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PubMed1994

Hypoxic fractions measured in murine tumors and normal tissues using the comet assay.

PURPOSE: To apply the alkaline comet assay to the detection of radiobiologically hypoxic cells in solid tumors and normal tissues of mice, and to examine the influence of strand break repair on the oxygen enhancement ratio measured using the alkaline comet assay. METHODS AND MATERIALS: In previous studies, we found that hypoxic fraction in squamous cell carcinomas growing in C3H mice could be reliably and easily measured using the alkaline comet assay. The comet assay applies fluorescence microscopy and image analysis to examine patterns of migration of deoxyribonucleic acid from individual cells embedded in agarose and exposed to an electric field. This method has sufficient resolution to detect subpopulations of hypoxic cells which show about 3 x fewer strand breaks than aerobic cells after irradiation. RESULTS: Fast rejoining kinetics in vitro are comparable to those measured in vivo, and rejoining of strand breaks in hypoxic tumor cells occurs at a similar rate as rejoining in aerobic cells. Little residual damage was detectable using the comet assay in tumors 4-24 h following 15 Gy, allowing repeat measurements to be performed. Bone marrow and testis, but not liver, spleen, or jejunum contained a small fraction of hypoxic cells when mice breathed 10% oxygen during irradiation. CONCLUSION: The comet assay confirms that some normal tissues may border on hypoxia. Rejoining of strand breaks occurs rapidly in both oxic and hypoxic cells so that the oxygen enhancement ratio remains relatively constant with time after irradiation. Interestingly, a smaller oxygen enhancement ratio was observed in tumors than was expected, probably as a result of the presence of acutely hypoxic cells.

Animals↗
PubMed1996

The comet assay: mechanisms and technical considerations.

The comet assay is frequently used to measure DNA damage in individual cells. In order to better understand the mechanisms behind the technique, we have studied the behaviour of DNA under different electrophoresis conditions in mammalian cells exposed to gamma radiation. The comet tails obtained after neutral electrophoresis seem to consist of DNA loops which are attached to structures in the nucleus, since the DNA cannot move in the second direction after two-dimensional electrophoresis. When the DNA is labelled by a short pulse, microautoradiography reveals that all label appear in the head of the comets when neutral electrophoresis is applied. After chase incubation, the label moves out into the tails. This gives further support to the view that the DNA loops are fixed to some structure in the nucleus where also the DNA synthesis takes place. Under alkaline electrophoresis conditions, however, the entire comet tails move in the new electrophoresis direction. Thus, it appears that the alkaline comet tails consist of free DNA fragments. Further, the effects of alkaline concentration and sodium chloride during unwinding and electrophoresis are discussed. Throughout the study, a protocol for drying and fixation of the comets has been used.

Animals↗
PubMed1998

Development and validation of the in vivo alkaline comet assay for detecting genomic damage in marine flatfish.

Biomonitoring is an important subject within environmental sciences. Biomonitoring tests are required to be quick, relatively inexpensive, accurate, and reproducible. No genetic test currently fulfils all of these requirements. The chromosome aberration and sister chromatid exchange tests are very time consuming, the DNA adduct technique is rather expensive, and the micronucleus test has not inconclusively proven its use as a reliable monitoring tool. This work is focused on the validation of the comet assay as a candidate for monitoring marine ecosystems. For the comet assay, this work deals with the effectiveness of tissue dissociation, storage of cells in lysing buffer and in liquid nitrogen, different electrophoretic conditions, neutralisation and fixation of slides, interindividual variation between samples, and responsiveness of four tissue types to ethyl methanesulphonate (EMS). The main conclusions are: (i) dissociation of solid tissues in a phosphate buffer supplemented with 200 mM N-t-butyl-alpha-phenylnitrone provides cells with an acceptable background DNA damage; (ii) freezing of cells or tissues in liquid nitrogen generally leads to an increase in DNA breakage, especially for liver, gill and kidney tissue; (iii) storage of slides in the lysing solution for up to one week gives minor changes in comet tails; (iv) differences in protocols for neutralisation and fixation may influence the results; (v) high intra- and interindividual variations in comets (length and DNA content) may obscure the interpretation of comet results; (vi) blood, gill, liver and kidney all showed a statistically significant increase of DNA damage after exposure to 50 mg EMS/l; (vii) electrophoresis at low voltage for longer periods is to be preferred to high voltage and short electrophoresis times. The simplicity and sensitivity of the comet assay make it an adequate test system for biomonitoring of chronic low level exposure. However, protocols and experimental conditions have to be chosen carefully.

Alkalies↗
PubMed2000

Identification of comet Hyakutake's extremely long ion tail from magnetic field signatures

Observations of the varying orientations of comet tails led to the suggestion of the existence of the solar wind--a continuous outflow of ionized material from the Sun. It is now well established that gas from comets is ionized by several processes and joins the solar wind, forming an ion (plasma) tail that points away from the Sun. The plasma environments of three comets have been measured in situ, but only in the upstream direction or less than 8,000 km downstream of the nucleus. Here we report a fortuitous crossing by a spacecraft of the plasma tail of comet Hyakutake (C/1996 B2), at a distance of more than 3.8 astronomical units (550 million kilometres) from its nucleus. This surpasses the tail length of 2 AU determined for the Great March Comet of 1843 (C/1843 D1). Our measurements reveal that, at this distance, the tail of comet Hyakutake was a structured entity at least 7 million kilometres in diameter.

Journal Article↗
PubMed2000

Discovery of a comet by its Lyman-alpha emission

Several searches for near-Earth objects have recently been initiated, as a result of increased awareness of the hazard of impacts on the Earth. These programs mainly search for asteroids, so amateur astronomers can still contribute to the discovery of comets, especially out of the orbital plane of the Solar System. An ideal way to search for comets would be to use a spaceborne instrument capable of imaging the whole sky on a daily basis in a systematic and repeatable way. Such an instrument already exists on the solar observatory SOHO; it operates at the Lyman-alpha wavelength of neutral hydrogen, which is the main component of the emission cloud of a comet. Here we report the discovery, using archival data from this satellite, of a hitherto unnoticed comet which reached a perihelion of 1.546 a.u. on 26 June 1997. We derive the water production rate of the comet as a function of time and find that it increases after perihelion, like that of comet Halley.

Journal Article↗
PubMed2001

Rapid collisional evolution of comets during the formation of the Oort cloud.

The Oort cloud of comets was formed by the ejection of icy planetesimals from the region of giant planets--Jupiter, Saturn, Uranus and Neptune--during their formation. Dynamical simulations have previously shown that comets reach the Oort cloud only after being perturbed into eccentric orbits that result in close encounters with the giant planets, which then eject them to distant orbits about 10(4) to 10(5) AU from the Sun (1 AU is the average Earth-Sun distance). All of the Oort cloud models constructed until now simulate its formation using only gravitational effects; these include the influence of the Sun, the planets and external perturbers such as passing stars and Galactic tides. Here we show that physical collisions between comets and small debris play a fundamental and hitherto unexplored role throughout most of the ejection process. For standard models of the protosolar nebula (starting with a minimum-mass nebula) we find that collisional evolution of comets is so severe that their erosional lifetimes are much shorter than the timescale for dynamical ejection. It therefore appears that collisions will prevent most comets escaping from most locations in the region of the giant planets until the disk mass there declines sufficiently that the dynamical ejection timescale is shorter than the collisional lifetime. One consequence is that the total mass of comets in the Oort cloud may be less than currently believed.

Journal Article↗
PubMed1996

Spectroscopic evidence for interstellar ices in comet Hyakutake.

Volatile compounds in comets are the most pristine materials surviving from the time of formation of the Solar System, and thus potentially provide information about conditions that prevailed in the primitive solar nebula. Moreover, comets may have supplied a substantial fraction of the volatiles on the terrestrial planets, perhaps including organic compounds that played a role in the origin of life on Earth. Here we report the detection of hydrogen isocyanide (HNC) in comet Hyakutake. The abundance of HNC relative to hydrogen cyanide (HCN) is very similar to that observed in quiescent interstellar molecular clouds, and quite different from the equilibrium ratio expected in the outermost solar nebula, where comets are thought to form. Such a departure from equilibrium has long been considered a hallmark of gas-phase chemical processing in the interstellar medium, suggesting that interstellar gases have been incorporated into the comet's nucleus, perhaps as ices frozen onto interstellar grains. If this interpretation is correct, our results should provide constraints on the temperature of the solar nebula, and the subsequent chemical processes that occurred in the region where comets formed.

Extraterrestrial Environment↗
PubMed1998

Use of the comet test in the evaluation of multidrug resistance of human cell lines.

The comet test is a reported method for measuring DNA damage in individual mammalian cells. In the present report, the ability of this test to detect multidrug resistance (MDR) was evaluated. For this purpose, two human leukemia, well-characterized parental cell lines, HL60 and CEM, and their derived multidrug-resistant cells, HL60/DNR and CEM/VBL, were cultured with or without different anti-cancer agents. To evaluate the comet test, two DNA-damaging agents were used: daunorubicin (DNR), which is involved in MDR, and ambamustine (AMBA), which is independent from MDR. Moreover, in order to evaluate the specificity of the comet test, the activity of vinblastine (VBL), an MDR-related, DNA-independent anti-cancer drug, was also tested. Finally, the specificity of the comet test in detecting MDR was confirmed by culturing parental or resistant cells with DNR with or without the revertant agent verapamil (VER). Results confirm that the comet test is able to predict cellular chemoresistance when DNA damaging agents are tested. Finally, experiments on the role of the comet test in evaluating certain aspects of DNA repair are discussed.

Antibiotics, Antineoplastic↗
PubMed1983

Aphelion clustering of "new" comets: Star tracks through Oort's cloud.

An explanation is proposed for the observed and often discussed clustering of long-period comet aphelia on the sky. Poisson and several multinomial distributions are applied to the most conspicuous cluster, considering only the 80 "new" and 59 "intermediate" comets with the best-determined orbits. The observed number of aphelion points in adjacent areas in two tests with 24 and 36 equal sky areas, respectively, shows a large deviation from a random distribution. The expected probability frequency for this to happen by chance is <0.1% for the "new" comets alone; it is further reduced when "new" and "intermediate" comets are combined. When these comets are analyzed separately from others, it is found that the clustering is the result of perturbations of their original orbits by the passage of a star, or a stellar system, through Oort's cloud a few million years ago. Because the statistical effect on the change of a comet's angular momentum about the sun is proportional to the square of the ratio of the mass to the velocity of a star relative to the sun, it is a priori probable that passages by a few stars should produce clustering of the aphelia, as is discussed in detail.

Journal Article↗
PubMed1997

Image analysis of comet assay measurements.

In the last decade the 'comet assay' or 'single cell gel electrophoresis assay' has been established as a sensitive method for the detection of DNA damage and the measurement of its recovery. The results published in the literature have often been obtained with different methods for comet structure measurement. In most cases these data are not comparable with each other. Even when using similar systems for the analysis, it is difficult to obtain matching data. This presentation will describe some technical aspects of our measurement equipment and evaluation software. It focuses on necessary experimental conditions to minimize errors in obtaining such data. The software developed here allows the rapid analysis of the microscopic samples (< 2 s per image). The image analysis was designed with respect to the morphological shapes of comet cells, which were investigated with a confocal laser microscope. The system is built with standard components which are commercially available. As a measure of the amount of DNA damage the ratio of fluorescence intensity was used inside the comet tail and the fluorescence intensity of the comet head. Other parameters such as DNA content, comet area, head radius, tail length and tail moment are also determined. The reproducibility of the system has been evaluated in several experiments over a period of 5 years.

Ataxia Telangiectasia↗
PubMed1995

The single cell gel electrophoresis assay for induced DNA damage (comet assay): measurement of tail length and moment.

Cultured hepatocytes have been treated with either DMSO or dimethylnitrosamine (NDMA) for either 1 or 2 h and the cells assessed for DNA-damage using the single cell alkaline gel electrophoresis assay (comet assay). A strong positive test response was observed producing comet tails of a length and DNA content not observed in either viable or dead control cells. A stronger test response was observed after a 2 h, as opposed to a 1 h, incubation of hepatocytes with NDMA. The method of processing the image of the comet is discussed and it is proposed that measurement of the length of the comet tail should commence at the estimated trailing edge of the cell, rather than at the leading edge or the estimated centre of the cell. Using this criterion, many control cells have no comet trails thereby enabling chemically induced tails to be more readily assessed. A simplified version of defining the comet tail moment is proposed.

Animals↗
PubMed1997

Evaluation of manual and image analysis quantification of DNA damage in the alkaline comet assay.

The alkaline comet assay or single cell microgel electrophoresis assay is a sensitive method of detecting DNA strand breaks and alkali labile sites in individual cells. The results of this assay can be analysed by different methods. In this study we compared analyses of the same slides by a manual method and by image analysis, post-treatment of clone 707 Friend erythroleukaemia cells with H2O2. The parameters which were found to be particularly useful were comet area and comet length (measured manually) and percentage tail DNA, tail moment, tail length and tail length/head radius (L/H), measured using image analysis. The manual method for comet analysis presented in this paper would appear to provide good and reliable comet data. However, the image analysis comet system described offers an alternative analysis method which avoids the need for photomicrographs and tedious manual analysis. The image analysis parameters: % tail DNA, tail moment, tail length and L/H give good consistent results and for large-scale analysis it will, therefore, conceivably be the method of choice.

Animals↗
PubMed1998

The ability of the Comet assay to discriminate between genotoxins and cytotoxins.

The Comet assay has been used widely in genetic toxicology, radiation biology and medical and environmental research. This assay detects single-strand breaks and alkali-labile sites in DNA and DNA degradation due to necrosis or apoptosis. It may also be modified to detect DNA cross-linking. Although a considerable number of chemicals have been tested in the assay there are many aspects of validation to be considered before the method could be considered to provide definitive evidence of genotoxic potential. For example, very few non-genotoxins have been tested to assess specificity of the Comet assay and there has been only one reported study which investigated whether the in vitro Comet assay is prone to false positive responses due to cytotoxicity. We have investigated the response of the alkaline Comet assay in TK6 human lymphoblastoid cells to cytotoxic damage and genotoxic damage. Several compounds which are toxic by different mechanisms were tested in the assay. Cycloheximide and trypsin gave a negative comet response at a highest dose of 5 mg/ml and no toxicity was observed. Sodium lauryl sulphate and potassium cyanide produced a significant increase in DNA migration at cell survival levels of < or = 75%. The distribution of damaged cells indicated that cells at various stages of necrotic cell death were present. Hydrogen peroxide, 4-nitroquinoline oxide, 9-aminoacridine, ethyl methanesulphonate, N-nitroso-N-ethylurea and glyoxal gave a positive comet response. Mitomycin C was negative at survival levels of approximately 70%. These results indicate that the maximum concentration of test substance tested should produce viabilities > 75% in order to avoid false positive responses due to cytotoxicity. The assay was able to detect DNA damage induced by an alkylating agent, an intercalating agent and oxidative damage. The cross-linking agent mitomycin C was not detected if a cut-off point of 75% viability is used as the criterion of a positive response.

4-Nitroquinoline-1-oxide↗
PubMed1998

Comet assay responses as indicators of carcinogen exposure.

Over 200 agents/factors have been examined in the single cell gel electrophoresis assay, more commonly known as the Comet assay, performed either in vitro or in vivo in a variety of species. Unequivocal carcinogenicity data are available for 119 of them, amongst which unequivocal Comet assay data exist for 95 agents. Of these 95 agents the prevalence of carcinogens was 88% (84/95). The carcinogens that were Comet positive (sensitivity) formed 88% (74/84), the non-carcinogens that were Comet negative (specificity) formed 64% (7/11). This simple analysis of the Comet assay has not taken account of the difference between in vitro and in vivo responses, species differences or organ and tissue differences. Also, limitations as to the conduct of the assay have not been examined in any depth. Thus, at the present time the Comet assay has high sensitivity for carcinogens, but its specificity is uncertain because few non-carcinogens have been tested.

Animals↗
PubMed1997

The comet assay as a repair test for prenatal diagnosis of Xeroderma pigmentosum and trichothiodystrophy.

Xeroderma pigmentosum (XP) and trichothiodystrophy (TTD) are autosomal recessive diseases associated with extreme cutaneous photosensitivity, a defect in nucleotide excision repair (NER), and genetic complexity. Severe prognosis and lack of treatment led families at risk to request genetic counseling. Unscheduled DNA synthesis (UDS) is the classic method for diagnosis and requires 4 to 5 wk before conclusion. The use of the alkaline comet assay (single cell gel electrophoresis assay) is proposed as a simple repair test for earlier prenatal diagnosis. Amniotic or chorionic villus cells in two pregnancies at risk for XP and one for TTD were examined in comparison with skin fibroblasts of family members or with repair-proficient or -deficient control cells. The comet assay and the UDS test were performed in parallel. In repair-proficient cells, DNA strand breaks due to the incision of UV-induced DNA damage result in increased migration of high molecular weight DNA in the comet assay. Fetal cells demonstrate repair capacity similar to that of fibroblasts. In incision repair-deficient XP and TTD cells, after post-UV incubation, migration does not occur and comet moments are reduced. Two fetuses belonging to two XP families responded normally and were diagnosed as unaffected. Fetal cells in a TTD family had reduced comet moments and a low UDS. This fetus was diagnosed and confirmed later as affected. Heterozygotes had normal responses with both assays. The comet assay offers discrimination similar to that of the UDS assay in identifying NER-deficient phenotypes. Practical advantages in view of prenatal diagnosis include the reduced number of cells required, a 24-h delay in obtaining results, and no need for radioactivity.

Cells, Cultured↗
PubMed2002

The mass disruption of Oort cloud comets.

We have calculated the number of dormant, nearly isotropic Oort cloud comets in the solar system by (i) combining orbital distribution models with statistical models of dormant comet discoveries by well-defined surveys and (ii) comparing the model results to observations of a population of dormant comets. Dynamical models that assume that comets are not destroyed predict that we should have discovered approximately 100 times more dormant nearly isotropic comets than are actually seen. Thus, as comets evolve inward from the Oort cloud, the majority of them must physically disrupt.

Journal Article↗
PubMedDate not supplied

[Dna comets as markers of cells death].

Unstimulated human peripheral blood lymphocytes gradually underwent death during incubation in vitro. According to morphological criteria, the type of death was identified as apoptosis. After immobilization in agarose, lysis, and electrophoresis, these lymphocytes formed DNA comets, which differed in DNA content, tail length, tail moment, and the fraction of DNA migrating in the comet tail. We classified the comets in 3 groups in accordance with the values of these parameters. There was a good correlation between the fraction of apoptotic cells (morphological data) and the fraction of "apoptotic" DNA comets. The results showed that DNA comets may be adequate markers of cell death (including apoptosis). The use of DNA comets as markers of spontaneous death made it possible to reveal an increased level of apoptosis in vitro lymphocytes from patients with systemic lupus erythematosus.

Apoptosis↗
PubMed1997

[The comet assay method: a new approach in genotoxicology research].

The comet assay is a fast, simple and sensitive genotoxicological technique for measuring DNA damage in an individual cell of virtually any cell type of animal or plant origin. Electrophoresis of complete cell genome (assuming a comet-like shape) combined with the image analysis systems for comet analysis provide densitometric and geometric parameters describing the complete comet as well as the head and tail. The comet optical density values are used to quantify the total comet fluorescence and hence indicate DNA content and the level of damage. The application of this method in different fields makes it a powerful tool in human genotoxic study as well as in the estimation of environmental pollution.

DNA Damage↗