Genomic instability is a defining characteristic of cancer and the analysis of DNA damage at the chromosome level is a crucial part of the study of carcinogenesis and genotoxicity. Chromosomal ...instability (CIN), the most common level of genomic instability in cancers, is defined as the rate of loss or gain of chromosomes through successive divisions. As such, DNA in cancer cells is highly unstable. However, the underlying mechanisms remain elusive. There is a debate as to whether instability succeeds transformation, or if it is a by-product of cancer, and therefore, studying potential molecular and cellular contributors of genomic instability is of high importance. Recent work has suggested an important role for ectopic expression of meiosis genes in driving genomic instability via a process called meiomitosis. Improving understanding of these mechanisms can contribute to the development of targeted therapies that exploit DNA damage and repair mechanisms. Here, we discuss a workflow of novel and established techniques used to assess chromosomal instability as well as the nature of genomic instability such as double strand breaks, micronuclei, and chromatin bridges. For each technique, we discuss their advantages and limitations in a lab setting. Lastly, we provide detailed protocols for the discussed techniques.
Ionizing radiations are harmful since they generate free radicals which through a series of reactions may cause damage to genetic material, imbalance in the antioxidant system, or lead to death. ...Lutein; a carotenoid compound because of its various medicinal properties has been chosen to evaluate its protection against radiation-induced damages. Swiss albino mice were divided into 8 groups of 6 mice each. Irradiation groups received whole-body radiation of 6Gy using an electron beam accelerator. Mice were fed with water/gallic acid/10% dimethyl sulfoxide (DMSO) (w/v)/ lutein respectively. The mice were sacrificed on the 16th day; whole blood was collected in 2% ethylene diamine tetraacetic acid (EDTA) tubes by cardiac puncture method for hematological studies and comet assay. The organs like the brain, lungs, and liver were dissected. Organ homogenates were prepared to perform the antioxidant assay. The femur of the leg was removed and flushed for micronucleus assay. Lutein post-treatment showed significantly increased superoxide dismutase activity in the lung homogenate in comparison to the radiation control. A significant increase in the levels of glutathione was observed in lutein post-treatment when compared to the lutein control in lung homogenate. Also in the lung homogenate of lutein post-treatment, a significant decrease in the levels of malondialdehyde was observed in comparison to the lutein control, and a similar effect was observed in the vehicle control groups. In the comet assay, it was observed that tail moment decreased significantly in lutein post-treatment when compared to its control group whereas no significant changes were observed with %DNA in tail and olive moment. Lutein treatment post-radiation has increased the polychromatic erythrocytes (PCE)/ (PCE+ normochromic erythrocytes (NCE) ratio. Significant changes were observed in the lutein post-treatment group with respect to antioxidant status. Micronucleus assay reveals that lutein treatment post-radiation increases cell multiplication. These results indicate a potent mitigator effect of lutein against radiation-induced antioxidant changes in vivo.
Humans ingest particles and fibers on daily basis. Non-digestible carbohydrates are beneficial to health and food additives are considered safe. However, titanium dioxide (E171) has been banned in ...the European Union because the European Food Safety Authority no longer considers it non-genotoxic. Ingestion of microplastics and nanoplastics are novel exposures; their potential hazardous effects to humans have been under the radar for many years. In this review, we have assessed the association between oral exposure to man-made particles/fibers and genotoxicity in gastrointestinal tract cells and secondary tissues. We identified a total of 137 studies on oral exposure to particles and fibers. This was reduced to 49 papers with sufficient quality and relevance, including exposures to asbestos, diesel exhaust particles, titanium dioxide, silver nanoparticles, zinc oxide, synthetic amorphous silica and certain other nanomaterials. Nineteen studies show positive results, 25 studies show null results, and 5 papers show equivocal results on genotoxicity. Recent studies seem to show null effects, whereas there is a higher proportion of positive genotoxicity results in early studies. Genotoxic effects seem to cluster in studies on diesel exhaust particles and titanium dioxide, whereas studies on silver nanoparticles, zinc oxide and synthetic amorphous silica seem to show mainly null effects. The most widely used genotoxic tests are the alkaline comet assay and micronucleus assay. There are relatively few results on genotoxicity using reliable measurements of oxidatively damaged DNA, DNA double strand breaks (γH2AX assay) and mutations. In general, evidence suggest that oral exposure to particles and fibers is associated with genotoxicity in animals.
•The review summarizes genotoxic effects of multi-walled carbon nanotubes.•Studies on NM-400, NM-401, NM-402, NM-403 and MWCNT-7 have been assessed.•Meta-analysis show generation of DNA strand breaks ...by MWCNT-7 in cell cultures.•Strong evidence of genotoxicity by MWCNT-7 in cell culture studies.•Knowledge gaps or inconclusive genotoxicity results by materials other than MWCNT-7.
Carbon nanotubes (CNTs) were the first nanomaterials to be evaluated by the International Agency for Research on Cancer (IARC). The categorization as possibly carcinogenic agent to humans was only applicable to multi-walled carbon nanotubes called MWCNT-7. Other types of CNTs were not classifiable because of missing data and it was not possible to pinpoint unique CNT characteristics that cause cancer. Importantly, the European Commission’s Joint Research Centre (JRC) has established a repository of industrially manufactured nanomaterials that encompasses at least four well-characterized MWCNTs called NM-400 to NM-403 (original JRC code). This review summarizes the genotoxic effects of these JRC materials and MWCNT-7. The review consists of 36 publications with results on cell culture experiments (22 publications), animal models (9 publications) or both (5 publications). As compared to the publications in the IARC monograph on CNTs, the current database represents a significant increase as there is only an overlap of 8 publications. However, the results come mainly from cell cultures and/or measurements of DNA strand breaks by the comet assay and the micronucleus assay (82 out of 97 outcomes). A meta-analysis of cell culture studies on DNA strand breaks showed a genotoxic response by MWCNT-7, less consistent effect by NM-400 and NM-402, and least consistent effect by NM-401 and NM-403. Results from other in vitro tests indicate strongest evidence of genotoxicity for MWCNT-7. There are too few observations from animal models and humans to make general conclusions about genotoxicity.
Pendimethalin and trifluralin are widely used dinitroaniline herbicides. Both compounds can be found as residue levels in agricultural products. This study was conducted in order to provide necessary ...information for the risk assessment of pendimethalin and trifluralin. In this study, reactive oxygen species (ROS) levels were measured to examine the potential of both compounds to induce oxidative damage in Chinese hamster lung fibroblast (V79) cells. Also, the genotoxic effects of pendimethalin and trifluralin at the concentration range of 1-500 μM was determined. Single cell gel electrophoresis (comet) and micronucleus assays were used on human peripheral lymphocytes and V79 cells for the genotoxicity assessment. The cell viability of two dinitroaniline herbicides were determined by the use of neutral red uptake assay on V79 cells. IC50 values were determined as 66 μM and 128 μM for pendimethalin and trifluralin, respectively. They significantly increased ROS levels on V79 cells for 1-24 h. Both herbicides significantly induced the DNA damage and showed genotoxicity on lymphocytes and V79 cells. Micronucleus frequency increased significantly after pendimethalin and trifluralin treatment of the lymphocytes and V79 cells. Therefore, we concluded that both of the herbicides induced the genotoxicity through the activation of oxidative stress pathway and chromosomal damage.
•IC50 values of pendimethalin in V79 cell lines were 66 μM.•IC50 values of trifluralin in V79 cell lines were 128 μM.•Dinitroaniline herbicides pendimethalin and trifluralin induced the DNA damage.•Oxidative stress and chromosomal damage might have a role on the genotoxicity.
Abstract
A novel in vitro 3D micronucleus assay was developed in China using the EpiSkin™ 3D human skin model. This EpiSkin™ Micronucleus Assay showed good predictivity and reproducibility during ...internal validation and is expected to contribute to in vitro genotoxicity testing as a follow-up for positive results from 2D micronucleus assay. Having developed the assay in one laboratory, further work focused on the transferability and inter-laboratory reproducibility in two additional Chinese authority laboratories (Guangdong Provincial Center for Disease Control and Prevention and Zhejiang Institute for Food and Drug Control). Formal training was provided for both laboratories, which resulted in good transferability based on the results of two positive compounds, such as mitomycin C and vinblastine. Independent experiments were then performed, and inter-laboratory reproducibility was checked using 2-acetylaminofluorene, 5-fluorouracil, 2,4-dichlorophenol, and d-limonene. The dose-responses of the positive control chemical, mitomycin C, were similar to those of the developing laboratory, and all test chemicals were correctly classified by all laboratories. Overall, there was a good transferability as well as intra- and inter-laboratory reproducibility of the EpiSkin™ Micronucleus Assay. This study further confirmed the assay’s robustness and provided confidence to enter following validation stages for scientific acceptance.
Bromophenols, the building blocks of polybrominated flame retardants (PBFRs) constitute an important class of pollutants as they had been reported from different types of ecosystems across the earth. ...The improper disposal of PBFRs containing electronic waste (e-waste) and wastes from industrial units is of concern due to their recalcitrant nature and acute toxicity. In the current study Pseudomonas sp. EN-4 was able to metabolise 4-bromophenol (4-BP), a model representative of bromophenols in presence of phenol as a co-substrate. The isolate withstand the extreme pH (5.0–10.0) and salinity conditions (0.5–2% w/v) and maintain its degradation potential in presence of different metal ions prevalent at contaminated sites. The HPLC and MS analysis of biotransformed samples revealed the formation of catechol, which might get channelized to the central metabolic pathway by catechol 1,2-dioxygenase mediated ortho-cleavage of the aromatic ring. Further, > 70 % transformation of 2-bromophenol (20 mg/L), 2,4,6-tribromophenol (20 mg/L) and 3,3’,5,5′-tetrabromobisphenol A (10 mg/L) indicates the metabolic versatility of strain EN-4. A significantly lower toxic potential of biologically treated samples on Vigna radiata and Channa punctatus validated the utility and efficacy of EN-4 cells. These findings lay the foundation for future studies in developing a site specific treatment system for bioremediation of waste generated by e-waste processing and other similar units.
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•Co-metabolic approach for efficient biotransformation of 4-BP was conducted.•4-BP degradation by Psdeudomonas sp. EN-4 was significantly enhanced with phenol.•Metabolic versatile EN-4, efficiently transform 2-BP, TBP and TBBPA.•A metabolic pathway of 4-BP degradation was proposed.•Significant lowering of phyto and genotoxicity in biotransformed samples of EN-4.
The species of Agrimonia and Filipendula have been traditionally used in folk medicine as anti-inflammatory herbs. This study extends the knowledge on bioactivities of F. palmata, A. eupatoria, A. ...procera, F. ulmaria and F. vulgaris by comprehensive characterization of their methanolic extracts. Antioxidant properties of extracts were evaluated by DPPH• (2,2-diphenyl-1-picrylhydrazyl), ABTS•+ 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) scavenging and oxygen radical absorbance capacities (ORAC). Genotoxicity of extracts was tested using alkaline single-cell gel electrophoresis (comet) and cytokinesis-block micronucleus assays in human lymphocytes in vitro and the Ames Salmonella/microsome test. All investigated Agrimonia and Filipendula extracts possessed strong antioxidant activity, which was comparable with that of a standard antioxidant trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid). Thirty five compounds belonging to the classes of phenolic acids, flavonoids, phenylpropanoids and ellagitanins were detected by ultra-performance liquid chromatography – mass spectrometry (UPLC-Q-TOF-MS). Agrimonia and Filipendula extracts induced an increase in a DNA damage in the comet assay expressed as mean percentage of DNA in the comet tail. However, these extracts did not produce reverse mutation in bacterial cells in the Ames test and were not genotoxic in the micronucleus test. However, a slight though significant decrease of nuclear division index values was determined. In general, this study proved that Agrimonia and Filipendula species are a good source of bioactive compounds; their extracts may be classified as non-mutagenic and non-clastogenic in vitro under conditions of the current study. Consequently, the plants may be a promising material for nutraceuticals and natural medicines.
•35 compounds were found in methanolic extracts of Agrimonia and Filipendula species.•The extracts were strong antioxidants in vitro, which was in a good correlation with their polyphenolic content.•The extracts were not mutagenic in the Ames Salmonella/microsome and micronucleus assays.•The extracts induced DNA damage evaluated by the comet assay.
The cytokinesis-block micronucleus assay is a well-established method to assess radiation-induced genetic damage in human cells. This assay has been adapted to imaging flow cytometry (IFC), allowing ...automated analysis of many cells, and eliminating the need to create microscope slides. Furthermore, to improve the efficiency of assay performance, a small-volume method previously developed was employed. Irradiated human blood samples were cultured, stained, and analyzed by IFC to produce images of the cells. Samples were run using both manual and 96-well plate automated acquisition. Multiple parameter-based image features were collected for each sample, and the results were compared to confirm that these acquisition methods are functionally identical. This paper details the multi-parametric analysis developed and the resulting calibration curves up to 10 Gy. The calibration curves were created using a quadratic random coefficient model with Poisson errors, as well as a logistic discriminant function. The curves were then validated with blinded, irradiated samples, using relative bias and relative mean square error. Overall, the accuracy of the dose estimates was adequate for triage dosimetry (within 1 Gy of the true dose) over 90% of the time for lower doses and about half the time for higher doses, with the lowest success rate between 5 and 6 Gy where the calibration curve reached its peak and there was the smallest change in MN/BNC with dose. This work describes the application of a novel multi-parametric analysis that fits the calibration curves and allows dose estimates up to 10 Gy, which were previously limited to 4 Gy. Furthermore, it demonstrates that the results from samples acquired manually and with the autosampler are functionally similar.
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