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PubMed2006

Evolution of DNA strand-breaks in cultured spermatocytes: the Comet Assay reveals differences in normal and gamma-irradiated germ cells.

In reproductive toxicity assessment, in vitro systems can be used to determine mechanisms of action of toxicants. However, they generally investigate the immediate effects of toxicants, on isolated germ cells or spermatozoa. We report here the usefulness of in vitro cultures of rat spermatocytes and Sertoli cells, in conjunction with the Comet Assay to analyze the evolution of DNA strand-breaks and thus to determine DNA damage in germ cells. We compared cultures of normal and gamma-irradiated germ cells. In non-irradiated spermatocytes, the Comet Assay revealed the presence of DNA strand-breaks, which numbers decreased with the duration of the culture, suggesting the involvement of DNA repair mechanisms related to the meiotic recombination. In irradiated cells, the evolution of DNA strand-breaks was strongly modified. Thus our model is able to detect genotoxic lesions and/or DNA repair impairment in cultured spermatocytes. We propose this model as an in vitro tool for the study of genotoxic injuries on spermatocytes.

Animals↗
PubMed2006

Evaluating the genotoxic effects of workers exposed to lead using micronucleus assay, comet assay and TCR gene mutation test.

To evaluate the genotoxic effects of lead (Pb) exposure, 25 workers in a workplace producing storage battery were monitored for three genetic end-points using micronucleus (MN) assay, comet assay and TCR gene mutation test. Twenty-five controls were matched with workers according to age, gender and smoking. The air Pb concentration in the workplace was 1.26 mg/m(3). All subjects were measured for Pb concentration of blood by atom absorption spectrophotometry. The mean Pb concentration of blood in workers (0.32 mg/l) was significantly higher than that in controls (0.02 mg/l). The results of MN test showed that the mean micronuclei rate (MNR) and mean micronucleated cells rate (MCR) in workers were 9.04+/-1.51 per thousand and 7.76+/-1.23 per thousand, respectively, which were significantly higher than those (2.36+/-0.42 per thousand and 1.92+/-0.31 per thousand) in controls (P<0.01). It was found in the comet assay that the mean tail length (MTL) of 25 workers and 25 controls were 2.42+/-0.09 and 1.02+/-0.08 microm, respectively, there was significant difference between workers and controls for MTL (P<0.01), also the difference of the mean tail moment (MTM) between workers (0.85+/-0.05) and controls (0.30+/-0.09) was very significant (P<0.01). However, in TCR gene mutation assay Mfs-TCR of workers and controls were 1.69+/-0.15 x 10(-4) and 1.74+/-0.17 x 10(-4), respectively, there was no significant difference between workers and controls (P>0.05). The results of our study indicated that the genetic damage was detectable in 25 workers occupationally exposed to lead.

Adult↗
PubMed2004

Protective activity of cedron (Aloysia triphylla) infusion over genetic damage induced by cisplatin evaluated by the comet assay technique.

Using the comet assay technique, this paper examines the protection from the cisplatin-induced genetic damage in mouse bone marrow cells provided by cedron-leaf infusion. Animals were separated into six groups: (I) untreated, (II) negative control, (III) treated with cedron-leaf infusion (5%), (IV) treated with cisplatin (6 mg/kg b.w.), (V) pretreated with infusion and treated with cisplatin and (VI) positive control (cyclophosphamide, 20 mg/kg b.w.). Based on the tail moment values found, four types of comets were distinguished. No statistical differences (P<0.01) were found between untreated animals, negative control and infusion treated mice. As expected, treatment of mice with a single dose of cis-DDP-induced genetic damage and the pretreatment with infusion prior to cis-DDP injection inhibited the capacity of cisplatin to induce genetic damage. Cell viability was up to 90% in all cases. The results suggest that infusion could exert its in vivo antigenotoxic action by enhancing the antioxidant status of bone marrow cells. The found could be attributed to its scavenging potency towards free radicals.

Animals↗
PubMed2006

Correlation between formamidopyrimidine DNA glycosylase (Fpg)-sensitive sites determined by a comet assay, increased MDA, and decreased glutathione during long exposure to thinner inhalation.

Thinner inhalation causes toxic effects in a variety of organs, principally in the central nervous system. Some studies have shown oxidative stress effects of thinner inhalation, such as: activation of free radical processes, decrease of antioxidants, and oxidation products of proteins and lipids but not of DNA. The aim of this study is to investigate the effect of thinner inhalation on DNA. We used the comet assay in conjunction with the enzyme formamidopyrimidine glycoslyase (Fpg). Our results show a significant increase in Fpg-sensitive sites in DNA of lymphocytes from rats exposed to thinner fumes compared to lymphocytes from control rats (p < 0.05). Moreover, DNA damage detected with Fpg shows a high correlation with increased malondialdehyde (MDA) and decreased glutathione (GSH), two widely used biomarkers of oxidative stress. The most abundant base oxidation product found in DNA is 8-oxoguanine; it is the main substrate of Fpg and the most commonly used biomarker for oxidative DNA damage. This suggests that oxidative DNA damage is at least partly responsible for the DNA damage detected by Fpg. We propose the comet assay in combination with Fpg as a sensitive biomarker to monitor exposure to thinner inhalation. Limitations of this method are discussed.

Administration, Inhalation↗
PubMed2005

Comparative evaluation of DNA damage by genotoxicants in primary rat cells applying the comet assay.

Various compounds known to cause DNA damage (hydrogen peroxide, visible light-excited methylene blue, N-nitrosomorpholine and benzo[a]pyrene) were tested with different primary rat cells (lymphocytes, testicular cells, type II pneumocytes and hepatocytes) to determine the range of induced DNA damage applying the comet assay. A dose-dependent increase of DNA breaks was observed after treatment with hydrogen peroxide in all cell types studied. The most prominent effect was observed in lymphocytes, whereas only a slight increase of DNA breaks was observed in hepatocytes. Visible light-excited methylene blue caused significant oxidative DNA damage, which did not significantly differ between the cell types used with the exception of hepatocytes, for which a lower level of DNA damage was observed. N-Nitrosomorpholine and benzo[a]pyrene induced a moderate but significant increase of DNA strand breaks in pneumocytes and hepatocytes while in lymphocytes no effect was observed. Our results clearly demonstrate that due to their differential function which is also expressed by the level of drug metabolizing and/or antioxidant enzymes, freshly isolated rat cells (lymphocytes, testicular cells, type II pneumocytes and hepatocytes) respond differently to the exposure to genotoxic agents as detected by comet assay.

Animals↗
PubMed2006

DNA damage and repair capacity by comet assay in lymphocytes of white-collar active smokers and passive smokers (non- and ex-smokers) at workplace.

The comet assay has been widely used to quantify DNA damage in isolated lymphocytes from subjects exposed to several environmental or occupational substances, especially for estimation of oxidative damage in the DNA, which is well-known to be induced by tobacco smoke. Passive smoking or environmental tobacco smoke (ETS) has been included among those substances that cause cancer with sufficient evidence in humans. In this study, we analyzed, by the alkaline version of comet assay, the lymphocyte DNA damage of white-collar active smokers and non- and ex-smokers exposed to ETS at the workplace. We investigated basal DNA damage, DNA oxidation by formamidopyrimidine glycosylase (Fpg), the repair capacity H2O2-induced DNA damage by kinetics studies and lymphocyte GSH levels, the major intracellular defense against exogenous oxidative stress imposed by cigarette smoking. Our results indicated high basal DNA damage with clear significant correlations with urinary nicotine and cotinine, number of cigarettes/day, and an inverse significant correlation with GSH cellular content in active smokers. Significant Fpg-sensitive sites were found in smokers (> 85%), considerably high but not significant in passive non- and ex-smokers (> 51% and 37%, respectively). The DNA repair capacity had seriously decreased in non-smokers > smokers > ex-smokers, while the same damage was repaired in a short time in never smokers.

Adult↗
PubMed2003

Enhanced DNA excision repair in CCRF-CEM cells resistant to 1,3-bis(2-chloroethyl)-1-nitrosourea, quantitated using the single cell gel electrophoresis (Comet) assay.

Enhanced DNA repair activity is important for the development of cellular resistance to alkylating agents. Here, we quantitated the kinetics of DNA excision repairs initiated by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in human leukemia CCRF-CEM cells. CEM cells that had been established resistant to BCNU (CEM-R) were evaluated in comparison with parental CEM cells (CEM-S). The excision repair kinetics were quantitated as the amount of DNA single strand breaks, which were generated from the incision/excision of the damaged DNA and were diminished by the rejoining of renewed DNA, using the single cell gel electrophoresis (Comet) assay. CEM-R cells were 10-fold more resistant to BCNU than CEM-S cells, and also showed cross-resistance to melphalan and cisplatin. In response to the treatment with BCNU, both CEM-S and CEM-R cells initiated an incision/excision reaction at the end of the incubation period, and completed the rejoining process within 4 hr. While CEM-S cells could not repair the damage induced by the high concentration of BCNU, CEM-R cells completed the repair process regardless of BCNU concentrations, suggesting enhanced excision repairs in CEM-R cells. The excision repair activity of CEM-R cells was increased with regard to the incision reaction and to the rate of the repair. Similar results were obtained using ultraviolet C, suggesting enhanced nucleotide excision repair in CEM-R cells. Thus, the enhanced DNA excision repairs were successfully quantitated in the resistant leukemic cell line using the Comet assay. The evaluation of the repair activity may predict the sensitivity of cancer cells to chemotherapy and provide a clue to overcome the resistance.

Antineoplastic Agents, Alkylating↗
PubMed2002

Detecting genotoxicity using the Comet assay following chronic exposure of Manila clam Tapes semidecussatus to polluted estuarine sediments.

Sediments frequently cause damage to biota due to the accumulation of toxic compounds and the bioavailability of sediment-bound contaminants. Damage can be assessed using biomarkers, such as the degree of genotoxic impact following in vivo exposure to pollutants. Genotoxic damage, expressed as single-strand DNA breaks, was measured in cells isolated from haemolymph, gill and digestive gland from the clam Tapes semidecussatus, using the single cell gel electrophoresis (Comet assay). Clams were exposed for three weeks to sediment samples collected from a polluted site and a 'clean' reference site. The level of DNA damage was assessed using an image analysis package and expressed as Tail Moment. Throughout the study, significant differences in DNA damage were recorded for each tissue type between clams exposed to the two sediment samples. We conclude that the Comet assay is a useful tool for the detection of DNA damage in clams chronically exposed to polluted sediments.

Animals↗
PubMed2001

Evaluation of the effect of 90Sr beta-radiation on human blood cells by chromosome aberration and single cell gel electrophoresis (comet assay) analysis.

Among various environmental genotoxins, ionizing radiation has received special attention because of its mutagenic, carcinogenic and teratogenic potential. In this context and considering the scarcity of literature data, the objective of the present study was to evaluate the effect of 90Sr beta-radiation on human cells. Blood cells from five healthy donors were irradiated in vitro with doses of 0.2-5.0Gy from a 90Sr source (0.2Gy/min) and processed for chromosome aberration analysis and for comet assay. The cytogenetic results showed that the most frequently found aberration types were acentric fragments, double minutes and dicentrics. The alpha and beta coefficients of the linear-quadratic model, that best fitted the data obtained, showed that 90Sr beta-radiation was less efficient in inducing chromosome aberrations than other types of low linear energy transfer (LET) radiation such as 3H beta-particles, 60Co gamma-rays, 137Cs and 192Ir and X-rays. Apparently, 90Sr beta-radiation in the dose range investigated had no effect on the modal chromosome number of irradiated cells or on cell cycle kinetics. Concerning the comet assay, there was an increase in DNA migration as a function of radiation dose as evaluated by an image analysis system (tail moment) or by visual classification (DNA damage). The dose-response relation adequately fitted the non-linear regression model. In contrast to the cytogenetic data, 90Sr beta-radiation induced more DNA damage than 60Co gamma-radiation when the material was analyzed immediately after exposures. A possible influence of selective death of cells damaged by radiation was suggested.

Adult↗
PubMed2002

Influence of mus201 and mus308 mutations of Drosophila melanogaster on the genotoxicity of model chemicals in somatic cells in vivo measured with the comet assay.

To check the possibilities of the recently developed comet assay, to be used in mechanistic studies in Drosophila melanogaster, neuroblast cells of third instar larvae are used to analyse in vivo, the effect of two repair deficient mutations: mus201, deficient on nucleotide excision repair, and mus308, deficient in a mechanism of damage bypass, on the genotoxicity of methyl methanesulphonate (MMS), ethyl methanesulphonate (EMS) and N-ethyl-N-nitrosourea (ENU). The obtained results reveal: (1) MMS-induced breaks are most probably consequence of N-alkylation damage mediated abasic (AP) site breakage; (2) MMS and at least part of the EMS induced damage leading to DNA strand breaks are efficiently repaired by the nucleotide excision repair mechanism; (3) ENU and part of EMS induced damage need a functional Mus308 protein to be processed, otherwise they can lead to DNA strand breaks. In addition, the results of this work confirm the validity of neuroblast cells to conduct the comet assay, and the usefulness of this assay in in vivo mechanistic studies related to DNA repair in D. melanogaster.

Animals↗
PubMed2001

Urban airborne particulate: genotoxicity evaluation of different size fractions by mutagenesis tests on microorganisms and comet assay.

The genotoxic effects of different size fractions of airborne particulate (Total, PM10 and PM25), extracted with acetone or toluene, were evaluated by: the Ames plate test (TA98 and TA100 strains, w/o S9), gene conversion and reversion (w/o endogenous metabolic activation) in the Saccharomyces cerevisiae D7 strain, and the comet assay on human leukocytes. The data on human leukocytes confirm the sensitivity of the comet assay and its applicability to assess genotoxicity in environmental samples. The PM2.5 fraction of airborne particulate generally shows the highest concentration of DNA-damaging compounds. Genotoxic response, in all the test systems applied, is highly dependent on extraction solvent used. Acetone seems to extract compounds with more similar genotoxic responses in the three test systems used than toluene extracts. Toluene appears to extract air pollutants genotoxic on yeast and leukocytes but is mainly cytotoxic on Salmonella.

Acetone↗
PubMed1999

The comet assay: a tool to study alteration of DNA integrity in developing plant leaves.

DNA integrity of Nicotiana tabacum L. and Vicia faba L. leaves in different stages of growth was analysed with the single cell gel electrophoresis (comet) assay. With this test DNA of individual cells is stretched by electrophoresis and the migration is measured, which gives an image of the nuclear DNA organisation. Nuclei were sampled when the plants had developed an apical bud, five true leaves and cotyledons. To get an idea of the kind of lesions observed, three different comet protocols were used. The neutral protocol with electrophoresis in a neutral buffer and the semi-alkaline or alkaline assay with alkaline unwinding followed by electrophoresis in neutral alkaline buffer, respectively. For V. faba there was a successive increased cellular DNA mobility with age of the leaves. The percentage DNA migration in control cells of fully developed leaves from N. tabacum almost reached the same level than after irradiation of not fully developed leaves with 50 Gy X-rays. The increased stretching of DNA with leaf age was most obvious if the DNA duplex was converted to single strands by alkali treatment before electrophoresis. Therefore, it could be concluded that with the ageing of leaves there is a decrease in DNA integrity, which could be the result of rising amounts of DNA single-strand breaks and 'alkali-vulnerable sites'.

Comet Assay↗
PubMed2003

Evaluation of the migration of mutagens/carcinogens from PET bottles into mineral water by Tradescantia/micronuclei test, Comet assay on leukocytes and GC/MS.

This study monitored the release of mutagenic/carcinogenic compounds into mineral water (natural and carbonated) from polyethylene terephthalate (PET) bottles, using a plant mutagenicity test which reveals micronuclei formation in Tradescantia pollen cells (Trad/MCN test), a DNA damage assay (Comet assay) on human leukocytes and gas chromatography/mass spectrometry (GC/MS) for the characterisation of migrants. The water samples were collected at a bottling plant and stored in PET bottles for a period ranging from 1 to 12 months. Every month some samples were randomly collected and lyophilised, the residual powders were extracted with organic solvents and then analysed by GC/MS and tested for DNA damage in human leukocytes, or reconstituted with distilled water to obtain concentrates for the exposure of Tradescantia inflorescences. Micronuclei increase in pollen was found only in natural mineral water stored for 2 months. DNA-damaging activity was found in many of the natural and carbonated water samples. Spring water was negative in the plant micronuclei test and the Comet assay, whereas distributed spring water showed DNA-damaging effects, suggesting a possible introduction of genotoxins through the distribution pipelines. GC/MS analysis showed the presence in mineral water of di(2-ethylhexyl)phthalate, a nongenotoxic hepatocarcinogenic plasticizer, after 9 months of storage in PET bottles.

Carcinogens↗
PubMedDate not supplied

Comet assays to assess DNA damage and repair in grass shrimp embryos exposed to phototoxicants.

Exposure of grass shrimp (Palaemonetes pugio) embryos to four compounds (anthracene, pyrene, alpha-terthienyl, methylene blue) along with solar exposure resulted in extensive DNA strand damage using the comet assay. DNA tail moments of embryos exposed to these chemicals in the dark ranged from 1.8 to 4.3, while exposure to chemicals and solar resulted in tail moments of 14.3-15.3. Reduction of DNA tail moments when solar exposed embryos were transferred to the dark, suggested DNA repair systems were active. The comet assay can be used to follow both DNA damage and repair following exposure to phototoxic chemicals.

Animals↗
PubMedDate not supplied

Use of the Comet assay to assess DNA damage in hemocytes and digestive gland cells of mussels and clams exposed to water contaminated with petroleum hydrocarbons.

Oil spills can result in the deposition of large quantities of petroleum hydrocarbons into intertidal and shallow waters seriously impacting bivalve populations. Petroleum hydrocarbons are enriched in polycyclic aromatic hydrocarbons (PAH) and PAH analogs many of which may have potential to damage DNA. The Comet assay is useful for assessing DNA damage and has been used to a limited degree with aquatic organisms, but mostly with studies in vitro. We have carried out studies with the Comet assay to assess the DNA damaging potential of complex mixtures of petroleum hydrocarbons for bivalves. Experiments were carried out with mussels (Mytilus edulis) and clams (Mya arenaria) with dispersions and water soluble fractions of an Arabian crude oil which was also chemically characterized in detail by GC-MS. Pilot studies were first conducted to evaluate test performance and reproducibility. An interindividual coefficient of variation ranging from 17 to 30% was established for the assay with hemocytes and digestive gland cells of both species. Exposure to hydrocarbon fractions had no significant impact on clams. However, an increase in DNA damage was observed at P < 0.1 with digestive gland cells of mussels exposed to aqueous fractions of a light crude oil. These studies have demonstrated a potential for DNA damage in bivalves exposed to oil spills in inshore waters as well as potential for interspecies sensitivity.

Animals↗
PubMed2003

Comet assay on gill cells and hemocytes from the blue mussel Mytilus edulis.

Gill cells and hemocytes from the blue mussel Mytilus edulis were examined for DNA damage using the comet assay after laboratory exposure in vitro and in vivo to methyl methansulfonate (MMS). Hydrogen peroxide and UV radiation were used as positive control. Comet assay was also carried out on hemocytes from blue mussels sampled at polluted and unpolluted coastal areas. After 60 min in vitro exposure of gill cells to MMS, the highest response, a tail moment of 6.70+/-4.25, was obtained at 1.0mg/L. At higher doses the response decreased. After 2 days in vivo exposure a dose response was seen at concentrations between 1.0 and 33.0mg/L MMS for both gill cells and hemocytes. However, after 4 days in vivo exposure using the same concentrations of MMS, a maximum effect was seen at a 10 times lower concentration of 3.3mg/L. At the higher doses, the effect decreased. Hemocytes from blue mussels sampled at four polluted sites in Køge Bay had a great variation in tail moments with the highest value of 5.38+/-4.39. The average of all samples from Køge Bay had tail moments of 2.75+/-1.00(n=19), which was significantly higher (P<0.05) than the average, 1.72+/-1.16(n=10), of samples from unpolluted coastal waters.

Animals↗
PubMed2001

Evaluation of the mutagenic and genotoxic activities of anti-hepatitis B analogs of beta-L-adenosine by the Ames test and the Comet assay.

beta-L-2'-deoxyadenosine (beta-L-dA), beta-L-2',3'-dideoxyadenosine (beta-L-ddA) and its two bis (S-acyl-2-thioethyl; SATE) phosphotriester derivatives, beta-L-2',3'-dideoxyadenosine-5'-monophosphate-bis(MeSATE) and beta-L-2',3'-dideoxyadenosine-5'-monophosphate-bis(tButylSATE) have been previously shown to exhibit potent and selective anti-hepatitis B activity in vitro. None of the four compounds was mutagenic up to 100 microg in the Ames test (microtechnique) using Salmonella typhimurium strains TA 97a, TA 98, TA 100 and TA 102, with and without metabolic activation. In addition, the genotoxicity of beta-LdA and the three other compounds was evaluated in human lymphocytes using the Comet assay, at doses up to 5 microg with or without the addition of a microsomal S9 fraction. None of the four compounds induced DNA strand breakage with and without metabolic activation. In summary, the data clearly demonstrate that the purine nucleoside beta-L-dA, beta-L-ddA and the two prodrugs, beta-L-ddAMP-bis(MeSATE) and beta-L-ddAMP-bis(tButylSATE) are not mutagenic in the Ames test and do not induce DNA damage in human lymphocytes, as assessed by the Comet assay.

Antiviral Agents↗
PubMed2000

Genotoxic effect of lead nitrate on mice using SCGE (comet assay).

Single stranded DNA breakage induced by lead nitrate in mice has been studied in vivo using alkaline single cell gel electrophoresis (comet assay). Mice were administered orally 0.7, 1.4, 2.8, 5.6, 11. 2, 22.4, 44.8 and 89.6 mg/kg body weight of lead nitrate and the assay was performed on whole blood at 24, 48, 72 h, 1st and 2nd week. Significant increase in mean tail-length of DNA was observed at all time intervals after treatment with lead nitrate when compared to controls. The mean tail-length did not show a dose-related increase and the elevation in the mean tail-length was of a fluctuating type. Increase in mean tail-lengths clearly gives evidence that lead nitrate causes DNA damage effectively. The study indicates that the alkaline comet assay is a sensitive and rapid method to detect DNA damage caused by heavy metals.

Animals↗