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At least 19 recordsLinked to original sources

PubMed2003

Miniaturizing the comet assay with 3D vertical comets.

BACKGROUND: The comet or single-cell gel electrophoresis assay is a sensitive method for the detection of DNA damage. The main drawback of comet sampling is the low cell density necessary to prevent nucleus overlap after electrophoresis, which limits large-scale high throughput screening. Another problem may be inconsistent comet focusing. We investigated whether an approach based on three-dimensional (3D) confocal microscopy might be beneficial for these concerns. METHODS: A vertical comet assay enabling three-dimensional confocal comet imaging of nuclei seeded at very high density was developed together with dedicated software algorithms to retrieve quantitative data at the single cell level. RESULTS: Three-dimensional confocal comet imaging greatly relieved the user interactions of our nonautomated two-dimensional comet sampling procedure. Batches of comets were blindly sampled, and confocal sectioning improved the clarity of the images and the accuracy of comet sampling. A 1-Gy dose response was readily established. The sampling speed was competitive with that of commercial packages. CONCLUSIONS: Vertical comet imaging is a new concept for fast and user-friendly comet sampling that allows miniaturization of the assay. It may become an essential step toward high throughput screening and exploit the benefits of confocal imaging.

Algorithms↗

PubMed2000

The comet assay with multiple mouse organs: comparison of comet assay results and carcinogenicity with 208 chemicals selected from the IARC monographs and U.S. NTP Carcinogenicity Database.

The comet assay is a microgel electrophoresis technique for detecting DNA damage at the level of the single cell. When this technique is applied to detect genotoxicity in experimental animals, the most important advantage is that DNA lesions can be measured in any organ, regardless of the extent of mitotic activity. The purpose of this article is to summarize the in vivo genotoxicity in eight organs of the mouse of 208 chemicals selected from International Agency for Research on Cancer (IARC) Groups 1, 2A, 2B, 3, and 4, and from the U.S. National Toxicology Program (NTP) Carcinogenicity Database, and to discuss the utility of the comet assay in genetic toxicology. Alkylating agents, amides, aromatic amines, azo compounds, cyclic nitro compounds, hydrazines, halides having reactive halogens, and polycyclic aromatic hydrocarbons were chemicals showing high positive effects in this assay. The responses detected reflected the ability of this assay to detect the fragmentation of DNA molecules produced by DNA single strand breaks induced chemically and those derived from alkali-labile sites developed from alkylated bases and bulky base adducts. The mouse or rat organs exhibiting increased levels of DNA damage were not necessarily the target organs for carcinogenicity. It was rare, in contrast, for the target organs not to show DNA damage. Therefore, organ-specific genotoxicity was necessary but not sufficient for the prediction of organ-specific carcinogenicity. It would be expected that DNA crosslinkers would be difficult to detect by this assay, because of the resulting inhibition of DNA unwinding. The proportion of 10 DNA crosslinkers that was positive, however, was high in the gastrointestinal mucosa, stomach, and colon, but less than 50% in the liver and lung. It was interesting that the genotoxicity of DNA crosslinkers could be detected in the gastrointestinal organs even though the agents were administered intraperitoneally. Chemical carcinogens can be classified as genotoxic (Ames test-positive) and putative nongenotoxic (Ames test-negative) carcinogens. The Ames test is generally used as a first screening method to assess chemical genotoxicity and has provided extensive information on DNA reactivity. Out of 208 chemicals studied, 117 are Ames test-positive rodent carcinogens, 43 are Ames test-negative rodent carcinogens, and 30 are rodent noncarcinogens (which include both Ames test-positive and negative noncarcinogens). High positive response ratio (110/117) for rodent genotoxic carcinogens and a high negative response ratio (6/30) for rodent noncarcinogens were shown in the comet assay. For Ames test-negative rodent carcinogens, less than 50% were positive in the comet assay, suggesting that the assay, which detects DNA lesions, is not suitable for identifying nongenotoxic carcinogens. In the safety evaluation of chemicals, it is important to demonstrate that Ames test-positive agents are not genotoxic in vivo. This assay had a high positive response ratio for rodent genotoxic carcinogens and a high negative response ratio for rodent genotoxic noncarcinogens, suggesting that the comet assay can be used to evaluate the in vivo genotoxicity of in vitro genotoxic chemicals. For chemicals whose in vivo genotoxicity has been tested in multiple organs by the comet assay, published data are summarized with unpublished data and compared with relevant genotoxicity and carcinogenicity data. Because it is clear that no single test is capable of detecting all relevant genotoxic agents, the usual approach should be to carry out a battery of in vitro and in vivo tests for genotoxicity. The conventional micronucleus test in the hematopoietic system is a simple method to assess in vivo clastogenicity of chemicals. Its performance is related to whether a chemical reaches the hematopoietic system. Among 208 chemicals studied (including 165 rodent carcinogens), 54 rodents carcinogens do not induce micronuclei in mouse hematopoietic system despite the positive finding with one or two in vitro tests. Forty-nine of 54 rodent carcinogens that do not induce micronuclei were positive in the comet assay, suggesting that the comet assay can be used as a further in vivo test apart from the cytogenetic assays in hematopoietic cells. In this review, we provide one recommendation for the in vivo comet assay protocol based on our own data.

Animals↗

PubMed2003

Recommendations for conducting the in vivo alkaline Comet assay. 4th International Comet Assay Workshop.

The in vivo alkaline single cell gel electrophoresis assay, hereafter the Comet assay, can be used to investigate the genotoxicity of industrial chemicals, biocides, agrochemicals and pharmaceuticals. The major advantages of this assay include the relative ease of application to any tissue of interest, the detection of multiple classes of DNA damage and the generation of data at the level of the single cell. These features give the Comet assay potential advantages over other in vivo test methods, which are limited largely to proliferating cells and/or a single tissue. The Comet assay has demonstrated its reliability in many testing circumstances and is, in general, considered to be acceptable for regulatory purposes. However, despite the considerable data published on the in vivo Comet assay and the general agreement within the international scientific community over many protocol-related issues, it was felt that a document giving detailed practical guidance on the protocol required for regulatory acceptance of the assay was required. In a recent meeting held in conjunction with the 4th International Comet Assay Workshop (Ulm, Germany, 22-25 July 2001) an expert panel reviewed existing data and recent developments of the Comet assay with a view to developing such a document. This paper is intended to act as an update to the more general guidelines which were published as a result of the International Workshop on Genotoxicity Test Procedures. The recommendations are also seen as a major step towards gaining more formal regulatory acceptance of the Comet assay.

Animals↗

PubMed1987

A proposed Comet Nucleus Penetrator for the Comet Rendezvous Asteroid Flyby mission.

Among the major objectives of NASA's program of space exploration is a better understanding of the origin and evolution of the solar system. Crucial to this objective is the study of comets, which are thought to be the most primitive, pristine bodies remaining in the solar system. The importance of the study of comets has led NASA to plan a mission to rendezvous with comet Tempel 2 in 1997. Critical to the understanding of comets will be measurements of the nucleus material to determine its elemental and isotopic composition, its mechanical properties, and its thermal state and properties. This paper describes a proposal for a Comet Nucleus Penetrator to accomplish these measurement goals. The Comet Nucleus Penetrator will implant instruments into the comet's nucleus beneath a probable volatile-depleted surface mantle into material more representative of the bulk composition of the nucleus.

Alpha Particles↗

PubMed1998

Emerging applications of the single cell gel electrophoresis (Comet) assay. I. Management of invasive transitional cell human bladder carcinoma. II. Fluorescent in situ hybridization Comets for the identification of damaged and repaired DNA sequences in individual cells.

ABSTRACT I: Management of invasive transitional cell human bladder carcinoma. The two main treatment options for invasive transitional cell bladder carcinoma are radiotherapy or primary cystectomy with urinary diversion or bladder substitution. Approximately 50% of patients fail to respond to radiotherapy and such patients so treated are disadvantaged by the absence of predictive information regarding their radiosensitivity, since the tumour gains additional time for metastatic spread before cystectomy is performed. The SF2 clonogenic assay, which measures the surviving fraction of tumour cells after 2 Gy X-ray irradiation, is regarded as a good measure of radiosensitivity. However, the assay is time consuming and provides results for only approximately 70% of human tumours. In this paper three bladder transitional cell carcinoma cell lines (HT1376, UMUC-3 and RT112) were exposed to X-irradiation (0-10 Gy). We have compared the responses obtained using a clonogenic assay and a more clinically feasible alkaline single cell gel electrophoresis (Comet) assay. A very good inverse correlation was obtained between cell survival (clonogenic assay) and mean tail moment (Comet assay) for the three cell lines, indicating that the Comet assay can be used to predict the radio-responsiveness of individual cell lines. The clinical usefulness of the assay for predicting response to radiotherapy in bladder cancer patients is currently being investigated. ABSTRACT II: Fluorescent in situ hybridization (FISH) Comets for the identification of damaged and repaired DNA sequences in individual cells. In mammalian cells the extent of DNA damage is partly and the rate of DNA repair very considerably dependent on DNA position and transcription. This has been established by biochemical techniques which are labour intensive and require large numbers of cells. The Comet assay for overall DNA damage and repair is relatively simple and allows individual cells to be examined. Here we present a protocol for combination of the Comet assay with fluorescent in situ hybridization (FISH) using a p53 gene probe which allows specific observation of p53 sequences within DNA comets. Chromosome-specific probes can also be used. Optimization of the FISH/Comet protocol to include automation of the analysis is currently underway to facilitate future application of the technique to study selective DNA damage and repair in defined sequences in single mammalian cells.

Carcinoma, Transitional Cell↗

PubMed1995

The comet moment as a measure of DNA damage in the comet assay.

The development of rapid assays of radiation-induced DNA damage requires the definition of reliable parameters for the evaluation of dose-response relationships to compare with cellular endpoints. We have used the single-cell gel electrophoresis (SCGE) or 'comet' assay to measure DNA damage in individual cells after irradiation. Both the alkaline and neutral protocols were used. In both cases, DNA was stained with ethidium bromide and viewed using a fluorescence microscope at 516-560 nm. Images of comets were stored as 512 x 512 pixel images using OPTIMAS, an image analysis software package. Using this software we tested various parameters for measuring DNA damage. We have developed a method of analysis that rigorously conforms to the mathematical definition of the moment of inertia of a plane figure. This parameter does not require the identification of separate head and tail regions, but rather calculates a moment of the whole comet image. We have termed this parameter 'comet moment'. This method is simple to calculate and can be performed using most image analysis software packages that support macro facilities. In experiments on CHO-K1 cells, tail length was found to increase linearly with dose, but plateaued at higher doses. Comet moment also increased linearly with dose, but over a larger dose range than tail length and had no tendency to plateau.

Animals↗

PubMed2005

Comparative study of the comet assay and the micronucleus test in amphibian larvae (Xenopus laevis) using benzo(a)pyrene, ethyl methanesulfonate, and methyl methanesulfonate: establishment of a positive control in the amphibian comet assay.

The present investigation explored the potential use of the comet assay (CA) as a genotoxicity test in the amphibian Xenopus laevis and compared it with the French standard micronucleus test (MNT). Benzo[a]pyrene (B[a]P), methyl methanesulfonate (MMS), and ethyl methanesulfonate (EMS) were used as model compounds for assessing DNA damage. Damage levels were measured as DNA strand breaks after alkaline electrophoresis of nuclei isolated from larval amphibian erythrocytes using the CA in order to establish a positive control for further ecotoxicological investigations. The results led to the selection of MMS as a positive control on the basis of the higher sensitivity of Xenopus laevis to this compound. The CA and MNT were compared for their ability to detect DNA damage with the doses of chemical agents and exposure times applied. EMS and MMS were shown to increase micronucleus and DNA strand break formation in larval erythrocytes concurrently. However, B[a]P increased micronucleus formation but not that of DNA strand breaks. Time-dose experiments over 12 days of exposure suggest that the CA provides an earlier significant response to genotoxicants than does the MNT. In Xenopus the CA appears to be a sensitive and suitable method for detecting genotoxicity like that caused by EMS and MMS. It can be considered a genotoxicity-screening tool. The results for B[a]P show that both tests should be used in a complementary manner on Xenopus.

Animals↗

PubMed1987

Comet Sugano-Saigusa-Fujikawa (1983V)--a small, puzzling comet.

Spectroscopic and infrared observations of Comet Sugano-Saigusa-Fujikawa (1983V) were obtained during its close approach to the Earth on 11-14 June 1983. The [O I] production rates of 1.8 +/- 0.9 x 10(26) atoms/s observed on 12.3 June and 7 +/- 3.5 x 10(26) atoms/s on 13.4 June lead to derived water-production rates of 3 x 10(27) mol/s on 12 June and 1.1 x 10(28) mol/s on 13 June. The abundances of the minor species NH2, CN, C2, and C3 are unusually low relative to [O I]. The upper limit to the average nuclear radius from our infrared and visual photometry on 12-13 June (assuming that the entire signal came from the nucleus) is approximately 370 m. The dust/gas mass ratio was <0.01 on June 12 and <0.005 on June 13.

Astronomical Phenomena↗

PubMed2000

Laser scanning cytometry for comet assay analysis.

BACKGROUND: The comet assay (single-cell gel electrophoresis) is a sensitive method for evaluating nuclear DNA damage. Previously used evaluation methods for the comet assay are time consuming and have an inherent risk of biased selection of comets due to manual selection and categorization of comet images. Laser scanning cytometry (LSC), the principle of which is equivalent to flow cytometry, enables quantification of fluorescence emitted from the cells on a microscope slide. In the present study, we explored whether LSC could be used to determine the degree of DNA damage demonstrated by the comet assay. METHODS: DNA damage was induced by ultraviolet A irradiation of keratinocytes and visualized by the comet assay. The evaluation included (a) LSC determination of DNA-specific fluorescence in 1,000 comet heads (undamaged DNA), (b) image acquisition of comets by rescanning of the microscope slide, and (c) digital image analysis and computation of tail moment and DNA content in the comet tails. RESULTS: Cells with damaged DNA were observed in a sub-G(1) area because the comet head loses DNA to the tail. We found a strong inverse correlation between tail moment and DNA content per nucleus. CONCLUSIONS: LSC enables an automated method for cell recognition and evaluation of the comets, thus providing quantitative information about nuclear DNA damage without subjective selection of analyzed comets.

Cells, Cultured↗

PubMed2001

Use of the alkaline comet assay for industrial genotoxicity screening: comparative investigation with the micronucleus test.

We evaluated the suitability of the alkaline comet assay as a screening test in industrial routine testing of new chemicals. Thirty-six pharmaceutical compounds with unknown genotoxic potential were tested comparatively in the comet assay and micronucleus test (MNT) using V79 Chinese hamster cells. The comparison of results is generally based on at least two independent experiments, each with two replicate cultures at a minimum of three concentrations. We found a high degree of concordance between results of the comet assay and MNT. All compounds with negative MNT results were also negative in the comet assay. All positive compounds in the comet assay were also positive in the MNT. However, 16 of 38 positive MNT results were negative in the comet assay. Some of the contrary findings may be due to aneugenic effects, which are detected in the MNT but not in the comet assay. However, the majority of the contrary results may be a consequence of cytotoxicity, which can induce elevated micronucleus frequencies but may not lead to positive effects in the comet assay. Additional data of 39 compounds tested in the Ames test and the comet assay were compared. Four of these compounds that were Ames positive were also positive in the comet assay. However, the comet assay also detected 16 compounds that were negative in the Ames test. We believe that the comet assay in vitro is a useful, fast screening system in mammalian cells that can be used in a test battery during drug development.

Animals↗

PubMed2003

Detection of radiation-induced apoptosis using the comet assay.

The electrophoresis pattern of apoptotic cells detected by the comet assay has a characteristic small head and spread tail. This image has been referred to as an apoptotic comet, but it has not been previously proven to be apoptotic cells by any direct method. In order to identify this image obtained by the comet assay as corresponding to an apoptotic cell, the frequency of appearance of apoptosis was examined using CHO-K1 and L5178Y cells which were exposed to gamma irradiation. As a method for detecting apoptosis, the terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay was used. When the frequency of appearance of apoptotic cells following gamma irradiation was observed over a period of time, there was a significant increase in appearance of apoptosis when using the TUNEL assay. However, there was only a slight increase when using the comet assay. In order to verify the low frequency of appearance of apoptosis when using the comet assay, we attempted to use the TUNEL assay to stain the apoptotic comets detected in the comet assay. The apoptotic comets were TUNEL positive and the normal comets were TUNEL negative. This indicates that the apoptotic comets were formed from DNA fragments with 3'-hydroxy ends that are generated as cells undergo apoptosis. Therefore, it was understood that the characteristic pattern of apoptotic comets detected by the comet assay corresponds to cells undergoing apoptosis.

Animals↗

PubMed2005

Genotoxicity of environmental agents assessed by the alkaline comet assay.

Generation of DNA damage is considered to be an important initial event in carcinogenesis. A considerable battery of assays exists for the detection of different genotoxic effects of compounds in experimental systems, or for investigations of exposure to genotoxic agents in environmental or occupational settings. Some of the tests may have limited use because of complicated technical setup or because they only are applicable to a few cell types. The single cell gel electrophoresis (comet) assay is technically simple, relatively fast, cheap, and DNA damage can be investigated in virtually all mammalian cell types without requirement for cell culture. The aim of this thesis was to evaluate the comet assay as a genotoxicity test in genetic toxicology of environmental agents, encompassing both experimental animal models and biomonitoring. The comet assay detects strand breaks (SB). The cells are embedded in agarose and lysed, generating nucleus-like structures in the gel (referred to as nucleoids). Following alkaline electrophoresis, the DNA strands migrate toward the anode, and the extent of migration depends on the number of SB in the nucleoid. The migration is visualized and scored in a fluorescence microscope after staining. Broad classes of oxidative DNA damage can be detected as additional SB if nucleoids are incubated with bacterial DNA glycosylase/endonuclease enzymes. Oxidized pyrimidines and purines can be detected by incubation with endonuclease III and formamidopyrimidine DNA glycosylase, respectively. The animal experimental studies indicated that the comet assay was able to detect genotoxic effects of diesel exhaust particles in lung tissue, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)-induced DNA damage in colon epithelial cells and liver tissue, and benzene-induced damage in bone marrow and liver cells. The strength of the comet assay was further outlined by application of repair enzymes, indicating no oxidative DNA base damage following IQ treatment. High levels of oxidative DNA lesions were detected after exposure to benzene or X-ray irradiation. The comet assay did not detect DNA damage in colon or liver following ingestion of diets containing of high contents of animal fat or sucrose, although other indices of DNA damage were found. Determined from the results of a large Japanese study, the discrimination between carcinogens and non-carcinogens appears to be similar between the comet assay and alkaline elution, which also detects SB. This suggests that the comet assay is a reliable genotoxicity test in animal experimental systems. In the biomonitoring studies, we investigated the effect of common exposures and lifestyle factors (rather than effects of known carcinogens) on the level of oxidative DNA damage in mononuclear blood cells of humans. In the first study, based on repeated measurements, it was shown that interindividual variation and seasonal variation were major determinants for the basal level of SB, whereas no effect of age, exercise, or antioxidant intake could be detected. The effect of exercise was further investigated under both normoxic and hypoxic circumstances, showing a strong effect of hypoxia, and only effect of exercise in terms of SB in hypoxia. In a placebo-controlled parallel dietary fruit and vegetable (or the corresponding amount of antioxidants) intervention study, no effects of the level of oxidative DNA damage or sensitivity to hydrogen peroxide were observed. Although this may seem in contrast to other antioxidant intervention studies, a critical literature survey of antioxidant intervention studies on oxidative DNA damage suggested that well-controlled studies tended to show no effect of antioxidant supplementation. In summary, the aggregated data from the publications included in this thesis, and other publications encompassing the comet assay, indicate that the comet assay is a reliable method for detection of DNA damage in tissues of experimental animals. Although not all types of genotoxic exposures should be expected to result in DNA damage in mononuclear blood cells, the comet assay seems to be a valuable tool for detection of genotoxic exposure in humans. The comet assay indicates that DNA damage is abundant in mammalian cells and affected by lifestyle and many environmental exposures, including diet, exercise, hypoxia, and sunlight.

Animals↗

PubMed1999

The DNA repair inhibitors hydroxyurea and cytosine arabinoside enhance the sensitivity of the alkaline single-cell gel electrophoresis ('comet') assay in metabolically-competent MCL-5 cells.

We have found previously that the metabolically-competent human MCL-5 cell line did not appear to be usefully sensitive to the DNA-damaging effects of several carcinogens, as measured by the alkaline single-cell gel electrophoresis ('comet') assay. We therefore sought to increase its sensitivity by inhibiting DNA repair during exposure to test compounds, using 10 mM hydroxyurea (HU) and 1.8 mM cytosine arabinoside (ara-C), which inhibit DNA resynthesis during nucleotide excision repair. The following compounds were tested, using a 30-min exposure, in the absence or presence of HU/ara-C: 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (8-MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4, 8-DiMeIQx), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-9H-pyrido[2,3-b]indole (A[alpha]C), 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeA[alpha]C), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), benzo[a]pyrene (B[a]P), 3-methylcholanthrene (3-MCA), 7, 12-dimethylbenz[a]anthracene (DMBA), 1-nitropyrene (1-NP), 2-nitrofluorene (2-NF), aniline, o-toluidine, benzene, lindane, bleomycin, cisplatin, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), sodium chromate, chromic chloride, and diethylstilboestrol (DES). We made the following observations. The background level of comet formation was reasonably constant over several months and was increased only slightly, but significantly, in the presence of the DNA-repair inhibitors. All compounds that induced comet formation did so without appreciable cytotoxicity as assessed by trypan blue exclusion. Of the compounds tested, the heterocyclic amines and polycyclic aromatic hydrocarbons (with the exceptions of PhIP and B[a]P) failed to induce convincing levels of comet formation in the absence of repair inhibitors. In their presence the heterocyclic amines tested induced comet formation (with the exception of 8-MeIQx), with widely differing potencies. 1-NP failed to elicit marked comet formation even in the presence of HU/ara-C. Aniline and o-toluidine produced significant levels of comet formation in the absence of HU/ara-C, but in their presence comet formation was markedly increased. Benzene, lindane, bleomycin, cisplatin, MNNG, sodium chromate and chromic chloride induced comet formation in the absence of HU/ara-C, but, with the exception of cisplatin, their presence enhanced comet formation. Neither sucrose nor DES elicited comet formation under the conditions used in this study. Many more agents need to be tested in order to determine how well the comet assay using MCL-5 cells (or modified versions of it) can distinguish genotoxins from non-genotoxins.

Amines↗

PubMed2002

Modification of lung cancer susceptibility by green tea extract as measured by the comet assay.

Green tea is widely consumed throughout the world and is known to possess various beneficial properties that may affect carcinogen metabolism, free radical scavenging, or formation of DNA adducts. Therefore, it is plausible that green tea extract may modify BPDE-induced DNA damage. In this report, we utilized the comet assay to (1) evaluate BPDE-induced DNA damage as a potential marker of cancer susceptibility and (2) assess the ability of green tea to modify BPDE-induced DNA damage. DNA damage in individual comet cells was quantified by (1) visually measuring the proportion of cells exhibiting migration versus those without and (2) the length of damaged DNA migration (comet tail). We detected a dose-response between BDPE concentration and mean comet tail length in EBV-immortalized lymphoblastiod (lymphoid) cell lines. As the concentration of BPDE increased from 0.5 to 3 microM, the length of the mean comet tail length increased proportionally in the 3590P (derived from a healthy subject) and 3640P (derived from a patient with head and neck cancer) cell lines. In separate experiments using lymphoid cells from 21 lung cancer cases and 12 healthy subjects, the mean comet tail length was significantly higher in the lung cancer cases (80.19 +/- 15.55) versus the healthy subjects (59.94 +/- 14.23) (P < 0.01). Similar findings were observed when analyzing the mean percentage of comet induced cells (84.57 +/- 8.85 and 69.04 +/- 12.50, respectively) (P < 0.01). When green tea extract was added in conjunction with BPDE, there was a notable reduction of the mean comet tail length (13.29 +/- 0.97) as compared to BPDE treatment alone (80.19 +/- 15.55) (P < 0.01) in lung cancer cases. There were no statistical differences between the baseline (no treatments) (12.74 +/- 0.63) and the green tea extract treatment (13.06 +/- 0.97) (P = 0.21). These data suggest the modification of lung cancer susceptibility by the green tea extract. Similar results were observed for the percentage of induced comet cells and the statistical trends were similar for the 12 healthy subjects. This preliminary study demonstrated that the detection of BPDE-induced DNA damage via the comet assay may be a useful biologic marker of lung cancer susceptibility. The differential effects in BPDE-induced DNA damage between lung cancer cases and healthy subjects suggests predisposed cancer susceptibility to lung cancer risk. This reports also demonstrated the chemopreventive effects of green tea extract on BPDE-induced DNA damage. These observations provide further support for the application of the comet assay in molecular epidemiologic studies.

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

PubMed1992

The seeding of life by comets.

The evidence that living organisms were already extant on the earth almost 4 Gyr ago and that early bombardment by comets and asteroids created a hostile environment up to about this time has revived the question of how it was possible for prebiotic chemical evolution to have provided the necessary ingredients for life to have developed in the short intervening time. The actual bracketed available temporal space is no more than 0.5 Gyr and probably much less. Was this sufficient time for an earth-based source of the first simple organic precursor molecules to have led to the level of the prokaryotic cell? If not, then the difficulty would be resolved if the ancient earth was impregnated by organic molecular seed from outer space. Curiously, it seems that the most likely source of such seeds was the same a one of the sources of the hostile enviroment, namely the comets which bombarded the earth. With the knowledge of comets gained by the space missions it has become clear that a very large fraction of the chemical composition of comet nuclei consists of quite complex organic molecules. Furthermore it has been demonstrated that comets consist of very fluffy aggregates of interstellar dust whose chemistry derives from photoprocessing of simple ice mixtures in space. Thus, the ultimate source of organics in comets comes from the chemical evolution of interstellar dust. An important and critical justification for assuming that interstellar dust is the ultimate source of prebiotic molecular insertion on the earth is the proof that comets are extremely fluffy aggregates, which have the possibility of breaking up into finely divided fragments when the comet impacts the earth's atmosphere. In the following we will summarize the properties of interstellar dust and the chemical and morphological structure of comets indicated by the most recent interpretations of comet observations. It will be shown that the suitable condition for comets having provided abundant prebiotic molecules as well as the water in which they could have further evolved are consistent with theories of the early earth environment.

Cosmic Dust↗

PubMed2004

Analysis of the action of the restriction endonuclease AluI using three different comet assay protocols.

BACKGROUND AND PURPOSE: The comet assay offers the opportunity to measure the amount of DNA damage and the effectiveness of DNA repair in single cells. In a first part, experiments are presented comparing three different protocols of the comet assay technique with respect to the analysis of the induction of DNA damage after X-irradiation in isolated human lymphocytes and CHO cells. In a second part, the restriction enzyme AluI, an agent producing DNA double-strand breaks exclusively, was introduced into CHO cells by electroporation and the effects were analyzed using the different comet assay protocols. The experiments were carried out in order to test the assertion that comet assay techniques can measure different types of DNA damages at different pH conditions of lysis and electrophoresis. MATERIAL AND METHODS: Three different comet assay protocols were used for the analysis of DNA damage in lymphocytes and CHO cells. RESULTS: The results clearly indicate that among the three protocols the modified comet assay technique used by the authors showed the highest sensitivity in the radiotherapy-relevant dose range between 0 and 2 Gy. All three protocols were capable of detecting an effect by AluI. This effect, however, was clearly different from radiation effects. Whereas after radiation exposure all cell nuclei show a dose-dependent increase in DNA content in the comet tail, most of the cell nuclei were unaffected by an AluI uptake. Nevertheless, there was an effect by AluI that could be detected in all three assay versions: between 5% and 15% of the nuclei showed clearly abnormal comet morphologies. CONCLUSION: Neither the strictly alkaline nor the strictly neutral comet assay is applicable in the radiation dose range of about 2 Gy. The restriction enzyme results show that other factors than just DNA strand breaks contribute to DNA migration into the tail of the comets.

Animals↗

PubMed2003

Comparative study with the alkaline Comet assay and the chromosome aberration test.

The alkaline Comet assay is becoming a useful tool for early genotoxicity testing of new pharmaceutical drug candidates. The aim of this study was to elucidate the predictive value of Comet assay results for the outcome of the chromosome aberration (CA) test. For this purpose, a validation exercise with 13 drug candidates was carried out utilizing V79 Chinese hamster cells and human lymphocytes. The study demonstrates that results of the Comet assay and the chromosome aberration test show a high degree of agreement, irrespective of the cell type used. In the Comet assay, seven compounds were positive and six were negative, while in the CA test, six were positive and seven were negative. The only discrepancy was found with one compound that was positive in the Comet assay with V79 cells, negative in the Comet assay with human lymphocytes and clearly negative in the CA test with human lymphocytes. For the selection of concentrations for testing in the Comet assay, cytotoxicity by means of cell count after incubation or viability by means of Trypan-blue dye exclusion (TBDE) were used. The results show that either parameter led to analysis of a concentration range in the Comet assay similar to that chosen in the CA test, in which cell count (when using V79 cells) or mitotic index (in case of lymphocytes) were used. However, since cell count after incubation of cells is much more labour-intensive, viability was preferred as the parameter to assess cytotoxicity and for selecting concentrations for analysis in the Comet assay. The data presented in this study may contribute the regulatory acceptance of the Comet assay, e.g. for mechanistic studies.

Animals↗

PubMed2004

Usefulness of ultrasound lung comets as a nonradiologic sign of extravascular lung water.

The "comet-tail" is an ultrasound sign detectable with ultrasound chest instruments; this sign consists of multiple comet-tails fanning out from the lung surface. They originate from water-thickened interlobular septa and would be ideal for nonradiologic bedside assessment of extravascular lung water. To assess the feasibility and value of ultrasonic comet signs, we studied 121 consecutive hospitalized patients (43 women and 78 men; aged 67 +/- 12 years) admitted to our combined cardiology-pneumology department (including cardiac intensive care unit); the study was conducted with commercially available echocardiographic systems including a portable unit. Transducer frequencies (range 2.5 to 3.5 MHz) were used. In each patient, the right and left chest was scanned by examining predefined locations in multiple intercostal spaces. Examiners blinded to clinical diagnoses noted the presence and numbers of lung comets at each examining site. A patient lung comet score was obtained by summing the number of comets in each of the scanning spaces. Within a few minutes, patients underwent chest x-ray, with specific assessment of extravascular lung water score by 2 pneumologist-radiologists blinded to clinical and echo findings. The chest ultrasound scan was obtained in all patients (feasibility 100%). The imaging time per examination was always <3 minutes. There was a linear correlation between echocardiographic comet score and radiologic lung water score (r = 0.78, p <0.01). Intrapatient variations (n = 15) showed an even stronger correlation between changes in echocardiographic lung comet and radiologic lung water scores (r = 0.89; p <0.01). In 121 consecutive hospitalized patients, we found a linear correlation between echocardiographic comet scores and radiologic extravascular lung water scores. Thus, the comet-tail is a simple, non-time-consuming, and reasonably accurate chest ultrasound sign of extravascular lung water that can be obtained at bedside (also with portable echocardiographic equipment) and is not restricted by cardiac acoustic window limitations.

Aged↗