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PubMed2000

Adaptation to alkylation damage in DNA measured by the comet assay.

The alkylating mutagens N-methyl-N-nitrosourea (MNU) and methyl methanesulfonate (MMS) were studied for their potential to induce DNA strand breaks and abasic (AP) sites in meristematic nuclei of Vicia faba root tips by the comet assay. The alkaline unwinding/neutral electrophoresis (A/N) and alkaline unwinding/alkaline electrophoresis (A/A) protocols were used for detection of DNA damage. With the A/N comet assay, less DNA damage was seen after conditioning pretreatment with a low dose prior to a high challenging dose of alkylating mutagens as compared to application of the high dose only, whereas a nearly additive effect was seen when the A/A comet assay was used. Adaptation was even more obvious when AP sites were revealed by the AP-endonuclease activity of exonuclease III. The adaptation observed with the A/N comet assay was abolished by pretreatment with the protein synthesis inhibitor cycloheximide. These data suggest that the comet assay is able to detect on molecular level a phenomenon resembling clastogenic adaptation.

Adaptation, Physiological↗
PubMed2002

Genotoxicity biomonitoring in regions exposed to vehicle emissions using the comet assay and the micronucleus test in native rodent Ctenomys minutus.

Exposure to motor vehicle emissions represents an important concern for possible long-term health effects. The present report describes: 1) the application and verification of the alkaline comet assay in Ctenomys minutus to detect the possible genotoxicity of automobile emissions; 2) a comparison of the comet assay results with peripheral blood micronucleus (MN) assay results performed in the same animals; and 3) the identification of agents involved in the responses and in the seasonal variation of the effects. Ctenomys minutus (Octodontidae-Rodentia) were captured in two different fields from both sides of RS/030, a highway on the coastal plain of the Brazilian state of Rio Grande do Sul. Reference animals were obtained from a nearby field that was about 3 km distant from any road. By the end of this study, 123 rodents (73 females and 50 males) were live-trapped. Our results indicate that there was an increase in cells with DNA damage for C. minutus environmentally exposed to automobile emissions, as demonstrated by the alkaline comet assay, but there was no increase in micronucleated cells. The alkaline comet assay showed age and gender differences in the response. The comet assay results suggest that adult females are the principal population affected by air pollutants from vehicle emissions. Chemical data were also collected from areas exposed to automobile exhaust and these indicated that elevated levels of hydrocarbons, metals, and NO(2) were associated with the elevated levels of damaged cells observed in the wild rodent C. minutus. Our results agree with previous data on engine and fuel components, where weak increases in damage for native rodents exposed to emissions have been observed. Other larger, controlled studies are needed to better understand how the metabolism of C. minutus affects its response to emission exposure.

Animals↗
PubMed2003

The comet assay: genotoxic damage or nuclear fragmentation?

The single cell gel electrophoresis (SCGE) or comet assay is based on the assumption that comet images result from genotoxic damage that ultimately generate DNA single- or double-strand breaks. A criticism of the assay is that some or all of the comet images may be the result of apoptosis-mediated nuclear fragmentation. The objective of this study was to determine if mutagen-induced DNA damage leading to strand breakage observed in the SCGE assay was repairable or was due to nonrepairable nuclear fragmentation. Chinese hamster ovary cells were treated with ethylmethanesulfonate, 2-acetoxyacetylaminofluorene, or H(2)O(2). These mutagens induce genetic damage by different molecular mechanisms. One group of SCGE slides was prepared immediately after treatment, while parallel treated cultures were repeatedly washed and allowed to undergo liquid holding recovery for DNA repair. It was hypothesized that cells with genotoxic damage can repair their genomic DNA, while apoptotic cells cannot reverse nuclear fragmentation. We found a significant decrease in the tail moments of nuclei from mutagen-treated cells after 4 hr of liquid holding. However, this measurement may represent only those cells capable of repair. Apoptotic cells may continue DNA fragmentation during the recovery time and this DNA may become so diffuse that the nuclei disappear after electrophoresis. To overcome this possible artifact, images of nuclei were captured before and after alkaline electrophoresis. Constellations of nuclei were located on SCGE slides by their coordinates on the microscope stage. We found that no nuclei were lost due to apoptotic nuclear fragmentation and DNA migration. Even the so-called "hedgehog" comet images with extreme DNA damage were not lost during liquid holding. These data support the conclusion that mutagen-induced DNA damage is the principal cause of the damage measured in the comet assay.

Animals↗
PubMed2006

A quantitative comet assay: imaging and analysis of virus plaques formed with a liquid overlay.

Although the plaque assay defines a "gold-standard" for measuring virus infectivity, its reliance on plaque counting limits its sensitivity. When the assay is performed with a liquid overlay, instead of agar overlay, spontaneous flows can promote a uni-directional spread of infection, creating elongated regions of cytopathology that resemble comets. As a model system comet and plaque cultures of vesicular stomatitis virus (VSV) on baby hamster kidney (BHK-21) cells were compared. Host-cell monolayers were infected with VSV particles, incubated 15 h in the presence of liquid or agar overlays and stained. VSV formed significantly larger comets than plaques, consistent with a mechanism of flow-enhanced spread. When antiviral drug (5-fluorouracil) was incorporated into the liquid overlay, comet sizes were reduced in a dose-dependent manner. Images of infected monolayers, acquired using a simple digital scanner, enabled a quantification of the inhibitory effect of the drug on infectivity. The resulting measure of drug susceptibility was found to be 18-fold more sensitive than the IC(50) measure attained by the traditional plaque-reduction assay. This quantitative comet assay has the potential to similarly enhance the sensitivity of infection measures for other plaque-forming viruses.

Animals↗
PubMed2005

A buccal cell model comet assay: development and evaluation for human biomonitoring and nutritional studies.

The comet assay is a widely used biomonitoring tool for DNA damage. The most commonly used cells in human studies are lymphocytes. There is an urgent need to find an alternative target human cell that can be collected from normal subjects with minimal invasion. There are some reports of buccal cells, collected easily from the inside of the mouth, being used in studies of DNA damage and repair, and these were of interest. However, our preliminary studies following the published protocol showed that buccal cells sustained massive damage and disintegrated at the high pH [O. Ostling, K.J. Johanson. Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem. Biophys. Res. Commun. 123 (1984) 291-298] used, but that at lower pH were extremely resistant to lysis, an essential step in the comet assay. Therefore, the aims of this study were to develop a protocol than enabled buccal cell lysis and DNA damage testing in the comet assay, and to use the model to evaluate the potential use of the buccal cell model in human biomonitoring and nutritional study. Specifically, we aimed to investigate intra- and inter-individual differences in buccal cell DNA damage (as strand breaks), the effect of in vitro exposure to both a standard oxidant challenge and antioxidant treatment, as well as in situ exposure to an antioxidant-rich beverage and supplementation-related effects using a carotenoid-rich food. Successful lysis was achieved using 0.25% trypsin for 30 min followed by proteinase K (1mg/ml) treatment for 60 min. When this procedure was performed on cells pre-embedded in agarose on a microscope slide, followed by electrophoresis (in 0.01 M NaOH, 1mM EDTA, pH 9.1, 18 min at 12 V), a satisfactory comet image was obtained, though inter-individual variation was quite wide. Pre-lysis exposure of cells to a standard oxidant challenge (induced by H2O2) increased DNA strand breaks in a dose related manner, and incubation of cells in Trolox (a water soluble Vitamin E analogue) conferred significant protection (P<0.05) against subsequent oxidant challenge. Exposure of buccal cell in situ (i.e. in the mouth) to antioxidant-rich green tea led to an acute decrease in basal DNA strand breaks. In a controlled human intervention trial, buccal cells from 14 subjects after 28 days' supplementation with a carotenoid-rich berry (Fructus barbarum L.) showed a small but statistically significant (P<0.05) decrease in DNA strand breaks. These data indicate that this buccal cell comet assay is a feasible and potentially useful alternative tool to the usual lymphocyte model in human biomonitoring and nutritional work.

Antioxidants↗
PubMed2006

Can individual repair kinetics of UVC-induced DNA damage in human lymphocytes be assessed through the comet assay?

The suitability of the comet assay for quantifying DNA repair capacity at individual level was studied following the kinetics of nucleotide excision repair (NER) in human lymphocytes from four healthy donors, at various time steps after a single dose of UVC. A significant increase of DNA migration was seen as soon as 20 min after UV exposure, reaching the peak within 60-90 min. Afterwards, a rapid decline was observed, approaching the basal level at 180-240 min. The increase could be ascribed to excision activity, while the reduction to gap filling and rejoining, as demonstrated by the effects of phase-specific inhibitors, novobiocin and aphidicolin. Therefore, the comet assay should allow following the biphasic kinetics of NER. Wide inter-individual differences were observed, although repeated tests on the same donor cells revealed a large experimental variation. To quantitatively compare the individual patterns, a mathematical model was developed that adequately fitted the experimental results and estimated appropriate descriptors for each phase and for each donor. A second approach was also used to directly compare the distributions of damaged cells and to assess the differences between donors and between experiments visualizing them as reciprocal distances on a two-dimensional space computed with a principal component analysis (PCA). The results confirmed the inter-individual differences, but also the strong influence of experimental factors of the comet assay. The two approaches provided the means of accurately comparing DNA repair kinetics at individual level, taking also into account the experimental variability which poses serious doubts on the suitability of the comet assay. Nevertheless, since this methodology allows a detailed analysis of repair kinetics and it is potentially very useful for identifying individual with reduced repair capacity, further efforts have to be addressed to improve the reproducibility of the comet assay.

Adult↗
PubMed2003

Detection of oxidative DNA damage in isolated marine bivalve hemocytes using the comet assay and formamidopyrimidine glycosylase (Fpg).

Organisms in polluted areas can be exposed to complex mixtures of chemicals; however, exposure to genotoxic contaminants can be particularly devastating. DNA damage can lead to necrosis, apoptosis, or heritable mutations, and therefore has the potential to impact populations as well as individuals. Single cell gel electrophoresis (the comet assay) is a simple and sensitive technique used to examine DNA damage in single cells. The lesion-specific DNA repair enzyme formamidopyrimidine glycoslyase (Fpg) can be used in conjunction with the comet assay to detect 8-oxoguanine and other damaged bases, which are products of oxidative damage. Fpg was used to detect oxidative DNA damage in experiments where isolated oyster (Crassostrea virginica) and clam (Mercenaria mercenaria) hemocytes were exposed to hydrogen peroxide. Standard enzyme buffers used with Fpg and the comet assay produced unacceptably high amounts of DNA damage in the marine bivalve hemocytes used in this study necessitating a modification of existing methods. A sodium chloride based reaction buffer was successfully used. Oxidative DNA damage can be detected in isolated oyster and clam hemocytes using Fpg and the comet assay when the sodium chloride reaction buffer and protocols outlined here are employed. The use of DNA repair enzymes, such as Fpg, in conjunction with the comet assay expands the usefulness and sensitivity of this assay, and provides important insights into the mechanisms of DNA damage.

Animals↗
PubMed2004

Comet assay using mullet (Mugil sp.) and sea catfish (Netuma sp.) erythrocytes for the detection of genotoxic pollutants in aquatic environment.

The development of comet assay for aquatic organisms is of particular relevance in light of the importance of coastal fisheries to several countries around the world. Two of the most common fish species native to southern Brazil are the gray mullet (Mugil sp.) and sea catfish (Netuma sp.) for which we have produced a standardized comet assay using whole erythrocytes taken from samples of these fish. We investigated the potential of the comet assay for monitoring genotoxicity in mullet and sea catfish and made a preliminary investigation of the baseline levels of DNA damage in the erythrocytes of samples of these fish from non-polluted areas as well as assessing the in vitro sensitivity of erythrocyte exposed to 2, 4 and 8 x 10(-5) M of methyl methanesulfonate (MMS) for 1, 2, 6 and 24h at 25 and 37 degrees C. Our results show that there was an increase in baseline DNA damage at higher temperatures and that the amount of MMS-induced DNA damage also increased at higher temperatures and that there was a clear dose/time response to treatment with MMS. To assess the possibility of using fish for environmental biomonitoring we also used the comet assay to investigate the in vitro genotoxic effect of MMS on whole blood cells from human donors and found a clear concentration-related effect at all exposure times, findings which agree with those of other workers. This study demonstrates the potential application of the comet assay to erythrocytes of mullets and sea catfish. However, these findings also suggest that temperature could alter both baseline DNA damage in untreated animals and in vitro cell sensitivity towards genotoxic pollutants.

Animals↗
PubMed2005

Assessment of DNA damage in peripheral blood of heavy smokers with the comet assay and the micronucleus test.

The comet assay (single-cell gel electrophoresis, SCG) is being increasingly used in human biomonitoring for the detection of genotoxic exposures. Cigarette smoking is a well-documented source of a variety of potentially mutagenic and carcinogenic compounds. Therefore, smoking should represent a relevant mutagenic exposure and lead to genotoxic effects in exposed cells. However, our previous investigations as well as several other published studies on human biomonitoring failed to show an effect of smoking on DNA migration in the comet assay, while some other studies did indicate such an effect. Although many factors can contribute to the generation of discrepant results in such studies, clear effects should be obtained after high exposure. We therefore performed a comparative study with healthy male heavy smokers (>20 cigarettes per day) and non-smokers (n=12 in each group). We measured the baseline comet assay effects in fresh whole blood samples and isolated lymphocytes. In addition, the amount of 'formamidopyrimidine DNA-glycosylase (FPG)-sensitive sites' was determined by a combination of the standard comet assay with the bacterial FPG protein. Furthermore, the influence of a repair inhibitor (aphidicolin, APC) on baseline DNA damage was comparatively analysed. Duplicate slides from each sample were processed and analysed separately. In all experiments, a reference standard (untreated V79 cells) was included to correct for assay variability. Finally, to compare the comet assay results with another genetic endpoint, all blood samples were investigated in parallel by the micronucleus test (MNT). Baseline and gamma radiation-induced micronucleus frequencies were determined. None of these approaches revealed a significant difference between heavy smokers and non-smokers with regard to a genotoxic effect in peripheral blood cells.

Adolescent↗
PubMed2005

Use of Comet assay to assess DNA damage in patients infected by Helicobacter pylori: comparisons between visual and image analyses.

Studies of DNA damage in gastric epithelial cells of Helicobacter pylori (H. pylori)-infected patients are conflicting, possibly due to different methods used for scoring DNA damage by Comet assay. Therefore, we compared the sensitivity of visual microscopic analysis (arbitrary units-scores and comets%) and image analysis system (tail moment), in the gastric epithelial cells from the antrum and corpus of 122 H. pylori-infected and 32 non-infected patients. The feasibility of cryopreserved peripheral blood lymphocytes and whole-blood cells for DNA damage biomonitoring was also investigated. In the antrum, the levels of DNA damage were significantly higher in H. pylori-infected patients with gastritis than in non-infected patients with normal mucosa, when evaluated by image analysis system, arbitrary units and comets%. In the corpus, the comets% was not sufficiently sensitive to detect the difference between H. pylori-infected patients with gastritis and non-infected patients with normal mucosa. The image analysis system was sensitive enough to detect differences between non-infected patients and H. pylori-infected patients with mild gastritis and between infected patients with moderate and severe gastritis, in both antrum and corpus, while arbitrary units and comets% were unable to detect these differences. In cryopreserved peripheral blood lymphocytes, the levels of DNA damage (tail moment) were significantly higher in H. pylori-infected patients with moderate and severe gastritis than in non-infected patients. Overall, our results indicate that the image analysis system is more sensitive and adequate to measure the levels of DNA damage in gastric epithelial cells than the other methods assayed.

Comet Assay↗
PubMed2006

Photo-chemically induced DNA effects in the comet assay with epidermal cells of SKH-1 mice after a single oral administration of different fluoroquinolones and 8-methoxypsoralen in combination with exposure to UVA.

UNLABELLED: Due to the need for in vivo photo-genotoxicity tests, the in vivo photo-comet assay was established in epidermal cells of the SKH-1 mouse. Groups of 10 male SKH-1 mice each were treated once orally with vehicle only, with three fluoroquinolones (25 mg/kg clinafloxacin, 20 mg/kg lomefloxacin, 200 mg/kg ciprofloxacin) or with 200mg/kg 8-methoxypsoralene (8-MOP). Thirty minutes after treatment half of the mice in each group were exposed to 23.8 J/cm2 UVA. Thereafter the mice were killed and their epidermal cells tested in the alkaline (pH >13) comet assay; at the same time after administration, compound-treated, non-irradiated mice were killed and analysed. A negative control group of ten male SKH-1 mice received the vehicle only; half of these animals were exposed to UVA, half were not. The comet tail lengths of epidermal cells of the mice were statistically significantly increased for all three fluoroquinolones (FQ) tested in combination with UV irradiation. Treatment with 8-methoxypsoralene+UV induced a significant reduction of comet tail length. Tail intensity and tail moment gave essentially the same results after combined exposure (compound+UV). Without irradiation, the tail lengths of controls and compound-treated mice were comparable under the conditions of this study. In contrast, tail intensity and tail moment were increased for all test compounds (including 8-MOP), without irradiation. Irradiated controls had a tail length comparable to non-irradiated controls, while tail intensity and tail moment were clearly increased in irradiated controls. IN CONCLUSION: under the present experimental conditions the in vivo photo-comet assay is able to detect photo-chemically induced DNA strand breaks as well as photo-chemically induced DNA cross-links.

Administration, Oral↗
PubMed2004

The use of the alkaline comet assay with lymphocytes in human biomonitoring studies.

We reviewed the data of 45 alkaline comet assay studies with lymphocytes published during the last three years with the objective of monitoring human exposure to genotoxic agents as a result of occupation, drug treatment, diseases or environmental pollution. The strengths of the studies were that: (i) a lot of data could be obtained within a relatively short period of time in a cost-effective manner, (ii) lymphocytes could be easily collected in a non-invasive way and proved to be good surrogate cells in that they picked up effects caused by agents with different cancer target organs and (iii) a remarkable concordance between comet assay and cytogenetic assay data was proved. However, our analysis revealed some shortcomings of the studies such as: (i) the inclusion of low number of study participants and bias in the number and gender of subjects between control and exposed groups, (ii) lack of qualitative and quantitative exposure data, (iii) lack of consideration of differences in physical activity and diet between control and exposed groups, (iv) difficulty in comparison of the studies due to lack of uniformity in the comet assay procedures such as duration of alkali unwinding and electrophoresis, slide scoring method and the metrics used to assess the extent of DNA damage and (v) controversy in the sensitivity of comet assay since it picked up DNA damage caused by agents such as wood dust, pesticides and hormone preparations which were found to be weak genotoxins or non-genotoxins in other tests, but gave inconsistent results with known mutagens/carcinogens such as tobacco smoke. We feel that for the alkaline comet assay to be an important tool in human biomonitoring studies, serious consideration should be given to the flaws in the design and performance of the assay.

Comet Assay↗
PubMed2004

Sensitivity of the FPG protein towards alkylation damage in the comet assay.

The comet assay (single cell gel electrophoresis) is widely used for the evaluation of DNA-damaging effects in genotoxicity testing and population monitoring. In its standard version at pH >13, DNA double strand breaks (DSB), DNA single strand breaks (SSB) and alkali-labile sites (ALS) lead to increased DNA migration. At reduced pH (12.5-12.1) the expression of ALS as SSB can be eliminated and the effect of SSB only can be identified. Specific endonucleases have been used to characterize specific classes of DNA damage. The formamido pyrimidine glycosylase (FPG) protein has been used to assess oxidative DNA base damage because it detects 8-OH guanine and other oxidatively damaged purines. Here, we show that the FPG protein also detects alkylation damage with high sensitivity in the comet assay. Human whole blood, isolated lymphocytes and V79 cells were treated with alkylating agents and post-incubated with FPG. FPG strongly enhanced MMS- and EMS-induced DNA damage but had no significant effect on ENU-induced DNA damage, indicating that the amount of N-7 guanine alkylation is responsible for the observed effect. Reducing the pH during alkali unwinding and electrophoresis to 12.5 to avoid the contribution of ALS to the comet assay effects, strongly decreased the sensitivity of the comet assay with and without FPG treatment and prevented DNA migration. We conclude that enhanced DNA effects in the comet assay by FPG after exposure to genotoxins with unknown mode of action should not directly be regarded as evidence for the presence of oxidative damage. Furthermore, reducing the pH leads to a considerable loss in sensitivity and should not be used in biomonitoring and other applications which require a sensitive protocol.

Alkylation↗
PubMed2001

Gamma ray induced DNA damage in human and mouse leucocytes measured by SCGE-Pro: a software developed for automated image analysis and data processing for Comet assay.

The studies reported in this communication had two major objectives: first to validate the in-house developed SCGE-Pro: a software developed for automated image analysis and data processing for Comet assay using human peripheral blood leucocytes exposed to radiation doses, viz. 2, 4 and 8 Gy, which are known to produce DNA/chromosome damage using alkaline Comet assay. The second objective was to investigate the effect of gamma radiation on DNA damage in mouse peripheral blood leucocytes using identical doses and experimental conditions, e.g. lyses, electrophoretic conditions and duration of electrophoresis which are known to affect tail moment (TM) and tail length (TL) of comets. Human and mouse whole blood samples were irradiated with different doses of gamma rays, e.g. 2, 4 and 8 Gy at a dose rate of 0.668Gy/min between 0 and 4 degrees C in air. After lyses, cells were electrophorased under alkaline conditions at pH 13, washed and stained with propidium iodide. Images of the cells were acquired and analyzed using in-house developed imaging software, SCGE-Pro, for Comet assay. For each comet, total fluorescence, tail fluorescence and tail length were measured. Increase in TM and TL was considered as the criteria of DNA damage. Analysis of data revealed heterogeneity in the response of leucocytes to gamma ray induced DNA damage both in human as well as in mouse. A wide variation in TM and TL was observed in control and irradiated groups of all the three donors. Data were analyzed for statistical significance using one-way ANOVA. Though a small variation in basal level of TM and TL was observed amongst human and mouse controls, the differences were not statistically significant. A dose-dependent increase in TM (P<0.001) and TL (P<0.001) was obtained at all the radiation doses (2-8 Gy) both in human and mouse leucocytes. However, there was a difference in the nature of dose response curves for human and mouse leucocytes. In human leucocytes, a linear increase in TM and TL was observed up to the highest radiation dose of 8 Gy. However, in case of mouse leucocytes, a sharp increase in TM and TL was observed only up to 4 Gy, and there after saturation ensued. In human samples, the dose response of both TM and TL showed best fits with linear model (r(TM)=0.999 and r(TL)=0.999), where as in mouse, the best fit was obtained with Sigmoid (Boltzman) model. From the present data on leucocytes with increase in TM and TL as the criteria of DNA damage, it appears that mouse is relatively more sensitive to radiation damage than humans.

Adult↗
PubMed2001

Comparison of genotoxicity of sevoflurane and isoflurane in human lymphocytes studied in vivo using the comet assay.

In the present paper, we report data on the possible genotoxic properties of two inhalation anaesthetics--sevoflurane (SVF) and isoflurane (ISF) - in peripheral blood lymphocytes of patients before, during and after anaesthesia as compared to an unexposed control group. Both anaesthetics were evaluated for genotoxic activity using the comet assay. The exposed groups consisted of 24 ASA grades 1-2 unpremedicated patients (aged 20-66 years, anaesthetized 115-162 min for elective lower abdominal surgery), while the control group consisted of 12 healthy individuals. After induction of anaesthesia (thiopenthone sodium 5-7 mg/kg, fentanyl citrate 0.1mg and vecuronium bromide 0.1mg/kg), anaesthesia was maintained with inhalation of SVF 1-1.5% (n=12) or ISF 1-1.5% (n=12) in oxygen-air mixture. Venous blood samples were obtained before the induction of anaesthesia, at 60 and 120 min of anaesthesia and on the first, third and fifth days following anaesthesia. The comet assay detects DNA damage which includes strand breaks and alkaline labile sites induced directly by genotoxic agents as well as DNA degradation due to cell death. One hundred cells from each sample were examined and graded as no tailed, short and long tailed nuclei. The mean comet response was not different between controls and patients before anaesthesia. However, similar significant increases were observed in the mean comet response in blood sampled from patients at 60 (36.5+/-11.2, 37.8+/-12.1), or 120 min (53.1+/-17.1, 50.0+/-12.2) of anaesthesia and on the first day (37.8+/-15.1, 35.2+/-15.7) after anaesthesia in SVF and ISF treated groups, respectively. Removal of the DNA damage was observed after the third day of anaesthesia and the repair was completed within 5 days. The DNA damage detected in lymphocytes of patients during anaesthesia with SVF or ISF showed similar results as demonstrated by an increased mean comet migration at 120 min of anaesthesia and the cells were able to repair the induced DNA damage completely on the fifth postoperative day.

Adult↗
PubMed2002

Assessment of genotoxic effects of chloropyriphos and acephate by the comet assay in mice leucocytes.

Two organophosphorus (OP) pesticides (chloropyriphos and acephate) and cyclophosphamide (CP) (positive control) were tested for their ability to induce in vivo genotoxic effect in leucocytes of Swiss albino mice using the single cell gel electrophoresis assay or comet assay. The mice were administered orally with doses ranging from 0.28 to 8.96 mg/kg body weight (b. wt.) of chloropyriphos and 12.25 to 392.00 mg/kg b.wt. of acephate. The assay was performed on whole blood at 24, 48, 72 and 96 h. A significant increase in mean comet tail length indicating DNA damage was observed at 24h post-treatment (P<0.05) with both pesticides in comparison to control. The damage was dose related. The mean comet tail length revealed a clear dose dependent increase. From 48 h post-treatment, a gradual decrease in mean tail length was noted. By 96 h of post-treatment the mean comet tail length reached control levels indicating repair of the damaged DNA. From the study it can be concluded that the comet assay is a sensitive assay for the detection of genotoxicity caused by pesticides.

Animals↗
PubMed2002

Comparative investigation of multiple organs of mice and rats in the comet assay.

Mice and/or rats are usually used to detect chemical carcinogenicity and it has been known that there are species differences in carcinogenicity. To know whether there are species difference in genotoxicity, we conducted comparative investigation of multiple organs of mice and rats in the comet assay. Since the sensitivity to xenobiotics is different for different species, we queried species difference in the genotoxic sensitivity at one equitoxic level but not at one equidose. Therefore, groups of four mice or rats were treated once intraperitoneally or orally with a chemical at highest dose without death and distinct toxic manifestation. When the death was not observed at 2000 mg/kg of a chemical, 2000 mg/kg was used for the comet study. The stomach, colon, liver, kidney, bladder, lung, brain, and bone marrow were sampled 3, 8, and 24h after treatment. Among chemicals tested, benzyl acetate, chlorodibromomethane and p-chloro-o-toluidine are carcinogenic to mice but not rats, and aniline, azobenzene, o-phenylphenol Na, and D-limonene are carcinogenic to rats but not mice. Although the two species differed in genotoxicity target organs and migration values, the judgement of a positive or negative response was the same for all chemicals studied except for 2,4-dimethoxyaniline, 2,5-diaminotoluene, and p,p'-DDT when chemicals with positive responses in at least one organ are judged to be comet assay-positive. 2,4-Dimethoxyaniline and 2,5-diaminotoluene that are Ames test-positive non-carcinogens in both species were positive in one organ (urinary bladder for 2,4-dimethoxyaniline and stomach for 2,5-diaminotoluene) in rats, but negative in all mouse organs. p,p'-DDT, which is an Ames test-negative but in vitro cytogenetic test-positive hepatic carcinogen in mice and rats, was positive in multiple rat organs, but not in any mouse organ. These results suggest that species differences in genotoxicity at one equitoxic level are not consistent with species difference in carcinogenicity and that the use of both species is appropriate to indicate a carcinogenic potential in the comet assay with multiple organs, when chemicals being positive in at least one organ are judged to be comet assay-positive.

Alkylating Agents↗
PubMed2002

A first step in visual identification of different cell populations by a modified alkaline comet assay.

Using a particular model of apoptosis, we here demonstrate the ability of the comet assay to differentiate between different cell populations. In our study, the natural killer Kurloff cells, used as effector cells, recognize and bind to the tumoral L2C target cells. Formation of such conjugates leads to the death of the target cells by apoptosis, as previously described by different conventional techniques. With the alkaline comet assay, a conjugate could directly be visualized as an association of an undamaged cell joined to a highly damaged cell. The modified comet assay used in this study comprises specific labelling of Kurloff cells with immunomagnetic beads, which are visible as grey-dull spheres against the bright-red staining of nuclear origin on the comet preparation. The use of such labelled effector cells suggest the potential of the comet assay to visually identify different cell populations in an unique test.

Animals↗