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PubMed2004

Evaluation of in vitro genotoxic activity of bleomycin and mitomycin C in human lymphocytes using the alkaline comet assay.

Although chemotherapy targets cancer tissue, it also damages the DNA of non-cancer cells. The aim of this study was to evaluate the in vitro genotoxic potential of therapeutic concentrations of bleomycin and mitomycin C, added alone or in combination to cultures of human peripheral lymphocytes. The levels of DNA damage and repair were assessed using the alkaline comet assay immediately after cell treatment as well as 24 and 48 hours following treatment. The results indicate that individual drugs and their combination induce a significant DNA damage to peripheral blood lymphocytes. Bleomycin alone induced the highest levels of primary DNA damage immediately after cell treatment. Although mitomycin C alone induced massive cross-linking and retarded DNA migration in resting cells, active proliferation and repair processes significantly increased DNA damage. Combined, they showed a synergetic effect, inducing complex patterns of DNA damage in peripheral blood lymphocytes and producing different types of lesions and a number of DNA alterations that directly or indirectly increased DNA migration. Our study has confirmed the sensitivity of the alkaline comet assay for assessing bleomycin and/or mitomycin C genotoxicity to human lymphocytes at concentration levels used in clinic. It has also demonstrated the utility of the alkaline comet assay as one of the primary screening methods for in vitro studies of drug-DNA interactions, especially in studying mechanisms of action of new drugs.

Adult↗
PubMed2004

Assessment of cellular damage by comet assay after photodynamic therapy in vitro.

The aim of this study was analysis of DNA damage in the cell line of the human melanoma G361 after photodynamic therapy (PDT) by comet assay. Photodynamic therapy is based on cytotoxic action of sensitizers (10 microM ZnTPPS4 fixed into 1 mM cyclodextrin hpbetaCD) and light with a suitable wavelength. Single-cell gel electrophoresis (SCGE, comet assay) is a rapid and sensitive method for detecting DNA strand breaks at the level of single cells. Great amount of DNA damage was detected with the dose of irradiation of 0.1; 0.5 J and 2.5 J x cm(-2). Only radiation dose of visible light in the presence of sensitizers can induce DNA breaks of tumour cells. Cells with DNA damage appear as fluorescent comets with tails of DNA fragmentation. In contrast, cells with undamage DNA appear as round spots, because their intact DNA does not migrate out of the cell.

Cell Line, Tumor↗
PubMed1996

Detection of DNA-crosslinking agents with the alkaline comet assay.

The single cell gel electrophoresis, or comet assay, under alkaline conditions is a sensitive, simple and rapid method for the detection of DNA damage at the individual cell level. Its applicability as an indicator for the DNA crosslinking potency of a test substance was investigated in human white blood cells by combined treatment with the DNA damaging agent methyl methanesulphonate (MMS) for 2 hr at 37 degrees C. The known crosslinking agents cisplatinum, mitomycin C and formaldehyde, and the formaldehyde releasers diazolidinyl urea and dimethylol urea, were shown to reduce MMS-induced DNA migration in the comet assay in a concentration-dependent manner. Two other protocols, adding MMS to the cells before or after treatment with a crosslinking agent, were carried out and achieved similar results. The results of this study indicate that the comet assay is a useful tool for the detection of crosslinking agents. Advantages and limitations of this method compared to the alkaline elution technique are discussed.

Adult↗
PubMed1996

An investigation of some Turkish herbal medicines in Salmonella typhimurium and in the COMET assay in human lymphocytes.

Medicinal plants play a major role in the life of Turkish people and of late medicinal plant usage has increased in many countries. Green plants in general contain mutagenic and carcinogenic substances, but there is little information about the biological activities of herbal medicine. In the present study, therefore, various Turkish medicinal herbs were investigated for their genotoxic potential in the Salmonella typhimurium microsomal activation assay and the alkaline single cell gel electrophoresis (COMET) assay. Extracts from these medicinal herbs and some fractions of these extracts were examined. The species investigated were Arctium minus, Ecballium elatterium, Momordica charantia, Plantago major, Urtica dioica, Viscum album, Salvia triloba, Euphorbia rigida, Stachys lavandulifolia, Acteoside, Abies nordmannia. They are used for various immune disorders and are applied either topically or taken orally as a herbal tea. Of the 19 samples of the extracts and fractions investigated, none produced a positive response in strains TA98 and TA100 with or without metabolic activation, but all produced an increase above negative control values in the COMET assay. Some extracts were investigated further and produced dose-related increases. In the case of Urtica and Euphorbia species, where two fractions from these plants were examined, one fraction produced a greater response than the other. It is suggested that the lesser response of the fractions might be due to less DNA strand-breaking agents in the fractions or they may have antigenotoxic properties. The breaks that are detected in the COMET assay could be alkali-labile AP-sites and intermediates in base- or nucleotide-excision repair and are difficult to interpret in terms of hazard for man. Further studies with additional genotoxicity assays would be required to make such a prediction.

DNA Damage↗
PubMed1994

Video enhanced laser scanning microscopic analysis of DNA comet formation.

Laser Scanning Microscopy is a sensitive tool that provides a unique method of analyzing biological systems. Coupled with the Single Cell Gel assay, it allows for accurate and reproducible detection of DNA strand breaks. An understanding of the theory of DNA comet formation is lacking. Using dexamethasone induced apoptosis in murine thymocytes as a model for double strand breaks, we used video enhanced laser scanning microscopy to evaluate the leading edge of DNA migration in the single cell gel assay. In this system, comet length increases significantly within the first thirty seconds of electrophoresis, the greatest increase in length is completed within the first minute, and the first two minutes are important in significant increases in DNA migration during DNA comet formation.

Animals↗
PubMed1995

Sizing highly fragmented DNA in individual apoptotic cells using the comet assay and a DNA crosslinking agent.

TK6 human B lymphoblast cells exposed to ionizing radiation undergo apoptosis in a time and dose-dependent manner. The resulting highly fragmented DNA is easily detected using the comet assay, a sensitive microscopic gel electrophoresis method capable of measuring DNA strand breaks in individual cells. The degree of DNA fragmentation may be indicative of different stages in the fragmentation process, responses to different agents, and/or cell type-dependent differences. In an effort to determine the number of breaks present in each apoptotic cell, we first applied a DNA-crosslinking agent, mechlorethamine, to TK6 cells containing a known number of DNA double-strand breaks produced by X rays. As the concentration of mechlorethamine increased, crosslinked DNA was less able to migrate during gel electrophoresis. Exposure of TK6 cells to 5 microM mechlorethamine prior to irradiation with 20 Gy was sufficient to "hide" the presence of these breaks by preventing DNA from migrating during electrophoresis. However, in apoptotic TK6 cells, it was necessary to apply a dose of mechlorethamine several times higher in order to produce a similar degree of inhibition of DNA migration. Calibrations using either the alkaline or neutral comet assays indicate that the average DNA fragment size in apoptotic TK6 cells is about 50 kb. Even in cells containing only 10-20% of the original amount of DNA, the remaining fragments still averaged about 50 kb, indicating that fragmentation to much smaller sizes occurs in some parts of the genome before others. When Chinese hamster V79 cells were exposed to hyperthermia (45 degrees C for 20 min), necrosis was induced over a period of several days. The size of DNA fragments in these cells was considerably larger (200-400 kb) and heterogeneity in appearance of comets was larger than observed for TK6 cells. This crosslinking method may be useful in discriminating cells dying by apoptosis from cells damaged or dying by other mechanisms.

Animals↗
PubMed1994

The collision of Jupiter and Comet Shoemaker-Levy 9.

A simple description of the disruption and deceleration of 100-m- to 5-km-diameter comets striking Jupiter is combined with numerical simulations of the subsequent explosions to predict the fate of Comet Shoemaker-Levy 9. Kilometer-size objects of density 1 g/cm3 explode at about the 10-bar level; a fragment of the same diameter but of density 0.3 g/cm3 explodes at about the 2-bar level. Detailed numerical simulations of the first 3 min of the explosion were performed using the astrophysical hydrodynamics program ZEUS-3D. Our numerical simulations begin either with hot cylinders with dimensions suggested by the disruption and deceleration model or with an initial wake constructed from a moving line charge. In all cases, extensive plumes of hot gas are expelled from the atmosphere. The models with wakes evolve about twice as fast as the initially confined models. Models of both types generate similar pressure waves into the planet. Temperatures and negative hydrogen ion opacities were computed by solving a battery of Saha equations. For atmospheric entry, light curves were computed assuming thermal radiation by clean jovian air with a surface area consistent with the (changing) cross-sectional area of the impactor. On entry the largest bolides could be very bright, possibly as bright as Jupiter for observers placed to see them, although for kilometer-size impactors the luminosity peak is obscured by clouds. The timescale is about 10 sec. For the fireball, light curves were computed from the numerical simulations assuming a grey atmosphere. Metals from the vaporized comet provide electrons that dramatically increase the opacity of Jovian air at low temperature; the resulting effective radiating temperature of the fireball is of order 3000 K. The fireball rises through and above the atmosphere, brightening at first as its surface area increases, but later fading to invisibility as its temperature drops and its opacity plummets. The timescale is about 100 sec.

Astronomy↗
PubMed1995

Comets, impacts, and atmospheres.

We are proposing a model for the delivery of volatiles to the inner planets by icy planetesimals (comets). Laboratory studies of the trapping of gases in ice forming at low temperatures simulate the formation of comet nuclei at various distances from the Sun in the solar nebula. The total gas content as well as the relative proportions of gases trapped in the ice are strong functions of temperature. As they trap N2 inefficiently, all planetesimals formed interior to Neptune are deficient in nitrogen, acquiring values of C/N resembling those found in the inner planet volatile inventories. A mixture of three basic types of comets appears capable of accounting for the observed volatile inventories on Venus, Earth, and Mars, with the caveat that impact erosion is necessary to explain the present condition of the martian atmosphere. The model includes the possibility of several epochs of clement conditions on early Mars. Some tests of these ideas are suggested, including measurements in Jupiter's atmosphere by the Galileo probe.

Atmosphere↗
PubMed1998

DNA damage in human and mouse spermatozoa after in vitro-irradiation assessed by the comet assay.

The comet assay is widely employed as a method to measure DNA damage in a wide variety of cell types following genotoxic insult. We have used this method in order to characterise DNA damage in spermatozoa following in vitro irradiation with 137Cs gamma rays. In contrast to somatic cells, the DNA of mammalian spermatozoa is bound by protamine molecules allowing a sixfold more highly compact structure and thus rendering conventional cell lysis protocols ineffective. Therefore, this new method uses an extensive lysis step to ensure effective removal of DNA-associated proteins allowing DNA damage to be scored reproducibly in both murine and human spermatozoa. Mouse spermatozoa collected from the vas deferens at post-mortem or human spermatozoa provided by donors were irradiated with doses of gamma-rays from 0-100 Gy using a 137Cs source and then processed for both alkaline and neutral comet assays. Under neutral electrophoresis conditions, which permits the measurement of double-stranded DNA breaks, a linear increase in the amount of DNA damage measured was observed with increasing radiation dose for both murine and human spermatozoa. Similarly, using alkaline electrophoresis conditions to examine DNA single-strand breaks and alkali-labile sites, a linear relationship was also observed for murine sperm but in contrast no such relationship was apparent for human spermatozoa subjected to the same radiation treatments. Interestingly, unirradiated sperm (both human and mouse) showed extensive DNA migration from the nucleus after alkaline assay. Since it is unlikely that the DNA of normal spermatozoa contains high numbers of single-strand breaks and damage was not detected for unirradiated sperm in the neutral assay, it is more likely that this DNA migration is due to the presence of high numbers of alkali labile sites within sperm DNA and that these may be related to the highly condensed structure of spermatozoal DNA. The large radiation doses used in these experiments to produce measurable amounts of DNA damage reflects the high radioresistance of spermatozoa compared to somatic cells and this may also be related to the differences in DNA packaging and conformation. In conclusion, this work shows that the comet assay represents a new method for examining DNA damage in spermatozoa and should be evaluated for use in reproductive toxicity testing.

Animals↗
PubMed1992

Computational study of radiation chemical processing in comet nuclei.

Cometary nuclei have been exposed to high levels of ionizing radiation since their formation. We present here some results of a computer model calculation of the effect of ionizing radiation on cometary material. The external (cosmic rays) and internal (embedded radionuclides) contributions in the processing of cometary nuclei are considered. As a first approximation we have used the available kinetic data of the liquid water system to model the radiation effects in a frozen cometary environment. Our [correction of out] data suggest that massive radiation chemical processing due to cosmic rays may have taken place only in the outer layers of comets. The internal contribution of radionuclides to the radiation processing of comet cores seems to be modest. Therefore, comets could be carriers of intact homochiral biomolecules.

Cosmic Radiation↗
PubMed1994

Carbonaceous components in the comet Halley dust.

Cometary grains containing large amounts of carbon and/or organic matter (CHON) were discovered by in situ measurements of comet Halley dust composition during VEGA and GIOTTO flyby missions. In this paper, we report the classification of these cometary grains by means of cluster analysis, discuss the resulting compositional groups, and compare them with substances observed or hypothesized in meteorites, interplanetary dust particles, and the interstellar medium. Grains dominated by carbon and/or organic matter (CHON grains) represent approximately 22% of the total population of measured cometary dust particles. They usually contain a minor abundance of rock-forming elements as well. Grains having organic material are relatively more abundant in the vicinity of the nucleus than in the outer regions of the coma, which suggests decomposition of the organics in the coma environment. The majority of comet Halley organic particles are multicomponent mixtures of carbon phases and organic compounds. Possibly, the cometary CHON grains may be related to kerogen material of an interstellar origin in carbonaceous meteorites. Pure carbon grains, hydrocarbons and polymers of cyanopolyynes, and multi-carbon monoxides are present in cometary dust as compositionally simple and distinctive components among a variety of others. There is no clear evidence of significant presence of pure formaldehyde or HCN polymers in Halley dust particles. The diversity of types of cometary organic compounds is consistent with the interstellar dust model of comets and probably reflects differences in composition of precursor dust. Preservation of this heterogeneity among submicron particles suggests the gentle formation of cometary nucleus by aggregation of interstellar dust in the protosolar nebula without complete mixing or chemical homogenization at the submicron level.

Astronomy↗
PubMed1986

Methods for computing comet core temperatures.

General analytic expressions are derived that relate the surface temperature to the temperature deep within the nucleus of a spherically symmetric layered comet in thermal equilibrium. The relation between the average surface temperature and the mean temperature at great depths depends entirely on the temperature dependence of the thermal conductivity. The core temperature is given by the inverse of the anti-derivative of the thermal conductivity, with respect to temperature, operating on the average value of the anti-derivative of the thermal conductivity evaluated at the surface temperature. Using these expressions detailed numerical models of the surface temperature of comets can be used to directly estimate the core temperature. For the special, albeit unphysical, case of an isothermal, low-conductivity comet nucleus, without sublimation, the core temperature can be determined analytically. To illustrate the dependence of core temperature on eccentricity this simple case is solved assuming that the temperatures dependence of the thermal conductivity is given by that of crystalline ice. For an eccentricity of approximately 0.5, the core temperature obtained is 3% colder than the corresponding value obtained assuming constant thermal conductivity an is 11% colder than the result of Klinger's (1981) formula. This method is also applied to a detailed numerical model with a complicated nonintegrable thermal conductivity.

Astronomy↗
PubMed1995

Origin of organic matter in the protosolar nebula and in comets.

Comet organics are traced to their origin in interstellar space. Possible sources of comet organics from solar nebula chemistry are briefly discussed. The infrared spectra of interstellar dust are compared with spectra of solar (space) irradiated laboratory organic residues and with meteorites. The spectra compare very favorably. The atomic composition of first generation laboratory organic residues compares favorably with that of comet Halley organics if divided into appropriate "volatile" (less refractory) and "refractory" (more refractory) complex organics.

Cosmic Dust↗
PubMed1995

Consequences of cometary aurora on the carbon chemistry at comet P/Halley.

Various experimental data acquired during the visit of Halley's comet in 1986 have shown that the amount of carbon produced due to photodissociation of parent carbon bearing species is not ample enough to explain the observations. This requires the presence of an additional source of atomic carbon. One of the possible source could be auroral-type activities resulting from the precipitation of high-energy "auroral electrons" of solar wind origin, the evidence of which have been inferred from many observations at comet Halley. We have developed a coupled chemistry-transport model to study the role of auroral and photoelectron impact as well as of chemistry on the modelling of carbon in the inner coma (< or = 10(4) km) of comet Halley. Our study suggest that electron impact dissociation of CO is the major source of carbon production in the inner coma, not the recombination of CO+ as suggested by earlier workers, while transport is the main loss process.

Carbon↗
PubMed1998

The distribution of the tail moments in single cell gel electrophoresis (comet assay) obeys a chi-square (chi2) not a gaussian distribution.

The parameter tail moment in single cell gel electrophoresis (comet assay) is calculated as the product of the two values: the percentage of DNA in the comet tail and the tail length in microm. Experiments were performed with cultured mammalian cells: B-Lymphoblasts, epithelial cells of a kidney tissue and a plate-epithelial cell line of a human carcinoma. They were irradiated in suspension with UV A at lambda = 343 nm, generated by an excimer laser-pumped dye laser. DNA migration was assessed and analysed. It is demonstrated that the distribution of the tail moments can be fitted by a chi2 (chi-square) distribution, whereas the factors of the product tail moment tend to be normally distributed. From this result, consequences for the statistical evaluation of the results can arise, especially for the computation of the confidence limits and for the valuation of the parameter tail moment from other comet assay experiments.

Cells, Cultured↗
PubMed1998

DNA strand breakage in aquatic organisms and the potential value of the comet assay in environmental monitoring.

This review considers the potential for DNA strand breaks, particularly as measured by the comet assay, to act as a biomarker of genetic toxicity in fish and other aquatic species. The background need for such biomarkers is introduced in relation to carcinogenicity, reproductive effects and other adverse effects of pollution. Sensitive measurements of DNA strand breakage can be achieved, e.g., by alkaline elution, alkaline unwinding or by single cell gel electrophoresis (comet) techniques. The DNA damage can be a reflection, not only of direct strand breakage, but also of alkali-labile sites and of repair enzyme-mediated breakage (i.e., is non-specific). A range of genotoxic chemicals (both with and without the requirement for metabolic activation) give positive effects in various cell types of vertebrate and invertebrate aquatic species, following in vitro and in vivo exposures under laboratory conditions. A limited number of analyses of organisms exposed to polluted waters or sediments in the field have implicated DNA strand breakage as a relatively sensitive, rapid and broad specificity indicator of genotoxic pollutant exposure. The comet assay deserves further exploitation to assess inter-individual and inter-cell variability in response to pollutants and naturally occurring genotoxic stimuli, and to assess the persistence of these effects.

Animals↗
PubMed1998

Flavonoids modulate comet assay responses to food mutagens in human lymphocytes and sperm.

The flavonoids, silymarin, myricetin, quercitin, kaempferol, rutin and kaempferol-3-rutinoside have been examined in combination with the food mutagens, 3-amino-1-methyl-5H-pyrido (4,3-b)indole (Trp-P-2) and 2-amino-3-methylimidazo-(4,5-f) quinoline (IQ), in the Comet assay in human lymphocytes from donor A and human sperm from donor B. These compounds alone have been shown to produce positive responses in the Comet assay, as have the food mutagens. However, in combination with the food mutagens, the flavonoids produced antigenotoxic effects since DNA damage was reduced in the Comet assay in lymphocytes and sperm. The assays were performed in the absence of metabolic activation, since when quercetin and kaempferol were examined in blood with metabolic activation, there was little or no difference in response to that obtained in its absence. In the blood, there was an exacerbation or synergy of response at the lowest doses of the flavonoids. In the sperm, with silymarin, myricetin and quercitin, antigenotoxic effects only were observed, but with kaempferol, in general, there were no protective effects. The food mutagen, 2-amino-1-methyl-6-phenylimadazo (4,5-b)pyridine (PhIP), was also examined in addition to Trp-P-2 and IQ in combination with silymarin and myricetin in donors A and C in human lymphocytes only. Similar exacerbation of effects were found at low doses of these flavonoids with antigenotoxic effects at high doses. This was confirmed in the Ames test. There were slightly different profiles in lymphocytes and sperm, but antigenotoxic effects were observed over a similar dose range. This would suggest that effects occur in somatic and germ cells on a one-to-one ratio. These results have implications for man in terms of risk assessment and in the modulation of isolated food constituents.

Adult↗
PubMed1997

The use of the same image analysis system to detect genetic damage in human lymphocytes treated with doxorubicin in the Comet and fluoresence in situ hybridisation (FISH) assays.

Two assays, the alkaline single cell gel electrophoresis (Comet) assay and the fluorescence in situ hybridisation (FISH) of a whole chromosome or 'chromosome painting' assay have gained importance in recent years as witnessed by the increasing yield of scientific literature using these techniques. Thus, it would be useful to have one system to measure both endpoints. In the present communication, a cost-effective electronic imaging system developed by Kinetic Imaging Ltd., UK, has been used to measure, after treatment of human lymphocytes with doxorubicin, DNA damage in the Comet assay (using software package KOMET) and chromosome damage with the FISH technique (using software package KROMASCAN). The chromosome damage has been detected using chromoprobe-M for chromosome 1 and compared with chromosome damage measured using the conventional Giemsa staining technique. In all three assays, cycling cells were treated, after phytohaemagglutinin stimulation, at 48 h for about 20 h, which resulted in statistically significant dose-related responses in each assay. In non-cycling cells there was no increase in damage in the Comet assay, but there was in the chromosome assays. The FISH assay was only conducted in cycling cells, since the probe used was metaphase-specific. At the highest doses of doxorubicin used, FISH and conventional chromosome assays had similar sensitivities.

Antibiotics, Antineoplastic↗