acoplamento Molecular , Mutagênicos/toxicidade
3.
Artigo em Inglês | MEDLINE | ID: mdl-36031332

RESUMO

The comet assay is used to measure DNA damage induced by chemical and physical agents. High concentrations of test agents may cause cytotoxicity or cell death, which may give rise to false positive results in the comet assay. Systematic studies on genotoxins and cytotoxins (i.e. non-genotoxic poisons) have attempted to establish a threshold of cytotoxicity or cell death by which DNA damage results measured by the comet assay could be regarded as a false positive result. Thresholds of cytotoxicity/cell death range from 20% to 50% in various publications. Curiously, a survey of the latest literature on comet assay results from cell culture studies suggests that one-third of publications did not assess cytotoxicity or cell death. We recommend that it should be mandatory to include results from at least one type of assay on cytotoxicity, cell death or cell proliferation in publications on comet assay results. A combination of cytotoxicity (or cell death) and proliferation (or colony forming efficiency assay) is preferable in actively proliferating cells because it covers more mechanisms of action. Applying a general threshold of cytotoxicity/cell death to all types of agents may not be applicable; however, 25% compared to the concurrent negative control seems to be a good starting value to avoid false positive comet assay results. Further research is needed to establish a threshold value to distinguish between true and potentially false positive genotoxic effects detected by the comet assay.


Assuntos
Dano ao DNA , Mutagênicos , Causas de Morte , Morte Celular , Ensaio Cometa
4.
Artigo em Inglês | MEDLINE | ID: mdl-36031336

RESUMO

Many conventional in vitro tests that are currently widely used for routine screening of chemicals have a sensitivity/specificity in the range between 60 % and 80 % for the detection of carcinogens. Most procedures were developed 30-40 years ago. In the last decades several assays became available which are based on the use of metabolically competent cell lines, improvement of the cultivation conditions and development of new endpoints. Validation studies indicate that some of these models may be more reliable for the detection of genotoxicants (i.e. many of them have sensitivity and specificity values between 80 % and 95 %). Therefore, they could replace conventional tests in the future. The bone marrow micronucleus (MN) assay with rodents is at present the most widely used in vivo test. The majority of studies indicate that it detects only 5-6 out of 10 carcinogens while experiments with transgenic rodents and comet assays seem to have a higher predictive value and detect genotoxic carcinogens that are negative in MN experiments. Alternatives to rodent experiments could be MN experiments with hen eggs or their replacement by combinations of new in vitro tests. Examples for promising candidates are ToxTracker, TGx-DDI, multiplex flow cytometry, γH2AX experiments, measurement of p53 activation and MN experiments with metabolically competent human derived liver cells. However, the realization of multicentric collaborative validation studies is mandatory to identify the most reliable tests.


Assuntos
Galinhas , Dano ao DNA , Animais , Carcinógenos , Ensaio Cometa , Feminino , Humanos , Testes para Micronúcleos , Testes de Mutagenicidade , Roedores , Sensibilidade e Especificidade
5.
Biomed Res Int ; 2022: 2822605, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36033573

RESUMO

Objective: To investigate the genotoxicity of metformin on planarian with different concentrations and exposure times. Methods: The planarians were treated, respectively, with 10 mmol/L and 50 mmol/L metformin for 1, 3, and 5 days, and then, the comet assay and random amplified polymorphic DNA (RAPD) analysis were performed. 13 random primers were used for PCR amplification with the genomic DNAs as templates. Planarians cultured in clear water were used as the control. Genomic template stability (GTS) was calculated by comparing and analyzing the RAPD patterns of the control group and the treatment groups. Results: In the comet assay, DNA damage of planarians treated with 10 mmol/L metformin for 1, 3, and 5 days was 10.2%, 25.4%, and 36.8%, respectively, and that of planarians treated with 50 mmol/L metformin was 40.6%, 62.8%, and 65.4%, respectively. GTS values of planarians exposed to 10 mmol/L metformin for 1, 3, and 5 days were 64.1%, 62.8%, and 52.6%, respectively, and those of planarians exposed to 50 mmol/L metformin for 1, 3, and 5 days were 52.6%, 51.3%, and 50%, respectively. DNA damage increased and GTS values decreased with the increasing metformin exposure concentration and exposure time. Conclusion: Metformin has certain genotoxicity on planarian in a dose- and time-related manner. The comet assay and RAPD analysis are highly sensitive methods for detecting genotoxicity with drugs.


Assuntos
Metformina , Planárias , Animais , Ensaio Cometa , Dano ao DNA , Água Doce , Instabilidade Genômica , Técnica de Amplificação ao Acaso de DNA Polimórfico
6.
Sci Rep ; 12(1): 13899, 2022 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-35974115

RESUMO

With the evolution of nanostructure materials, silver nanoparticles (Ag-NPs) emerged as the predominantly exploited nanomaterial in multifarious sectors due to their versatile properties. Along with the heightening applications of Ag-NPs, however, there is increasing concern over their indubitable toxicity towards the ecosystem, which indeed affects surrounding organisms and human health. In this study, we evaluated the detrimental effects of Ag-NPs in relation to Egyptian wild female beetles, Blaps polychresta, after injection with a single dose of Ag-NPs at different doses and monitoring for 30 days to determine the sublethal dose. Accordingly, the sublethal dose revealed the lowest negative influence was found at 0.03 mg/g body weight. The adverse impacts of Ag-NPs on the ovaries of female beetles were investigated by estimating the enzyme activities, DNA damage using a comet assay, and apoptosis by means of flow cytometry. Besides, the ultrastructural abnormalities were surveyed adopting transmission electron microscopy (TEM). The results manifested comet cells of 7.67 ± 0.88% and 22.33 ± 0.51 for Ag-NPs treated and control groups, respectively. Similarly, the data from flow cytometry demonstrated a substantial reduction in viable cells associated with a significant rise in apoptotic cells for the Ag-NPs treated group in comparison with the control group. Moreover, significant disturbances in enzyme activities for the treated group were perceived correlated with evident diminutions in antioxidant enzymes. Remarkably, the ultrastructural investigation emphasized these findings, exposing considerable deformities of the ovaries in the Ag-NPs treated group compared with the control group. To the best of our knowledge, this is the first report discussing the influence of Ag-NPs at the lowest dose on ovaries of B. polychresta. Collectively, our findings would significantly contribute to considering the critical effects of Ag-NPs at low levels, in addition to the potential use of B. polychresta as a good bio-indicator in ecotoxicological analyses.


Assuntos
Besouros , Nanopartículas Metálicas , Animais , Ensaio Cometa , Ecossistema , Feminino , Humanos , Nanopartículas Metálicas/química , Prata/química , Prata/toxicidade
7.
Int J Mol Sci ; 23(15)2022 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-35955556

RESUMO

In the last years, radiofrequency (RF) has demonstrated that it can reduce DNA damage induced by a subsequent treatment with chemical or physical agents in different cell types, resembling the adaptive response, a phenomenon well documented in radiobiology. Such an effect has also been reported by other authors both in vitro and in vivo, and plausible hypotheses have been formulated, spanning from the perturbation of the cell redox status, to DNA repair mechanisms, and stress response machinery, as possible cellular mechanisms activated by RF pre-exposure. These mechanisms may underpin the observed phenomenon, and require deeper investigations. The present study aimed to determine whether autophagy contributes to RF-induced adaptive response. To this purpose, SH-SY5Y human neuroblastoma cells were exposed for 20 h to 1950 MHz, UMTS signal, and then treated with menadione. The results obtained indicated a reduction in menadione-induced DNA damage, assessed by applying the comet assay. Such a reduction was negated when autophagy was inhibited by bafilomycin A1 and E64d. Moreover, CRISPR SH-SY5Y cell lines defective for ATG7 or ATG5 genes did not show an adaptive response. These findings suggest the involvement of autophagy in the RF-induced adaptive response in human neuroblastoma cells; although, further investigation is required to extend such observation at the molecular level.


Assuntos
Neuroblastoma , Vitamina K 3 , Autofagia , Linhagem Celular Tumoral , Ensaio Cometa , Humanos , Neuroblastoma/metabolismo , Ondas de Rádio
8.
Aging Cell ; 21(9): e13698, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35996812

RESUMO

It is known that chondrocytes from joints with osteoarthritis (OA) exhibit high levels of DNA damage, but the degree to which chondrocytes accumulate DNA damage during "normal aging" has not been established. The goal of this study was to quantify the DNA damage present in chondrocytes obtained from cadaveric donors of a wide age range, and to compare the extent of this damage to OA chondrocytes. The alkaline comet assay was used to measure the DNA damage in normal cartilage from the ankle (talus) and the knee (femur) of cadaveric donors, as well as in OA chondrocytes obtained at the time of total knee replacement. Chondrocytes from younger donors (<45 years) had less DNA damage than older donors (>70 years) as assessed by the percentage of DNA in the comet "tail". In donors between 50 and 60 years old, there was increased DNA damage in chondrocytes from OA cartilage as compared to cadaveric. Talar chondrocytes from 23 donors between the ages of 34 and 78 revealed a linear increase in DNA damage with age (R2  = 0.865, p < 0.0001). A "two-tailed" comet assay was used to demonstrate that most of the accumulated damage is in the form of strand breaks as opposed to alkali-labile base damage. Chondrocytes from young donors required 10 Gy irradiation to recapitulate the DNA damage present in chondrocytes from older donors. Given the potential for DNA damage to contribute to chondrocyte dysfunction and senescence, this study supports the investigation of mechanisms by which hypo-replicative cell types accumulate high levels of damage.


Assuntos
Cartilagem Articular , Osteoartrite , Adulto , Idoso , Envelhecimento/genética , Cadáver , Cartilagem Articular/metabolismo , Células Cultivadas , Condrócitos/metabolismo , Ensaio Cometa , Dano ao DNA/genética , Humanos , Pessoa de Meia-Idade , Osteoartrite/genética , Osteoartrite/metabolismo
9.
Nanotoxicology ; 16(3): 393-407, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35818303

RESUMO

The use of metal oxide nanoparticles (NPs) is steadily spreading, leading to increased environmental exposures to many organisms, including humans. To improve our knowledge of this potential hazard, we have evaluated the genotoxic risk of cerium oxide (CeO2NPs) and magnesium oxide (MgONPs) nanoparticle exposures using Drosophila as an in vivo assay model. In this study, two well-known assays, such as the wing somatic mutation and recombination test (wing-spot assay) and the single-cell gel electrophoresis test (comet assay) were used. As a novelty, and for the first time, changes in the expression levels of a wide panel of DNA repair genes were also evaluated. Our results indicate that none of the concentrations of CeO2NPs increased the total spot frequency in the wing-spot assay, while induction was observed at the highest dose of MgONPs. Regarding the comet assay, both tested NPs were unable to induce single DNA strand breaks or oxidative damage in DNA bases. Nevertheless, exposure to CeO2NPs induced significant increases in the expression levels of the Mlh1 and Brca2 genes, which are involved in the double-strand break repair pathway, together with a decrease in the expression levels of the MCPH1 and Rad51D genes. Regarding the effects of MgONPs exposure, the expression levels of the Ercc1, Brca2, Rad1, mu2, and stg genes were significantly increased, while Mlh1 and MCPH1 genes were decreased. Our results show the usefulness of our approach in detecting mild genotoxic effects by evaluating changes in the expression of a panel of genes involved in DNA repair pathways.


Assuntos
Cério , Nanopartículas Metálicas , Nanopartículas , Animais , Proteínas de Ciclo Celular , Cério/toxicidade , Ensaio Cometa , Proteínas do Citoesqueleto , DNA , Dano ao DNA , Drosophila , Humanos , Óxido de Magnésio/toxicidade , Nanopartículas Metálicas/toxicidade , Nanopartículas/toxicidade , Óxidos
10.
Artigo em Inglês | MEDLINE | ID: mdl-35649683

RESUMO

This short narrative review describes the use of the comet assay to evaluate the formation of genotoxic compounds in the gut lumen in human studies. The fecal water genotoxicity assay is based on ability of the gut content to induce genotoxicity in a cellular model, employing the aqueous component of the feces (fecal water) as this is supposed to contain most of the reactive species and to convey them to the intestinal epithelium. This non-invasive and low-cost assay has been demonstrated to be associated with colon cancer risk in animal models, and although the final validation against human tumors is lacking, it is widely used as a colo-rectal cancer risk biomarker in human nutritional intervention studies. The contribution given to the field of nutrition and cancer by the FW genotoxicity assay is highlighted, particularly in conjunction with other risk biomarkers, to shed light on the complex relationship among diet, microbiota, individual subject characteristics and the formation of genotoxic compounds in the gut.


Assuntos
Neoplasias do Colo , Animais , Biomarcadores , Neoplasias do Colo/genética , Ensaio Cometa , Humanos , Água
11.
J Toxicol Sci ; 47(6): 221-235, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35650139

RESUMO

Flavonoids such as quercetin and its glucosides, especially isoquercitrin are well known as anti-inflammatory, anti-allergic, and anti-carcinogenic, etc. The safety of isoquercitrin formulations needs to be established prior to their use in functional food applications. The mutagenicity and genotoxicity of the IQC-γCD inclusion complex were assessed with three standard assays of the bacterial reverse mutation assay (Ames test) and using a combined in-vivo micronucleus and comet assay under the Organisation for Economic Co-operation and Development (OECD) guidelines. In combined rat bone marrow micronucleus and rat liver comet assay performed in male Sprague Dawley (SD) rats, the various doses of IQC-γCD inclusion complex (max. 2000 mg/kg bw) and positive controls ethyl methanesulfonate (EMS) and mitomycin C (MMC), respectively, and negative control (vehicle) were administrated. The results of the Salmonella typhimurium mutagenicity assay (strains TA100, TA1535, WP2uvrA, TA98, and TA1537) after exposure to the IQC-γCD inclusion complex with the absence and presence of the metabolic activation system (S9 fraction from rat liver) revealed a weakly positive response but with no biologically relevant mutagenicity at the conditions examined according to recommended regulatory guidelines. The combined micronucleus and comet assay results reveal that the IQC-γCD inclusion complex did not induce in-vivo genotoxic potential or indication of any oxidative DNA damage in rat liver tissues. Altogether, considering the results of the study, it is unlikely that the consumption of IQC-γCD inclusion complex as food or supplement would present any concern for humans regarding the mutagenicity and genotoxicity.


Assuntos
Mutagênicos , gama-Ciclodextrinas , Animais , Ensaio Cometa , Dano ao DNA , Masculino , Testes para Micronúcleos/métodos , Testes de Mutagenicidade/métodos , Mutagênicos/toxicidade , Quercetina/análogos & derivados , Quercetina/toxicidade , Ratos , Ratos Sprague-Dawley
12.
Int J Toxicol ; 41(4): 297-311, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35658642

RESUMO

DNA damage is an established initiating event in the mutagenicity and carcinogenicity of genotoxic chemicals. Accordingly, assessment of this endpoint is critical for chemicals which are being developed for use in humans. To assess the ability of the Chicken Egg Genotoxicity Assay (CEGA) to detect genotoxic pharmaceuticals, a set of 23 compounds with different pharmacological and reported genotoxic effects was tested for the potential to produce nuclear DNA adducts and strand breaks in the embryo-fetal livers using the 32P-nucleotide postlabeling (NPL) and comet assays, respectively. Due to high toxicity, two aneugens, colchicine and vinblastine, and an autophagy inhibitor, hydroxychloroquine, could not be evaluated. Out of the 20 remaining pharmaceuticals, 10 including estrogen modulators, diethylstilbestrol and tamoxifen, antineoplastics cyclophosphamide, etoposide, and mitomycin C, antifungal griseofulvin, local anesthetics lidocaine and prilocaine, and antihistamines diphenhydramine and doxylamine, yielded clear positive outcomes in at least one of the assays. The antihypertensive vasodilator hydralazine and antineoplastics streptozotocin and teniposide, produced only DNA strand breaks, which were not dose-dependent, and thus, the results with these 3 pharmaceuticals were considered equivocal. No DNA damage was detected for 7 compounds, including the purine antagonist 6-thioguanine, antipyretic analgesics acetaminophen and phenacetin, antibiotic ciprofloxacin, antilipidemic clofibrate, anti-inflammatory ibuprofen, and sedative phenobarbital. However, low solubility of these compounds limited dosages tested in CEGA. Overall, results in CEGA were largely in concordance with the outcomes in other systems in vitro and in vivo, indicating that CEGA provides reliable detection of DNA damaging activity of genotoxic compounds. Further evaluations with a broader set of compounds would support this conclusion.


Assuntos
Galinhas , Dano ao DNA , Animais , Ensaio Cometa/métodos , Adutos de DNA , Humanos , Testes de Mutagenicidade/métodos , Mutagênicos/toxicidade , Preparações Farmacêuticas
13.
Molecules ; 27(12)2022 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-35744887

RESUMO

In this paper, we present for the first time the evaluation of cytotoxicity and genotoxicity of de novo synthesized pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine sulfonamides MM129, MM130, and MM131 in human tumor cell lines: HeLa, HCT 116, PC-3, and BxPC-3. Cytotoxic and genotoxic properties of the tested compounds were estimated using the MTT assay, comet assay (alkaline and neutral version), and γ-H2AX immuno-staining. Examined sulfonamides exhibited strong anticancer properties towards tested cells in a very low concentration range (IC50 = 0.17-1.15 µM) after 72 h exposure time. The results of the alkaline and neutral version of the comet assay following 24 h incubation of the cells with tested compounds demonstrated the capability of heterocycles to induce significant DNA damage in exposed cells. HCT 116 cells were the most sensitive to the genotoxic activity of novel tricyclic pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine sulfonamides in the neutral version of the comet assay. Immunocytochemical detection of γ-H2AX showed an increase in DNA DSBs level in the HCT 116 cell line, after 24 h incubation with all tested compounds, confirming the results obtained in the neutral comet assay. Among all investigated compounds, MM131 showed the strongest cytotoxic and genotoxic activity toward all tested cell types. In conclusion, our results suggest that MM129, MM130, and MM131 exhibit high cytotoxic and genotoxic potential in vitro, especially towards the colorectal cancer cell line HCT 116. However, further investigations and analyses are required for their future implementation in the field of medicine.


Assuntos
Antineoplásicos , Triazinas , Antineoplásicos/química , Linhagem Celular Tumoral , Ensaio Cometa , Dano ao DNA , Humanos , Sulfanilamida , Sulfonamidas/farmacologia , Triazinas/química , Triazinas/farmacologia
14.
Environ Mol Mutagen ; 63(4): 204-214, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35527348

RESUMO

The study explicates the genotoxic effects of commercial grade cypermethrin on peripheral erythrocytes of Catla catla, chronically exposed to two environmentally relevant concentrations. The fish was treated with sub-lethal concentrations 0.12 µg/L and 0.41 µg/L (1/10th and /1/3rd of 96 h LC50 ) of cypermethrin for 45 days. DNA damage in the exposed fish was assessed using alkaline comet assay, presence of micronuclei (MN), erythrocyte nuclear and cytoplasmic abnormalities. Exposure to cypermethrin induced a dose-dependent increase in percent DNA damage, micronucleus frequency and erythrocyte abnormalities. Nuclear anomalies such as notched nuclei, lobed nuclei, bridged nuclei, and deformed nuclei; and cytoplasmic anomalies like anisochromasia, vacuolated cytoplasm, lobed cells, and echinocytes were observed. The findings revealed the genotoxic potential of commercial formulations pyrethroid cypermethrin at concentrations found in the environment and their potential deleterious effects on nontarget aquatic organisms.


Assuntos
Cyprinidae , Piretrinas , Poluentes Químicos da Água , Animais , Ensaio Cometa , Cyprinidae/genética , Dano ao DNA , Eritrócitos , Testes para Micronúcleos , Piretrinas/toxicidade , Poluentes Químicos da Água/toxicidade
15.
Chemosphere ; 303(Pt 2): 134914, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35588874

RESUMO

During the decommissioning and removal of radioactive material in nuclear facilities, fine, tritiated dusts of stainless steel, cement or tungsten are generated that could be accidently released to the environment. However, the potential radio- and ecotoxicological effects these tritiated particles may have are unknown. In this study, stainless steel particles (SSPs) representative of those likely to be tritiated are manufactured by hydrogenation and their tissue-specific bioaccumulation, release (depuration) and subsequent genotoxic response have been studied in the marine mussel, Mytilus galloprovincialis, as a baseline for future assessments of the potential effects of tritiated SSPs. Exposure to 1000 µg L-1 of SSPs and adopting Cr as a proxy for stainless steel revealed relatively rapid accumulation (∼5 h) in the various mussel tissues but mostly in the digestive gland. Over longer periods up to 18 days, SSPs were readily rejected and egested as faecal material. DNA strand breaks, as a measure of genotoxicity, were determined at each time point in mussel haemocytes using single cell gel electrophoresis, or the comet assay. Lack of chemical genotoxicity was attributed to the rapid processing of SSP particles and limited dissolution of elemental components of steel. Further work employing tritiated SSPs will enable radio-toxicology to be studied without the confounding effects of chemical toxicity.


Assuntos
Mytilus , Aço Inoxidável , Animais , Bioacumulação , Ensaio Cometa/métodos , Dano ao DNA
16.
Microsc Res Tech ; 85(9): 3095-3103, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35608124

RESUMO

The present study aimed to evaluate the genotoxic potential of cerium oxide (CeO2 ), magnesium oxide (MgO) nanoparticles and their ionic forms by alkaline comet assay. Eisenia hortensis were exposed to different series of concentrations (25, 50, 100, 200, and 400 µg/ml) of chemicals for 48 h to find LC50 . The LC50 for MgO and CeO2 NPs were 70 and 80 µg/ml. Whereas, the LC50 for their ionic forms were 50 and 70 µg/ml. To assess the potential DNA damage caused by the chosen chemicals, E. hortensis was further exposed for 48 h to the following concentrations, based on their respective LC50s : LC50/2 , LC50 , and 2xLC50 . Comet scores demonstrated the significant increase (p < 0.05) in DNA damage at all concentrations, both for NPs and ionic forms in a concentration-dependent manner. Findings of the present study revealed the genotoxic effects of CeO2 NPs, MgO NPs and their ionic forms on E. hortensis. RESEARCH HIGHLIGHTS: Genotoxic assessment of CeO2 and MgO NPs and their ionic forms was conducted. Characterization of NPs through electron microscopy and alkaline comet assay was performed on E. Hortensis. Highest DNA damage of CeO2 and MgO NPs was observed on earthworm.


Assuntos
Cério , Nanopartículas , Oligoquetos , Animais , Cério/toxicidade , Ensaio Cometa , Dano ao DNA , Magnésio/toxicidade , Óxido de Magnésio/farmacologia , Nanopartículas/toxicidade , Oligoquetos/genética
17.
Toxicol Appl Pharmacol ; 446: 116065, 2022 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-35568224

RESUMO

Despite of the global contamination and ubiquitous exposure to nitenpyram (NIT), little knowledge is available on the adverse effects to human health, with some evidence referring to its genotoxic potency to non-target organisms and esophageal squamous papilloma in rats. Human bone marrow mesenchymal stem cells (hBMSCs) was employed as an in vitro model more relevant to humans to assess the potential genotoxicity of NIT and to understand the underlying mechanisms at cellular and molecular levels. Noncytotoxic concentrations of NIT, 50-2500 µg/mL, dose-dependently elevated micronucleus (MN) and nuclear bud (NB) frequencies to 8.7-29‰ and 15-35‰, respectively. Additional metabolism by rat liver S9 fraction decreased chromosome impairment by 27-52% on MN frequencies and 63-76% on NB frequencies. A commercial NIT product, containing 20% of NIT and 60% of pymetrozine, caused higher cytotoxicity and chromosome impairment in comparison with NIT alone. Expressions of genes responses to DNA damage, ATM, ATR, p53, p21, Bax, H2AX, and GADD45A were disturbed by NIT treatment. Reactive oxygen species (ROS) amount and superoxide dismutase (SOD) activity were enhanced by NIT. Comet assay showed that lower concentrations of NIT, 12.5-100 µg/mL, induced the DNA damage. Transcriptomic analysis identified 468 differentially expressed genes (p < 0.05, |log2(Foldchange)| ≥ 1), from which 22 pathways were enriched. Multiple affected pathways were related to cancer including viral carcinogenesis and bladder cancer. NIT may produce genotoxicity via inducing oxidative stress and deregulating PI3K/Akt, AMPK and mTOR signaling pathways, associated with carcinogenetic potency. While environmental levels of NIT alone may pose little risk to human health, attention should be paid to the health risk arose from the synergistic or additive effects that may exist among NEOs and other types of pesticides.


Assuntos
Células-Tronco Mesenquimais , Neonicotinoides , Transcriptoma , Animais , Ensaio Cometa , Dano ao DNA/efeitos dos fármacos , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Testes para Micronúcleos , Neonicotinoides/farmacologia , Neonicotinoides/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Fosfatidilinositol 3-Quinases , Ratos , Transcriptoma/efeitos dos fármacos
18.
Clin Exp Immunol ; 209(1): 83-89, 2022 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-35511492

RESUMO

STAT3 plays an important role in various complex and sometimes contradictory pathways such as proliferation, differentiation, migration, inflammation, and apoptosis. The transcriptional activity of the STAT3 gene is controlled by a transcription factor called ZNF341. There is insufficient data on radiation sensitivity and post-radiation DNA repair in STAT3- loss-of-function (LOF) patients. We aimed to investigate the radiosensitivity in patients with STAT3-LOF and ZNF341 deficiency. Twelve patients with STAT3-LOF and four ZNF341-deficiency patients were recruited from three clinical immunology centers in Turkey and evaluated for radiosensitivity by the Comet assay, comparing to 14 age- and sex-matched healthy controls. The tail length (TL) (µm), percentage of DNA in the tail (TDNA%), and olive tail moment (OTM) (arbitrary units) were evaluated at the same time for baseline (spontaneous), initial (immediately after 2 Gy irradiation), and recovery (2 h after irradiation) periods by using a computerized image-analysis system, estimating DNA damage. Except for a patient with ZNF341 deficiency who developed nasal cell primitive neuroendocrine tumor and papillary thyroid cancer during the follow-up, there was no cancer in both groups. During the recovery period of irradiation, TL, TDNA%, and OTM values of healthy controls decreased rapidly toward the baseline, while these values of patients with STAT3-LOF and ZNF341 deficiency continued to increase, implying impaired DNA repair mechanisms. Increased radiosensitivity and impaired DNA repair were demonstrated in patients diagnosed with STAT3-LOF and ZNF341 deficiency, potentially explaining the susceptibility to malignant transformation.


Assuntos
Reparo do DNA , Tolerância a Radiação , Fator de Transcrição STAT3 , Fatores de Transcrição , Ensaio Cometa , Dano ao DNA/genética , Reparo do DNA/genética , Regulação da Expressão Gênica , Humanos , Mutação com Perda de Função , Tolerância a Radiação/genética , Fator de Transcrição STAT3/genética , Fatores de Transcrição/genética
19.
Drug Deliv Transl Res ; 12(9): 2243-2258, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35612707

RESUMO

Due to their unique chemical and physical properties, nanobiomaterials (NBMs) are extensively studied for applications in medicine and drug delivery. Despite these exciting properties, their small sizes also make them susceptible to toxicity. Whilst nanomaterial immunotoxicity and cytotoxicity are studied in great depth, there is still limited data on their potential genotoxicity or ability to cause DNA damage. In the past years, new medical device regulations, which came into place in 2020, were developed, which require the assessment of long-term NBM exposure; therefore, in recent years, increased attention is being paid to genotoxicity screening of these materials. In this article, and through an interlaboratory comparison (ILC) study conducted within the Horizon 2020 REFINE project, we assess five different NBM formulations, each with different uses, namely, a bio-persistent gold nanoparticle (AuNP), an IR-780 dye-loaded liposome which is used in deep tissue imaging (LipImage™815), an unloaded PACA polymeric nanoparticle used as a drug delivery system (PACA), and two loaded PACA NBMs, i.e. the cabazitaxel drug-loaded PACA (CBZ-PACA) and the NR668 dye-loaded PACA (NR668 PACA) for their potential to cause DNA strand breaks using the alkaline comet assay and discuss the current state of genotoxicity testing for nanomaterials. We have found through our interlaboratory comparison that the alkaline comet assay can be suitably applied to the pre-clinical assessment of NBMs, as a reproducible and repeatable methodology for assessing NBM-induced DNA damage. Workflow for assessing the applicability of the alkaline comet assay to determine nanobiomaterial (NBM)-induced DNA strand breaks, through an interlaboratory comparison study (ILC).


Assuntos
Ouro , Nanopartículas Metálicas , Ensaio Cometa/métodos , DNA , Dano ao DNA , Nanopartículas Metálicas/toxicidade
20.
J Vis Exp ; (183)2022 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-35635461

RESUMO

Cells are continually exposed to agents arising from the internal and external environments, which may damage DNA. This damage can cause aberrant cell function, and therefore DNA damage may play a critical role in the development of, conceivably, all major human diseases, e.g., cancer, neurodegenerative and cardiovascular disease, and aging. Single-cell gel electrophoresis (i.e., the comet assay) is one of the most common and sensitive methods to study the formation and repair of a wide range of types of DNA damage (e.g., single- and double-strand breaks, alkali-labile sites, DNA-DNA crosslinks, and, in combination with certain repair enzymes, oxidized purines, and pyrimidines), in both in vitro and in vivo systems. However, the low sample throughput of the conventional assay and laborious sample workup are limiting factors to its widest possible application. With the "scoring" of comets increasingly automated, the limitation is now the ability to process significant numbers of comet slides. Here, a high-throughput (HTP) variant of the comet assay (HTP comet assay) has been developed, which significantly increases the number of samples analyzed, decreases assay run time, the number of individual slide manipulations, reagent requirements, and risk of physical damage to the gels. Furthermore, the footprint of the electrophoresis tank is significantly decreased due to the vertical orientation of the slides and integral cooling. Also reported here is a novel approach to chilling comet assay slides, which conveniently and efficiently facilitates the solidification of the comet gels. Here, the application of these devices to representative comet assay methods has been described. These simple innovations greatly support the use of the comet assay and its application to areas of study such as exposure biology, ecotoxicology, biomonitoring, toxicity screening/testing, together with understanding pathogenesis.


Assuntos
Dano ao DNA , Reparo do DNA , Ensaio Cometa/métodos , DNA/análise , Humanos , Testes de Toxicidade
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