Topic
Genotoxicity
About: Genotoxicity is a research topic. Over the lifetime, 10950 publications have been published within this topic receiving 330929 citations. The topic is also known as: genotoxic & genetic toxicology.
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TL;DR: Human lymphocytes were exposed to X-irradiation or treated with H2O2 and the extent of DNA migration was measured using a single-cell microgel electrophoresis technique under alkaline conditions and this technique appears to be sensitive and useful for detecting damage and repair in single cells.
10,170 citations
TL;DR: The expert panel reached a consensus that the optimal version of the Comet assay for identifying agents with genotoxic activity was the alkaline (pH > 13) versions of the assay developed by Singh et al.
Abstract: Atthe International Workshop on Genotoxicity Test Procedures (IWGTP) held in Washington, DC, March 25-26, 1999, an expert panel met to develop guidelines for the use of the single-cell gel (SCG)/Comet assay in genetic toxicology. The expert panel reached a consensus that the optimal version of the Comet assay for identifying agents with genotoxic activity was the alkaline (pH > 13) version of the assay developed by Singh et al. [1988]. The pH > 13 version is capable of detecting DNA single-strand breaks (SSB), alkali-labile sites (ALS), DNA-DNA/DNA-protein cross-linking, and SSB associated with incomplete excision repair sites. Relative to other genotoxicity tests, the advantages of the SCG assay include its demonstrated sensitivity for detecting low levels of DNA damage, the requirement for small numbers of cells per sample, its flexibility, its low costs, its ease of application, and the short time needed to complete a study. The expert panel decided that no single version of the alkaline (pH > 13) Comet assay was clearly superior. However, critical technical steps within the assay were discussed and guidelines developed for preparing slides with agarose gels, lysing cells to liberate DNA, exposing the liberated DNA to alkali to produce single-stranded DNA and to express ALS as SSB, electrophoresing the DNA using pH > 13 alkaline conditions, alkali neutralization, DNA staining, comet visualization, and data collection. Based on the current state of knowledge, the expert panel developed guidelines for conducting in vitro or in vivo Comet assays. The goal of the expert panel was to identify minimal standards for obtaining reproducible and reliable Comet data deemed suitable for regulatory submission. The expert panel used the current Organization for Economic Co-operation and Development (OECD) guidelines for in vitro and in vivo genetic toxicological studies as guides during the development of the corresponding in vitro and in vivo SCG assay guidelines. Guideline topics considered included initial considerations, principles of the test method, description of the test method, procedure, results, data analysis and reporting. Special consideration was given by the expert panel to the potential adverse effect of DNA degradation associated with cytotoxicity on the interpretation of Comet assay results. The expert panel also discussed related SCG methodologies that might be useful in the interpretation of positive Comet data. The related methodologies discussed included: (1) the use of different pH conditions during electrophoreses to discriminate between DNA strand breaks and ALS; (2) the use of repair enzymes or antibodies to detect specific classes of DNA damage; (3) the use of a neutral diffusion assay to identify apoptotic/necrotic cells; and (4) the use of the acellular SCG assay to evaluate the ability of a test substance to interact directly with DNA. The alkaline (pH > 13) Comet assay guidelines developed by the expert panel represent a work in progress. Additional information is needed before the assay can be critically evaluated for its utility in genetic toxicology. The information needed includes comprehensive data on the different sources of variability (e.g., cell to cell, gel to gel, run to run, culture to culture, animal to animal, experiment to experiment) intrinsic to the alkaline (pH > 3) SCG assay, the generation of a large database based on in vitro and in vivo testing using these guidelines, and the results of appropriately designed multilaboratory international validation studies.
4,583 citations
TL;DR: A possible mechanism of toxicity is proposed which involves disruption of the mitochondrial respiratory chain by Ag-np leading to production of ROS and interruption of ATP synthesis, which in turn cause DNA damage.
Abstract: Silver nanoparticles (Ag-np) are being used increasingly in wound dressings, catheters, and various household products due to their antimicrobial activity. The toxicity of starch-coated silver nanoparticles was studied using normal human lung fibroblast cells (IMR-90) and human glioblastoma cells (U251). The toxicity was evaluated using changes in cell morphology, cell viability, metabolic activity, and oxidative stress. Ag-np reduced ATP content of the cell caused damage to mitochondria and increased production of reactive oxygen species (ROS) in a dose-dependent manner. DNA damage, as measured by single cell gel electrophoresis (SCGE) and cytokinesis blocked micronucleus assay (CBMN), was also dose-dependent and more prominent in the cancer cells. The nanoparticle treatment caused cell cycle arrest in G2/M phase possibly due to repair of damaged DNA. Annexin-V propidium iodide (PI) staining showed no massive apoptosis or necrosis. The transmission electron microscopic (TEM) analysis indicated the presen...
3,261 citations
TL;DR: The comet assay (single-cell gel electrophoresis) is a simple method for measuring deoxyribonucleic acid (DNA) strand breaks in eukaryotic cells that has applications in testing novel chemicals for genotoxicity, monitoring environmental contamination with genotoxins, human biomonitoring and molecular epidemiology, and fundamental research in DNA damage and repair.
Abstract: The comet assay (single-cell gel electrophoresis) is a simple method for measuring deoxyribonucleic acid (DNA) strand breaks in eukaryotic cells. Cells embedded in agarose on a microscope slide are lysed with detergent and high salt to form nucleoids containing supercoiled loops of DNA linked to the nuclear matrix. Electrophoresis at high pH results in structures resembling comets, observed by fluorescence microscopy; the intensity of the comet tail relative to the head reflects the number of DNA breaks. The likely basis for this is that loops containing a break lose their supercoiling and become free to extend toward the anode. The assay has applications in testing novel chemicals for genotoxicity, monitoring environmental contamination with genotoxins, human biomonitoring and molecular epidemiology, and fundamental research in DNA damage and repair. The sensitivity and specificity of the assay are greatly enhanced if the nucleoids are incubated with bacterial repair endonucleases that recognize specific kinds of damage in the DNA and convert lesions to DNA breaks, increasing the amount of DNA in the comet tail. DNA repair can be monitored by incubating cells after treatment with damaging agent and measuring the damage remaining at intervals. Alternatively, the repair activity in a cell extract can be measured by incubating it with nucleoids containing specific damage.
2,580 citations
TL;DR: In its current basic form the CBMN assay can provide the following measures of genotoxicity and cytotoxicity: chromosome breakage, chromosome loss, chromosome rearrangement, cell division inhibition, necrosis and apoptosis.
Abstract: The study of DNA damage at the chromosome level is an essential part of genetic toxicology because chromosomal mutation is an important event in carcinogenesis. The micronucleus assays have emerged as one of the preferred methods for assessing chromosome damage because they enable both chromosome loss and chromosome breakage to be measured reliably. Because micronuclei can only be expressed in cells that complete nuclear division a special method was developed that identifies such cells by their binucleate appearance when blocked from performing cytokinesis by cytochalasin-B (Cyt-B), a microfilament-assembly inhibitor. The cytokinesis-block micronucleus (CBMN) assay allows better precision because the data obtained are not confounded by altered cell division kinetics caused by cytotoxicity of agents tested or sub-optimal cell culture conditions. The method is now applied to various cell types for population monitoring of genetic damage, screening of chemicals for genotoxic potential and for specific purposes such as the prediction of the radiosensitivity of tumours and the inter-individual variation in radiosensitivity. In its current basic form the CBMN assay can provide, using simple morphological criteria, the following measures of genotoxicity and cytotoxicity: chromosome breakage, chromosome loss, chromosome rearrangement (nucleoplasmic bridges), cell division inhibition, necrosis and apoptosis. The cytosine-arabinoside modification of the CBMN assay allows for measurement of excision repairable lesions. The use of molecular probes enables chromosome loss to be distinguished from chromosome breakage and importantly non-disjunction in non-micronucleated binucleated cells can be efficiently measured. The in vitro CBMN technique, therefore, provides multiple and complementary measures of genotoxicity and cytotoxicity which can be achieved with relative ease within one system. The basic principles and methods (including detailed scoring criteria for all the genotoxicity and cytotoxicity end-points) of the CBMN assay are described and areas for future development identified.
2,287 citations