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PubMed2004

In vivo genotoxic effect of nickel chloride in mice leukocytes using comet assay.

DNA damage induced by nickel chloride (NiCl2) in leucocytes of Swiss albino mice has been studied in vivo. The comet assay or the alkaline single cell gel electrophoresis (SCGE) assay was used to measure the DNA damage. The mice were administered orally with acute doses of 3.4, 6.8, 13.6, 27.2, 54.4 and 108.8 mg/kg body weight (b.wt.) NiCl2. Samples of whole blood were collected at 24, 48 and 72 h, first week and second week post-treatment for alkaline SCGE assay to study single/double strand breaks in DNA. A significant increase in mean comet tail length indicating DNA damage was observed with NiCl2 at 24, 48 and 72 h post-treatment (P<0.05). A gradual decrease in the mean tail length was observed at 72 h post-treatment indicating repair of the damaged DNA. The mean tail length showed a dose-related increase and time dependent decrease after treatment with NiCl2 when compared to controls. The study also confirms that the comet assay is a sensitive and rapid method to detect DNA damage caused by heavy metals like nickel (Ni).

Administration, Oral↗
PubMedDate not supplied

Assessing DNA damage in cnidarians using the Comet assay.

The assessment of DNA damage by the Comet assay has been described as a useful non-specific general biomarker of stress in many marine organisms. In field situations it has successfully been employed to distinguish between reference and polluted sites and in the laboratory it has been widely used as a mechanistic tool to determine pollutant effects and mechanisms of DNA damage. To date a wide range of marine vertebrates and invertebrates have been used, however, the usefulness of this assay as a biomarker in cnidarians has not yet been assessed. The aims of this study were to optimize the Comet assay for cnidarian cells and to assess its utility for detecting genotoxic damage in these cells. Cells were isolated from the North American pacific coast temperate sea anemone Anthopleura elegantissima using a non-enzymatic dissociation procedure and viability was determined to be in excess of 90%. Cells were incubated either with (1 h acute exposures) hydrogen peroxide (H(2)O(2)), ethylmethanesulphonate (EMS) or benzo(a)pyrene (B[a]P). In comparison to other marine species, anemone cells exhibited high control or background levels of DNA strand breaks. Despite this, however, we observed dose responses for each of the study chemicals with no reduction in cell viability. This study demonstrates that anemone cells respond to known DNA damaging agents, including B[a]P which requires metabolism to exert its genotoxic effect, and that the Comet assay may prove to be a useful biomarker of stress in cnidarian species.

Animals↗
PubMed2006

Comet assay for the detection of genotoxicity in blood cells of flounder (Paralichthys olivaceus) exposed to sediments and polycyclic aromatic hydrocarbons.

To investigate the genotoxic effect of marine sediments on aquatic organism, sediment samples were collected from 13 sites along the coast of Gwangyang Bay (Korea). Concentrations of polycyclic aromatic hydrocarbons (PAHs) in sediments were determined and the relationship between exposure of flounder blood cells to sediment extracts and DNA single-strand breakage in the blood cells was examined using the comet assay. Levels of DNA damage were proportionally increased by exposure concentration and the highest sediment-associated DNA damage was observed at the station showing the highest PAHs contamination. DNA damage in blood cells exposed to five types of PAHs (benzo[a]pyrene, fluoranthene, anthracene, pyrene and phenanthrene) and in flounder (Paralichthys olivaceus) exposed to benzo[a]pyrene (BaP) for 0, 2 and 4 days were assessed by measuring comet tail length. The tail lengths of five PAHs-exposed groups at 50 and 100 ppb were significantly different from the non-exposed group, and the genotoxic effect of BaP correlated with both concentration and duration of exposure. Throughout the study, significant differences in DNA breakage were recorded between cells exposed to sediment extracts or PAHs and non-exposed control. This study demonstrated the comet assay as a successful tool in monitoring contamination of marine sediments and assessing genotoxicity of PAHs in marine organisms, either in vitro or in vivo.

Animals↗
PubMed2006

Formamidopyrimidine adducts are detected using the comet assay in human cells treated with reactive metabolites of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK).

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the most lung-specific of the carcinogens present in tobacco smoke. Its bioactivation in cells leads to a small amount of methylation or pyridyloxobutylation DNA damage. Considering its great sensitivity, the comet assay seems a technique of choice to investigate NNK-related damage. Several strategies were used to impart some specificity to the assay: (1) using analogs that produce a limited variety of DNA lesions, as they mimic either the methylation or the pyridyloxobutylation pathway; (2) using cells with different bioactivation abilities; (3) using alkali conversion and/or enzymes specific for cleaving particular classes of damage; (4) using different lysis conditions to convert a specific class of DNA lesions into enzyme-sensitive lesions. We determined that several NNK-associated lesions can be detected with some specificity with the comet assay. For the methylation pathway, they are AP sites and the more frequent formamidopyrimidine (fapy) adducts. These fapy adducts correspond to N7-methylguanines generated in the cells that were ring-opened during the assay by the lysis solution at pH 10. For the pyridyloxobutylation pathway, alkylphosphotriesters and a roughly equal frequency of fapy sites were detected. By analogy to the methylation damage, these fapy adducts are thought to be the ring-opened form of N7-pyridyloxobutylguanines (N7-pobG). N7-pobG are unstable and this constitutes the first indirect demonstration of their formation in cells. But contrary to N7-m-fapy, the lysis time or pH did not influence the frequency of N7-pob-fapy adducts detected, suggesting that they already exist in the cells and are not related to the experimental conditions. These N7-pob-fapy have a strong mutagenic potential and we think that the comet assay, in spite of its limitations, is a good way to study them considering their low frequency and the inherent instability of the adduct from which they originate.

Carcinogens↗
PubMed2003

Investigations on the effect of cigarette smoking in the comet assay.

The comet assay (single-cell gel electrophoresis, SCG) is widely accepted as an in vitro and in vivo genotoxicity test. Because of its demonstrated ability to detect various kinds of DNA damage and its ease of application, the technique is being increasingly used in human biomonitoring. However, the assessment of small genotoxic effects as typically obtained in biomonitoring may be limited by the different sources of assay variability and the lack of an optimal protocol with high sensitivity. To better characterize the suitability of the comet assay for biomonitoring, we are performing a comprehensive investigation on blood samples from smokers and non-smokers. Because tobacco smoke is a well-documented source of a variety of potentially mutagenic and carcinogenic compounds, smokers should be a suitable study group with relevant mutagen exposure. Here, we report our results for the first sample of 20 healthy male smokers and 20 healthy male non-smokers. Baseline and benzo[a]pyrene diolepoxide (BPDE)-induced effects were analysed by two investigators using two image analysis systems. The study was repeated within 4 months. Furthermore, the influence of a repair inhibitor (aphidicolin, APC) on baseline and BPDE-induced DNA damage was comparatively analysed. In all experiments, a reference standard (untreated V79 cells) was included to correct for assay variability. None of these approaches revealed significant differences between smokers and non-smokers. Although more data is needed for a final conclusion, this study indicates some limitations of the comet assay with regard to the detection of DNA damage induced by environmental mutagens in peripheral blood cells.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed2004

Assessment of DNA damage by comet assay on frozen total blood: method and evaluation in smokers and non-smokers.

This study was undertaken with the aim to develop an optimised protocol for the evaluation of DNA damage in frozen whole blood. This was achieved by use of the single-cell gel electrophoresis (SCGE) or comet assay in its alkaline version. After collection of blood, the total blood sample was mixed with dimethyl sulfoxide (DMSO), a cryoprotectant commonly used for prevention of freezing-induced damage to living cells, and then stored at -80 degrees C. We observed no statistically significant differences in the level of DNA damage between fresh blood samples and frozen blood samples, as assessed by the comet assay. Considering the absence of effects of the freezing step, a frozen blood sample was included as a control sample in subsequent experiments. Thus the protocol was applied to blood samples of twenty healthy subjects including smokers and non-smokers. The comparative analysis indicated that the level of DNA damage was 56% higher in smokers than in non-smokers (P = 0.01). Altogether, this study strongly suggests that frozen whole blood could be utilised in association with the comet assay in human epidemiological bio-monitoring for the assessment of genetic damage in populations at risk.

Blood↗
PubMed2004

Sensitivity to DNA-damage induction and chromosomal alterations in mucosa cells from patients with and without cancer of the oropharynx detected by a combination of Comet assay and fluorescence in situ hybridization.

In addition to exogenous risk factors, the development of head and neck cancer is based on genetic alterations and individual sensitivity to mutagens. The DNA-damaging effect of xenobiotics and the location of chromosomal changes warrant further investigation. The aim of this study was to evaluate variance in structural genetic changes in human epithelia as target cells for head and neck carcinogenesis. The combination of the single-cell gel electrophoresis (Comet) assay with the fluorescence in situ hybridization (FISH) technique is presented to examine differences in sensitivity to DNA-damage induction and in alterations of chromosomes 1, 3, 5 and 8 in patients with and without squamous cell carcinoma of the oropharynx. Macroscopically healthy biopsies from the mucosa, taken at a distance from the tumor of 10 patients with oropharyngeal carcinoma and from 10 patients without tumor were harvested during surgery. Cells were isolated by enzymatic digestion and incubated with benzo[a]pyrene-diolepoxide (BPDE), causing DNA-adduct formation by covalent binding of BPDE with DNA bases. The cells were subsequently analyzed by means of the Comet assay to separate DNA fragments and to visualize the DNA-damage. A hybridization mixture with whole-chromosome paints for Chr1, Chr3, Chr5 and Chr8 was added. After fluorescent staining, the entire DNA and the DNA of chromosomes 1, 3, 5 and 8 were evaluated by digital analysis. BPDE caused significant DNA damage in oropharyngeal mucosa cells of patients with and patients without carcinoma. No differences in the amount of DNA damage could be observed between patients suffering from sqamous cell carcinoma and patients without malignancy. Evaluation of chromosomal alterations, however, revealed significantly higher damage levels in chromosomes 3, 5 and 8 compared with chromosome 1 in tumor patients. In contrast, for patients without oropharyngeal carcinoma no differences in chromosomal alterations could be observed. The Comet assay could be combined with FISH to examine the sensitivity to DNA-damage induction and chromosomal alterations in human epithelial cells exposed to a genotoxic agent. Chromosomal breakage is increased for chromosomes 3, 5 and 8 as compared with chromosome 1, indicating a higher sensitivity of these chromosomes in epithelial cells of tumor patients. Using Comet/FISH on human epithelia, selected genetic alterations can be detected, which supports description of endogenous risk factors in carcinogenesis of the upper aerodigestive tract.

Adult↗
PubMed2006

Effect of different organotins on DNA of mollusk (Scapharca inaequivalvis) erythrocytes assessed by the comet assay.

The alkaline comet assay, employing a single-cell gel-electrophoresis, is a rapid, simple and sensitive technique for visualizing and measuring DNA damage leading to strand breakage in individual cells. In this study, we report data about the effect of different organotin compounds (MBTC, DBTC and TBTC) on DNA from erythrocytes of the Scapharca inaequivalvis bivalve mollusc. Our results show significant DNA damage after 30 min in vitro incubation with 10microM of organotins. Since TBTC turned out to be the most genotoxic compound, followed by MBTC and DBTC, we exposed the molluscs to 50ppb of TBTC for 11 days. A significant increase of comet parameters was measured in our experimental conditions. The use of the comet test as a high-throughput screening assay to monitor the effect of environmental pollutants on marine organisms has been proposed.

Animals↗
PubMed2004

Optimisation of the comet genotoxicity assay in freshly isolated murine hepatocytes: detection of strong in vitro DNA damaging properties for styrene.

While the comet assay is used to detect DNA damage in isolated cells following exposure to chemicals in vitro, few publications report the use of the procedure in liver cells isolated from mice. Our initial efforts to use the assay to assess DNA damage in mouse hepatocytes maintained on collagen-coated dishes were hampered by high levels of baseline damage in controls, which appeared to result from mechanical damage sustained during the dislodgement of adherent cells in the early stages of the assay protocol. Here we describe an efficient version of the comet assay in cultured mouse hepatocytes that involves careful recovery of cells using a "scraping" buffer supplemented with 10% high purity grade DMSO. Use of this buffer strongly diminished the frequency of false positives. Using the industrial reagent styrene as a positive control in the optimised procedure, non-cytotoxic concentrations of this substance (2.5-10 mM) significantly increased mean comet tail length, area, and moment. Co-incubation with the CYP inhibitor SKF-525A strongly attenuated these effects of styrene. Collectively, these findings confirm this method is highly suitable for the detection of DNA damage by bioactivation-dependent compounds in freshly isolated mouse hepatocytes.

Adult↗
PubMed2005

The effects in mice of combined treatments to X-rays and antineoplastic drugs in the Comet assay.

The Comet assay is a rapid, easy and reproducible method to detect genotoxic activity of chemical and physical agents in vitro and in vivo. In the present study the effects of exposure to irradiation or chemicals: cyclophosphamide (CP) and mitomycin C (MMC) or combined exposure to low doses of both agents (0.25 Gy+3.15 mg/kgbw CP and 0.25 Gy+0.25 mg/kgbw MMC) were examined for the induction of DNA damage in the Comet assay measured simultaneously in somatic (bone marrow lymphocytes) and haploid germ cells. The male mice were treated in vivo and sacrificed at 24 h after exposure. The percentage contents of DNA in the "comet tail" increased with increasing doses of X-rays and chemicals. After combined exposure to X-rays and CP and to X-rays and MMC weak increases of DNA damage in bone marrow lymphocytes and in germ cells were observed by comparison with the results obtained for each agent acting alone. There were slightly different responses in bone marrow lymphocytes and in germ cells, but effects were observed over a similar dose range.

Animals↗
PubMed2004

Enhancement of genotoxic effects in the comet assay with human blood samples by aphidicolin.

The comet assay (single cell gel electrophoresis) has become increasingly used in human biomonitoring. In its standard version at pH > 13, DNA lesions such as DNA double-strand breaks (DSB), DNA single strand breaks (SSB) and alkali-labile sites (ALS) lead to increased DNA migration. Besides DNA damage, strand break formation during excision repair can also increase DNA migration. Inhibitors of DNA repair have been shown to enhance the DNA effects of mutagens and the use of repair inhibitors has been proposed for human biomonitoring studies to increase the sensitivity of the comet assay. To further evaluate the usefulness of such an approach we performed an experimental study with human blood and tested the enhancing effect of aphidicolin (APC) on DNA effects induced by different mutagens. Our results clearly show that APC enhances the genotoxic effects of benzo[a]pyrene diolepoxide (BPDE), bischloroethylnitrosurea (BCNU) and methyl methanesulfonate (MMS), but has no significant effect on gamma radiation-induced DNA effects. The enhancing effect is seen in unstimulated and PHA-stimulated blood, indicating repair activity under both conditions but the effect is stronger in stimulated blood. Our results indicate that APC can be used to increase the sensitivity of the comet assay towards a broad spectrum of induced primary DNA lesions and support the usefulness of this approach. However, for human biomonitoring, a sensitive protocol still has to be established.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed2003

The Comet assay for the detection of DNA damage in Mus spretus from Doñana National Park.

Doñana Park (Spain), a protected area in Europe, was affected by an environmental disaster in April 1998 that caused the spreading of acidic water and mud full of toxic metals from the Aznalcollar pyrite mine. In order to assess the contamination in the area and to monitor the possible biological effects of the toxic spill, a series of coordinated studies was performed utilizing several animal species living in that area. We performed genotoxicity monitoring using the Comet assay on peripheral blood leukocytes of the Algerian mouse (Mus spretus), a nonprotected rodent suitable as a bioindicator. The mice were sampled in different areas 6 months after the ecological disaster and again 1 year later. Our results showed that in 1998 all the areas examined were contaminated, as determined by an increase in the Comet assay parameters in the analyzed animals, whereas a significant decrease in the values of these parameters was observed in the 1999 samples, which were collected in a riverside area subject to tide flows. Thus, the Comet assay has proven to be an interesting and sensitive tool in studies of environmental genotoxicity.

Accidents↗
PubMed2000

Assessment of DNA damage induced by high-LET ions in human lymphocytes using the comet assay.

The alkaline single-cell gel electrophoresis assay (comet assay) was used to analyze DNA damage induced in human lymphocytes by irradiation with high linear energy transfer (LET) ions. Our aim was to measure DNA breaks and to demonstrate the heterogeneity of the damage levels in a lymphocyte population irradiated with ions of different energies and LETs. Four experiments with heavy ions (Ar, C and U), as well as gamma-ray exposure, were conducted to enable comparisons. We demonstrated that the comet assay is able to assess the variability in DNA damage induced at the single cell level. The amount of DNA damage and its heterogeneity increased with particle fluence and LET, but saturated at high LETs. However, when expressed in terms of the mean dose, gamma-rays were more efficient than most of the ions used. The comet assay also allowed the detection of highly damaged cells (HDC), which were previously described as cells in late apoptotic stages. The rapid emergence of HDC in this study suggests that they were generated following ion irradiation-induced creation of DNA break clusters induced by ion exposure. Another clue was that the proportion of HDC increased with LET and fluence. We hypothesized that the LET threshold observed and the higher efficiency of low-LET radiation might be linked to the impossibility of measuring small DNA fragments in HDC.

Comet Assay↗
PubMed1999

Preservation of comet assay slides: comparison with fresh slides.

The single cell gel electrophoresis assay (comet assay) is an inexpensive, rapid and highly sensitive method for the determination of DNA damage, crosslinks, and alkaline-labile lesions in individual cells. A limitation of the procedure is that the microelectrophoretic gels must be scored rapidly as the comet configuration deteriorates on storage due to dehydration of the agarose and diffusion of DNA. The objectives of this study were firstly to evaluate drying regimes as rapid and simple methods of preservation of the microgels as close to their original fresh state as possible, and secondly to examine the effects of storage of the slides. Human hepatoma (HepG2) cells challenged for 30 min with hydrogen peroxide (H(2)O(2)) were used in the study. Microgel slides were prepared and evaluated immediately, or after drying with or without a methanol fixation step. Microgels that were dried at a variety of temperatures (22-50 degrees C) and re-hydrated did not differ in the values obtained for H(2)O(2)-induced DNA damage when compared to fresh samples. Samples could also be continually dried and re-hydrated over a period of up to 3 months with no obvious loss of information. In conclusion, drying of microgels represents a simple and inexpensive method of preserving comet assay slides.

Cell Nucleus↗
PubMed2001

Application of the comet and micronucleus assays to butterfish (Pholis gunnellus) erythrocytes from the Firth of Forth, Scotland.

This report describes an investigation of genotoxic effects in an inter-tidal fish species sampled along a pollution gradient in the Firth of Forth, Scotland, UK. The comet assay is an electrophoretic technique for measuring DNA breakage in nuclei from individual cells and has only recently been applied to field investigations of genotoxicity. The measurement of nuclear anomalies (NA), such as the presence of micronuclei (MN) and 'lobes', has been successfully utilised in many field studies of genotoxic effects of contaminated sediments. These two techniques were applied to nucleated red blood cells (RBC) from the butterfish, Pholis gunnellus. The comet assay was adapted and validated for use in this species. Fish were sampled from the inner Firth of Forth, which has a legacy of industrial contamination and the outer Firth of Forth which is comparatively clean. The analysis of DNA strand breakage using this technique did not reveal any significant differences between animals sampled from inner and outer zones of the Firth. In contrast, MN and NA frequencies were elevated in the inner polluted zone of the Firth compared to the outer zone. This study suggests: (1) there are genotoxic effects associated with contaminants in the inner Firth of Forth, and (2) the comet assay may not be a suitable genotoxicity biomarker in fish.

Animals↗
PubMed2003

Application of the micronucleus and comet assays to mussel Dreissena polymorpha haemocytes for genotoxicity monitoring of freshwater environments.

Assessment of DNA damage is of primary concern when determining the pollution-related stress in living organisms. To monitor genotoxicity of the freshwater environments we used micronucleus (MN) and comet assay on Dreissena polymorpha haemocytes. Caged mussels, collected from the river Drava, were transplanted to four monitoring sites of different pollution intensity in the river Sava. Exposition lasted for a month. The baseline level of MN frequencies in the haemocytes of mussels from reference site (river Drava) was 0.5 per thousand. No increase in MN frequency was found in mussels from the medium-polluted site (Zagreb) in the river Sava while other, more polluted sites showed higher MN frequencies ranging from 2.7 per thousand (Lukavec) and 3.1 per thousand (Oborovo) to 5.2 per thousand (Sisak). Results from comet assay showed concordance with MN assay in indicating intensity of DNA damage. The use of haemocytes from caged, non-indigenous mussels in MN and comet assay proved to be a sensitive tool for the freshwater genotoxicity monitoring.

Animals↗
PubMed2001

The Correction of Myopia Evaluation Trial (COMET): design and general baseline characteristics.

The Correction of Myopia Evaluation Trial (COMET) is a multicenter, randomized, double-masked, controlled clinical trial evaluating whether there is a difference in the progression of myopia between children wearing progressive addition lenses (PALs) versus conventional single vision lenses (SVLs), as measured by cycloplegic autorefraction. Axial length, measured by A-scan ultrasonography, is an additional outcome measure. To meet the recruitment goal of 450 participants, eligible children ages 6-11 years (inclusive) with myopia in both eyes (spherical equivalent between -1.25 diopters (D) and -4.50 D, astigmatism < or = 1.50 D, and anisometropia < 1.00 D) were recruited at four clinical centers between September 1997 and September 1998. Children who participated were assigned to receive PALs (Varilux Comfort with a +2.00 D addition) or SVLs. Measures include standardized cycloplegic autorefraction (Nidek ARK700A autorefractor), axial length (Sonomed A2500 ultrasound), subjective refraction (Marco TRS system), visual acuity (modified Early Treatment Diabetic Retinopathy Study protocol), accommodation (Canon R-1), and phoria (cover test and Maddox rod). Outcome measures are collected annually; adherence is assessed and prescriptions updated semiannually. Participants are being followed for at least 3 years. COMET enrolled 469 children. Their mean age is 9.3 years (range 6-11 years); 52% are female. COMET children are ethnically diverse, according to a self-report with 46% White, 26% African American, 14% Hispanic, and 8% Asian. Best-corrected visual acuity is better than 20/32 in both eyes. Baseline mean (+/-SD) cycloplegic refractive correction is -2.38 D (+/-0.81) in the right eye and -2.40 D (+/-0.82) in the left eye; mean (+/-SD) axial length is 24.1 mm (+/-0.7) in both eyes. Follow-up of these children will provide a first step in answering the important question of whether there are effective means to slow myopia progression. Study results should be applicable to a large proportion of children with myopia. The study will also provide useful information on myopia progression in children wearing conventional single vision lenses.

Child↗
PubMed2003

DNA damage in workers exposed to lead using comet assay.

Lead (Pb) is a ubiquitous and toxic metal. Secondary Pb recovery unit workers are prone to possible occupational Pb exposure. Hence, this investigation was conducted to assess the genotoxic effect of Pb exposure in these workers. In the study, 45 workers were monitored for DNA damage in blood leucocytes. Simultaneously 36 subjects were used as control group in this study. All the subjects were estimated for Pb content in whole blood by ICP-MS. The alkali single cell gel electrophoresis assay (comet assay) was adopted for detecting the DNA damage. The air inside the premises of the unit had Pb concentrations of 4.2 microg/m(3). The level of DNA damage was determined as the percentage of cells with comets. The mean Pb content was found to be significantly higher in the study group (248.3 microg/l) when compared with the controls (27.49 microg/l). Significantly more cells with DNA damage (44.58%) were observed in the study group than in the control persons (21.14%). Smoking had a significant effect on DNA damage in the control group whereas an insignificant effect was noticed in the exposed workers. Study as well as the control group failed to show a significant effect on DNA damage with age (P>0.05). Pb content and years of exposure significantly correlated with DNA damage in the study group (r=0.602, r=0.690; P<0.01). The increased levels of DNA damage observed in the exposed workers, justifies the use of the comet assay for the evaluation of genotoxic effects in humans exposed to Pb.

Adult↗