Use Of Comet Assay Research Articles

Four Decades of the Comet Assay: pH Optimum of Lysis Buffer Still Needs to be Elucidated.

The proper course and reproducibility of diagnostic techniques depend on narrowly defined reaction conditions, including the reaction pH. Nevertheless, numerous assays are affected by an inaccurately defined reaction pH. Buffers are sometimes suggested for use outside their useful pH ranges, which complicates the reproducibility of results because the buffering capacity is insufficient to retain the disclosed pH. Here, we focus on the comet assay lysis buffer. Comet assay is broadly used for quantifying DNA breaks in eukaryotic cells. The most widespread comet assay protocols employ lysis of the cells before electrophoresis in a buffer containing Triton X-100, a high concentration of NaCl, sodium sarcosinate, EDTA, and Tris, with some modifications. However, nearly all researchers report that they use Tris buffer at pH 10, and some report the pH of the Tris additive alone. Alternatively, others report the pH of the final lysis buffer. However, the lysis solution used in the comet assay is buffered at a pH outside the useful range of Tris. Tris-based buffers have a useful pH range of 7.0 - 9.0. The buffer composed of 10 mM Tris has pKa 8.10 at 25°C and 8.69 at 4°C. The cell lysis conditions used in nearly all modifications of comet assay protocols remain imprecise and uncritically employed. Despite the pH of the lysis buffer likely has negligible effect on the detection of DNA breaks, precise lysis conditions are highly important for the use of comet assay in the detection of base modifications, which are often unstable and sensitive to pH.

Use of Comet Assay and Micronucleus test as noninvasive biomarkers in workers occupationally exposed to TiO2

Use of Comet Assay and Micronucleus test as noninvasive biomarkers in workers occupationally exposed to TiO2

DNA damage in buccal cells in oral PMDs and malignant disorders by comet assay

Aim: DNA damage associated with Oral Squamous Cell Carcinoma (OSCC) and potentially malignant disorders (PMDs) is produced due to carcinogenic agents or increased oxidative stress. Comet assay can assist in early detection and evaluation of the amount of DNA damage; lymphocytesare the most commonly used cells for performing comet assay. Utilisation of buccal epithelial cells in comet assay can be a minimally invasive and rapid method. The present study compared the efficacy of comet assay in assessing DNA damage in buccal cells over peripheral blood leucocytes (PBLs) in oral potentially malignant and malignant disorders. Methods: The study included fifty five patients each of Leukoplakia, Oral Submucous Fibrosis (OSMF) and OSCC along with fifty five healthy individuals as control. Buccal epithelial cells were collected from all the selected subjects. DNA damage was evaluated bymeasuring the mean tail length (µm). Results: A significantly increased mean tail length (µm) and higher DNA damage were found in OSCC (26.1096 + 1.84355) and there was a progressive stepwise increase in mean tail length from control(8.4982 + 0.93307) to PMD [leukoplakia (14.6105 + 0.71857); OSMF (12.5009 + 1.12694)] to OSCC.The mean tail length in different habit groups was greater than controls, though no significant difference was noted between habit groups. The mean tail length of buccal cells was significantly greater than the mean tail length of PBLs in all study groups and controls. Conclusion: Hence, use of comet assay on buccal epithelial cells can prove to be beneficiary for evaluation of DNA damage.

DNA damage and repair measured by comet assay in cancer patients

The last decade witnessed an increase in the use of comet assay for DNA damage monitoring in cancer patients and controls. Apart from case-control studies, reports described the determination of DNA damage prior to (baseline value) and after chemo-/radiotherapy, the treatment resulted in significantly elevated DNA damage. However, studies on DNA damage as a factor reflecting cancer prognosis and therapy prediction are scarce. In most cases, DNA damage was analysed in surrogate tissues. The data on DNA damage are available for 17 types of cancer. The reviewed data unambiguously pinpoint the usefulness of the comet assay in human cancer research due to its sensitivity and cost-effectiveness in evaluating DNA damage associated with the disease and with the treatment.DNA repair capacity (DRC) represents a complex marker for functional evaluation of multigene DNA repair processes in cancer onset with future prospects in personalized prevention and/or cancer treatment. A comparison between studies and more general conclusions are precluded by a variable design of the studies and a lack of standard protocol for both DNA damage and DRC determination. Since cancer is a heterogeneous complex disease, numerous points have to be considered: a) DNA damage and DRC measured in surrogate/target tissues, b) changes in the levels of DNA damage and DRC may be a cause or a consequence of the disease, c) changes in DRC alter sensitivity of tumour cells to antineoplastic drugs, d) one time point-sampling of patients provides insufficient information on the role of DNA damage and its repair in carcinogenesis. Finally, systemic cancer therapy is targeted at DNA damage and its repair. A proper understanding of these processes is a key precondition for the optimisation of therapy regimens, prediction of therapeutic response and prognosis in cancer patients.

A systematic review of the use of the alkaline comet assay for genotoxicity studies in human colon-derived cells

This review describes the use of the comet assay for assessment of DNA damage in human colon cells. We screened 98 papers, which employed human colon -derived cells to analyse DNA damage induced by different insults with the comet assay. In most cases tumour cell lines were used, and only a few studies were performed with primary colon cells. The comet assay was mostly applied to test chemotherapeutics and natural products. We could not find a clear difference between the susceptibility of cell lines to genotoxic insults and they were all suitable for comet assay experiments. Further comparisons between cell lines, and with primary cells and stem cells would be desirable to understand the relevance of the established cell lines as model for the human target tissue better.

Genotoxicity assessment of pesticide profenofos in freshwater fish Channa punctatus (Bloch) using comet assay and random amplified polymorphic DNA (RAPD)

The present study explored the induced genotoxicity (DNA damage) due to organophosphate pesticide profenofos (PFF) after in vivo exposure in freshwater fish Channa punctatus by the use of Comet assay and Random amplified polymorphic DNA (RAPD). The fish specimens were exposed to sub-lethal concentration of 1.16 ppb (50% of LC50) in a semi-static system and the DNA damage was assessed in exposed and control fish. The DNA damage was measured in erythrocytes as the percentage of DNA damage in Comet tails and RAPD technique using oligonucleotide primers of fish specimens exposed to the sublethal concentrations of PFF. The most informative primers in terms of variation in RAPD profile were found to be OPA-01, OPA-03, OPB-02, OPB-01 and OPA-13. Appearance/disappearance of bands and increase/decrease in the band intensity were evident in the RAPD profile of fish specimens exposed to PFF as compared to the control. Findings from the present study suggest that the potential impacts of assessment of the genotoxic impact of pesticide on fish.

Detection of oxidative stress and DNA damage in freshwater snail Lymnea leuteola exposed to profenofos

Extensive production and use of organophosphate pesticide in agriculture, has risen concerned about its ecotoxicity and risk assessment of insecticides, which are more important. Therefore, the present investigation was aimed to study the induction of oxidative stress and DNA damage by organophosphate insecticide profenofos (PFF) in freshwater snail Lymnea luteola (L. luteola). The median lethal value (96 h LC50) of PFF was estimated as 1.26 mg/L for L. luteola in a semi-static system and on the basis of LC50 value three concentrations viz., 0.126 (1/10 of LC50, Sublethal I), 0.63 (1/2 of LC50, Sublethal II) and 0.84 mg/L (2/3 of LC50, Sublethal III) were determined. Snails were exposed to above-mentioned concentrations of PFF along with solvent control (acetone) and negative control for 96 h. The haemolymph was collected at 24 and 96 h of after treatment. In heamolymph of PFF exposed snail, lipid peroxide, glutathione reduced glutathione S transferase and superoxide dismutase activities at the tested concentrations significantly differ from those in the control. The genotoxicity induced in hemocytes of treated snails was measured by alkaline single cell gel electrophoresis assay. The data of this experiment demonstrated significantly enhancement of oxidative stress and DNA damage in the treated snails as compared to controls. Also, we observed statistically significant correlations of ROS with DNA damage (% tail DNA) (R2 = 0.9708) for 24 h and DNA damage (R2 = 0.9665) for 96 h. Results of the current experiment can be useful in risk evaluation of PFF among aquatic organisms. The study confirmed the use of comet assay for in vivo laboratory experiments using freshwater snail for selecting the toxic potential of industrial chemicals and environmental contaminants.

Human Papillomaviruses Preferentially Recruit DNA Repair Factors to Viral Genomes for Rapid Repair and Amplification.

ABSTRACTHigh-risk human papillomaviruses (HPVs) activate the ataxia telangiectasia mutated-dependent (ATM) DNA damage response as well as the ataxia telangiectasia mutated-dependent DNA-related (ATR) pathway in the absence of external DNA damaging agents for differentiation-dependent genome amplification. Through the use of comet assays and pulsed-field gel electrophoresis, our studies showed that these pathways are activated in response to DNA breaks induced by the viral proteins E6 and E7 alone and independently of viral replication. The majority of these virally induced DNA breaks are present in cellular DNAs and only minimally in HPV episomes. Treatment of HPV-positive cells with inhibitors of both ATM and ATR leads to the generation of DNA breaks and the fragmentation of viral episomes, indicating that DNA breaks are introduced into HPV genomes. These breaks, however, are rapidly repaired through the preferential recruitment of homologous recombination repair enzymes, such as RAD51 and BRCA1, to viral genomes at the expense of cellular DNAs. When HPV-positive cells are treated with hydroxyurea, this recruitment of RAD51 and BRCA1 to viral genomes is greatly enhanced with little recruitment to damaged cellular DNAs and with retention of the ability of viral genomes to amplify. Overall, our studies demonstrated that human papillomaviruses induce breaks into cellular and viral DNAs and that the preferential repair of these lesions in viral episomes leads to genome amplification.

Evaluating γH2AX in spermatozoa from male infertility patients

To investigate whether γH2AX levels were different in the spermatozoa of healthy men compared with infertility patients, and to assess the possible correlations between γH2AX and conventional semen parameters and double-stranded breaks (DSBs) identified with the use of comet assay. Prospective study. Clinical laboratory. Semen from 100 male infertile patients and 100 healthy sperm donors. Human sperm samples were analyzed in terms of World Health Organization parameters. The γH2AX levels were detected by means of flow cytometry. DSBs of sperm were detected by means of comet assay. Morphology slides were made and the sperm morphology assessed according to strict criteria. Conventional semen analyses, γH2AX levels in sperm, DNA DSBs in sperm, and correlations among γH2AX, conventional semen analyses, and DSBs. Concentration, viability, motility, and normal sperm morphology were significantly lower in male infertility patients compared with healthy men. Also, γH2AX levels and the number of DSBs were significantly higher in the sperm of infertile subjects compared with healthy men. γH2AX levels correlated negatively with conventional semen parameters and positively with DSBs. A threshold γH2AX level of 18.55% was identified as a cutoff value to discriminate infertile subjects from fertile control subjects with a specificity of 86.0% and a sensitivity of 83.0%. The positive and negative predictive values of the 18.55% γH2AX threshold were high: 87.7% and 85.5%, respectively. γH2AX levels were higher in the sperm of male infertility patients than in healthy men. γH2AX levels in sperm, as evaluated with the use of flow cytometry, might be a useful biomarker for evaluating DSBs in human spermatozoa.

The use of comet assay in plant toxicology: recent advances.

The systematic study of genotoxicity in plants induced by contaminants and other stress agents has been hindered to date by the lack of reliable and robust biomarkers. The comet assay is a versatile and sensitive method for the evaluation of DNA damages and DNA repair capacity at single-cell level. Due to its simplicity and sensitivity, and the small number of cells required to obtain robust results, the use of plant comet assay has drastically increased in the last decade. For years its use was restricted to a few model species, e.g., Allium cepa, Nicotiana tabacum, Vicia faba, or Arabidopsis thaliana but this number largely increased in the last years. Plant comet assay has been used to study the genotoxic impact of radiation, chemicals including pesticides, phytocompounds, heavy metals, nanoparticles or contaminated complex matrices. Here we will review the most recent data on the use of this technique as a standard approach for studying the genotoxic effects of different stress conditions on plants. Also, we will discuss the integration of information provided by the comet assay with other DNA-damage indicators, and with cellular responses including oxidative stress, cell division or cell death. Finally, we will focus on putative relations between transcripts related with DNA damage pathways, DNA replication and repair, oxidative stress and cell cycle progression that have been identified in plant cells with comet assays demonstrating DNA damage.

Use of Comet Assay in Human Lymphocytes as a Molecular Biomarker for Simultaneous Monitoring of Genetic Damage and Genotoxicity in Residents Who Lived Nearby the Santiago River, Mexico, in 2012

Most aquatic and health management authorities need assessing the potential genotoxicity of contaminated effluent to avoid the effects of a lot chemical agents found as environmental contaminants. Previous studies report the presence of some pesticides and heavy metals in waters of Santiago River and suggest a genetic risk for residents, who lived nearby this river, so it is important determine its mutagenic risk. The objective of this study was to use of comet assay in human lymphocytes as a molecular biomarker of genetic damage in residents who lived nearby the Santiago River, Mexico, in 2012 and monitoring simultaneous of genotoxicity of these waters in unexposed persons. The genotoxic activity of water samples taken from the Santiago River was evaluated in human lymphocytes from persons unexposed and the evaluation of genetic damage were studied in people living near the river.

Evaluation of DNA damage and cytotoxicity induced by triazole fungicide in cultured bovine lymphocytes

The aim of the present study was to investigate the effect of triazole fungicide Prosaro® (tebuconazole/prothioconazole) on the induction of micronuclei (MNs) and DNA damage in bovine peripheral lymphocytes. Whole blood cultures or isolated lymphocytes were exposed to 1.5, 3, 7.5, 15 and 30 μg ml−1 concentrations of triazole formulation for 24 and 48 h, or 2 h for MN and Comet assays. For 24 and 48 h of incubation with the fungicide no significant elevations in the MN frequencies in bovine lymphocytes were found. However, a clear reduction of cytochalasin-blocked proliferation index (CBPI) was observed in the donors (p < 0.05, p < 0.01, and p < 0.001). After the prolonged time of exposure the highest concentrations resulted in nearly total inhibition of cell proliferation (30 μg ml−1). After the use of Comet assay, only a moderate increase in DNA damage without statistical significance was recorded, except the highest concentration (30 μg ml−1; p < 0.05).

Genotoxic potency of mercuric chloride in gill cells of marine gastropod Planaxis sulcatus using comet assay.

In vivo and in vitro exposures were used to investigate the genotoxicity of mercuric chloride (HgCl2) to the marine snail, Planaxis sulcatus. The comet assay protocol was validated on gill cells exposed in vitro to hydrogen peroxide (H2O2, 0-50 μM). Snails were exposed in vivo for 96 h to HgCl2 (10, 20, 50, and 100 μg/l). Our results showed significant concentration-dependent increase in the tail DNA (TDNA) and olive tail moment (OTM) in exposed snails for all doses compared with controls. In vitro exposure to HgCl2 (10-100 μg/l) resulted in significantly higher values for TDNA at all concentrations. Our results showed that DNA damage increased in the gill cell with increasing exposure time. This study demonstrates the usefulness of comet assay for detection of DNA damage after exposure to HgCl2 and the sensitivity of marine snail P. sulcatus as a good candidate species for metal pollution.

Use of comet assay for risk assessment of benzene exposure in gas stations from Rio de Janeiro, Brazil.

Use of comet assay for risk assessment of benzene exposure in gas stations from Rio de Janeiro, Brazil.

The use of comet assay to assess global DNA methylation in human biomonitoring studies.

This work was supported by The Portuguese Science Foundation (FCT) through CESAM (UID/AMB/50017/2013) and CNRS/INEE - National Center for Scientific Research/Institute of Ecology and Environment, via OHMI – International Observatory Hommes-Millieux. Carla Costa and Marta S. Monteiro are supported by the grants SFRH/BPD/96196/2013 and SFRH/BPD/45911/2008, respectively, funded by FCT (QREN – POPH – Type 4.1 – Advanced training, subsidized by the European Social Fund and national funds of MEC).

Use of comet assay for the risk assessment of oil- and chemical-industry workers

The comet assay is a fluorescent microscopic method that is able to detect DNA strand-breaks even in non-proliferative cells in samples with low cell counts. The aim of the authors was to measure genotoxic DNA damage and assess oxidative DNA damage caused by occupational exposure in groups exposed to benzene, polycyclic aromatic carbohydrates and styrene at the workplace in order to clarify whether the comet assay can be used as an effect marker tool in genotoxicology monitoring. In addition to the basic steps of the comet assay, one sample was treated with formamido-pirimidine-DNA-glycolase restriction-enzyme that measures oxidative DNA damage. An increase was observed in tail moments in each group of untreated and Fpg-treated samples compared to the control. It can be concluded that occupational exposure can be detected with the method. The comet assay may prove to be an excellent effect marker and a supplementary technique for monitoring the presence or absence of genotoxic effects.

Monitoring of genotoxicity in marine zooplankton induced by toxic metals in Ennore estuary, Southeast coast of India

The present study provides preliminary in-situ data on genetic integrity of marine zooplankton. Paracalanus parvus, Oithona rigida and Euterpina acutifrons were collected during four different seasons (summer, pre-monsoon, monsoon and post-monsoon) from 2011 to 2012 in Ennore and Kovalum estuaries. DNA damage levels in different zooplankton were analyzed by comet assay and were correlated with different environmental stressors. Spatial and temporal variations in DNA damage was observed in all the species. Zooplankton from Ennore estuary showed significantly lower genetic integrity. Particulate, sediment, and zooplankton fractions of Pb, Ni, Cu, Cr and Co were associated with high DNA damage during the period of lowest pH, salinity and dissolved oxygen. Zn and Cd showed lower genotoxic impact than the other metals. Feeding modes strongly influenced the genetic integrity in the zooplankton species studied. These results support the use of comet assay as a tool in effectively monitoring genotoxicity in marine plankton communities.

Use of the comet assay technique for quick and reliable prediction of in vitro response to chemotherapeutics in breast and colon cancer.

BackgroundDetermination of response to chemotherapy is a major requirement of personalized medicine. Resistance, whether developed or native, critically affects a treatment’s success. Single Cell Gel lectrophoresis - also known as a comet assay - is used to detect DNA damage at the level of individual eukaryotic cells. We assessed the use of comet assays in determining response to chemotherapeutic drugs that are widely used in breast and colon cancer.ResultsWe treated human breast and colon cancer cell lines with melphalan, cisplatin, mechlorethamine or doxorubicin, as monotherapies. Drug activities varied even in the same cancer types, further demonstrating the heterogeneity of different cancer types.ConclusionThe comet assay technique can provide reliable and quick results with minimum requirements and is applicable to a wide variety of drugs.

Determination of lymphocyte DNA damage by use of comet assay in painters

Determination of lymphocyte DNA damage by use of comet assay in painters

In situ evaluation of the genotoxic potential of the river Nile: II. Detection of DNA strand-breakage and apoptosis in Oreochromis niloticus niloticus (Linnaeus, 1758) and Clarias gariepinus (Burchell, 1822)

This work is part of a wider eco-toxicological study proposed to evaluate the biological impact of contaminants along the whole course of the river Nile, Egypt. Here we present data on the presence of DNA strand-breaks and apoptotic cells assessed by use of comet and diffusion assays in erythrocytes of Nile tilapia (Oreochromis niloticus niloticus) and African catfish (Clarias gariepinus). The results showed high degrees of DNA damage and increased frequencies of apoptotic nuclei in blood of fish collected from downstream compared with those sampled from upstream river Nile. Qualitative analysis revealed a shift in the frequency of DNA-damage classes towards higher damage levels correlating with the increasing pollution gradient. The degree of DNA damage measured by use of comet assay and diffusion assay exhibited seasonal variations. Both fish species showed significant increases in DNA damage during the summer. The results of our study indicated that the alkaline comet assay seems to be a useful technique for in situ genotoxic monitoring. At the same time the diffusion assay is sensitive enough to detect low frequencies of apoptotic nuclei. The results reveal species-specific differences in sensitivities, suggesting that Nile tilapia may serve as a more sensitive test species compared with the African catfish. Based on the outcome of the comet and diffusion assays, it can be concluded that the water quality of the river Nile with respect to the presence of genotoxic compounds needs to be improved, especially in its estuaries. As far as we know this is the first time that the comet and diffusion assays are used for genotoxic monitoring of the river Nile.