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PubMedDate not supplied

1-amino-4-phenyl-1,2,3,6-tetrahydropyridine and 1-amino-4-phenylpyridinium salt, the 1-amino analogues of neurotoxins, MPTP and MPP+, induce apoptosis in PC12 cells: detection of apoptotic cells by Comet assay and flow cytometric analysis.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is known to induce parkinsonism in humans when it is oxidized to the 1-methyl-4-phenylpyridinium salt (MPP+). We previously reported the syntheses of 1-amino-4-phenyl-1,2,3,6-tetrahydropyridine (APTP) and 1-amino-4-phenyl-pyridinium salt (APP+), the 1-amino analogues of the dopaminergic neurotoxins, MPTP and MPP+, respectively, and demonstrated that both APTP and APP+ are cytotoxic to PC12 cells. In this study, we found that both APTP and APP+ induce apoptotic cell death in PC12 cells. Apoptosis was determined by the Comet assay and flow cytometric analysis. Prior to using the Comet assay for detection of apoptotic PC12 cells, Comet images of apoptotic and necrotic cells were first distinguished by using several standards. Comet images were classified into four groups (A to D) according to their shapes. Class D consisted of the apoptotic cells and was easily distinguished. We also demonstrated that apoptotic and necrotic PC12 cells can be easily differentiated and quantified using the convenient Comet assay.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗

PubMedDate not supplied

[Use of the comet test in occupational medicine and industrial toxicology: considerations and prospects].

The comet test (Single Cell Gel Electrophoresis or SCGE) is an easy to perform, rapid and highly sensitive genotoxicity assay; it requires small amounts of biological substrate and is applicable in vivo and in vitro to a wide variety of cells and tissues. Modified versions of the comet test able to detect oxidative damage or the effects of agents inducing DNA-DNA or DNA-protein crosslinks are available. Similarly to other genotoxicity assays, the comet test is not predictive far individual cancer risk. Limitations and critical features presently linked to comet test applications, with particular regard to the biomonitoring of individuals exposed to genotoxic agents, include: lack of sensitivity with respect to aneugens (agents inducing numerical chromosomal aberrations), possible underestimation of genotoxic potency of agents with mixed action mechanisms, sensitivity depending on the genotoxic agent itself, dependence an biological substrate with regard to the influence of cytotoxicity on the assay results, influence of age, tobacco smoke, alcohol and drug consumption, diet, kinetics of DNA adducts and DNA repair mechanisms. Consequently, a routine use of the comet test in biological monitoring of individuals that are occupationally and environmentally exposed to genotoxic agents is submitted to its validation by multicentric studies on large population samples with different exposure patterns, with a suitable characterisation of the role played by the above mentioned factors. Human studies should also be increasingly focused on the direct cellular targets of exposure to genotoxicants (e.g. epithelial cells of oral cavity and airways).

Age Factors↗

PubMed2000

Detection of specific DNA lesions by a combination of comet assay and FISH in plants.

We have studied comet formation on Vicia faba nuclei embedded in agarose and treated with the endonucleases DNase I (to produce SSBs or DSBs at random sites), FokI (to produce DSBs preferentially within FokI repeats), or EcoRI (to produce DSBs at random sites but not within FokI elements). DNase I-induced SSBs were detected when enzyme treatment was followed by alkaline denaturation. DSBs efficiently mediated comet formation using neutral conditions. FISH with DNA probes, detecting specific chromosomal domains such as FokI element-containing heterochromatin, NORs, or telomeres, was done on comets. The distribution of FISH signals between the head and tail of comets indicated to which degree these domains were damaged and reflected the distribution of cleavage sites for the applied restriction endonucleases within these domains. The data confirmed the expectation that the observed comet formation was based on enzyme-specific DNA breakage.

DNA, Plant↗

PubMed1982

Comets: chemistry and chemical evolution.

Lasting commitment to cosmic chemistry and an awareness of the fascinating role of comets in that study was a consequence of an association with Harold Urey early in my astronomical career. Urey's influence on cometary research spread as colleagues with whom I was associated, in turn, developed their own programs in cometary chemistry. One phase of the Chicago research shows that Whipple's icy nucleus would be below about 250 K. This property, combined with their small internal pressure, means cometary interiors remain essentially unchanged during their lifetime. Observations of cometary spectra indicate that they are rich in simple organic species. Experiments on comet-like ice mixture suggests that the extensive array of interstellar molecules also may be found in comets. The capture of cometary debris by the earth or the impact of comets would have been an early source of biochemically significant molecules. Recent hypotheses on radiogenic heating and melting of water ice in the central zone of nuclei do not seem consistent with recent observations or ideas of structure. Thus comets are not a likely place for life to develop.

Astronomical Phenomena↗

PubMed1999

Radiation-induced comet-formation in human skin fibroblasts from radiotherapy patients with different normal tissue reactions.

BACKGROUND: In clinical radiotherapy most patients tolerate the applied dosage with no or moderate side effects. However, 5 to 10% of all individuals show increased acute and/or late reactions. In-vitro test systems are investigated for their suitability for predictive purposes. This paper attempts a correlation between the induction and repair of DNA damage measured in the comet assay and the clinical observed reaction in order to evaluate the suitability of the comet assay for prediction of radiation sensitivity. PATIENTS AND METHODS: Skin fibroblasts of 30 patients with average tissue reactions or acute and/or late increased side effects and cell lines of 4 individuals carrying the heritable disease ataxia telangiectasia (AT) were irradiated in vitro. The induction and repair of DNA damage was measured at different time points after irradiation in the comet assay (single cell gel electrophoresis). These results were compared to the acute and late clinical reactions classified according to the RTOG grading system. RESULTS: The radiation induced DNA damage decreased over time reflecting DNA repair. Cells of the AT individuals showed an elevated damage induction and a reduced repair capacity compared to patients with average tissue reactions. Fibroblasts of patients with increased acute and late side effects exhibited slower DNA repair. In addition to the known lack of cell cycle control, our results indicate that AT cells show reduced DNA repair capacity. CONCLUSIONS: The comet assay seems to be able to detect some types of increased individual radiation sensitivity. In contrast to other predictive in-vitro tests, the comet assay needs less time and fewer cells, which would be useful in a clinical setting.

Ataxia Telangiectasia↗

PubMed1989

Thermal evolution of Comet P/Tempel 1--representing the group of targets for the CRAF and CNSR missions.

The properties of the outer layers of comets considered for the future Comet Rendezvous and Asteroid Flyby and Comet Nucleus Sample Return missions are studied, by following numerically the thermal evolution of spherically symmetric models of the nucleus, in the orbit of Comet P/Tempel-1. The evolution starts from isothermal (10 degrees K) and homogeneous nuclei, composed of amorphous ice and dust. The crystallization of amorphous ice at 137 degrees K is taken into account. As the ice sublimates, a permanent dust mantle is allowed to accumulate, at a rate which is proportional to the sublimation rate. Evolutionary sequences are computed for different values of the density, the dust/ice mass ratio, and the (constant) fraction of the dust which is not carried away with the sublimating ice. The main conclusions are (a) the temperatures at the outer and inner surfaces of the dust mantle are not very sensitive to changes in the parameters; (b) although the dust is assumed permeable to water vapor the rate of erosion of the nucleus slows down as the dust mantle grows and its insulating effect increases; (c) the temperature at a depth of 10 m is approximately 160 degrees K for all models considered and hence, the ice at this depth is crystalline; (d) the total thickness of the crystalline ice layer, between the dust mantle and the amorphous ice core, varies from 40 to 240 m, depending on the parameters assumed. Consequently, it should be difficult for the probes of the two comet missions to sample pristine amorphous ice, unless they are aimed at the bottom of an active crater.

Astronomical Phenomena↗

PubMed1995

Comparative studies by comet test and SCE analysis in human lymphocytes from 200 healthy subjects.

The comet test (single cell gel electrophoresis, SCGE) appears to be a promising tool to estimate DNA damage at the single cell level and it provides information on the presence of damage among individual cells. Previously, we analyzed the degree of DNA damage in peripheral human lymphocytes from 100 healthy subjects living in Pisa (Italy) taking into account age, gender and smoking habit, and we also reported some results aiming at the assessment of the comet test (Betti el al., 1994). In addition, SCE analysis was carried out in order to compare the two endpoints. Because of the interesting results obtained, the present study was extended to 200 individuals, and data analyzed included information concerning number of cigarettes smoked a day, tar/cigarette and job. Data obtained confirmed that the SCGE is more sensitive than SCE in revealing smoking habit effects but comet induction did not seem to be related to the amount of cigarette tar inhaled. Moreover, sampling time was found to play a greater role in the comet assay as compared to SCE. Job position did not significantly influence SCE mean/subject or comet length mean/subject.

Adolescent↗

PubMed1995

Determination of radiation-induced damage in lymphocytes using the micronucleus and microgel electrophoresis 'Comet' assays.

DNA damage assays may be useful as rapid predictors of normal tissue radiosensitivity in clinical samples. We measured in vitro radiation-induced (2 Gy) damage to lymphocytes from cancer patients and normal healthy donors using both the micronucleus and microgel electrophoresis (Comet) assays simultaneously. For damage assessment, there was a good correlation (P < 0.001) between the mean comet lengths and the fraction of cells with comets. There was no correlation with initial damage, determined as the proportion of cells within a sample that formed comets, in comparison with the mean frequency of micronuclei per binucleate cell. However, there appeared to be an association between the determination of repair proficiency in the Comet assay and the mean frequency of micronuclei per binucleate cell in lymphocytes from cancer patients.

DNA↗

PubMed1997

The comet assay: what can it really tell us?

A range of applications of the alkaline comet assay is covered, from investigations of the physicochemical behaviour of DNA, through studies of cellular responses to DNA damage, to biomonitoring of human populations. The underlying principles of this assay are discussed, and new evidence presented which supports the concept of relaxation of supercoiled loops, rather than alkaline unwinding, as the primary reason for comet tail formation. DNA-damaging agents that do not induce strand breaks directly can be detected when cellular repair processes convert lesions to transient strand breaks; an approach is outlined here which maximises this effect and thus widens the scope of the assay. Purified repair enzymes, applied to DNA during the course of the comet assay procedure, greatly increase the sensitivity and specificity of the assay; recent developments with formamidopyrimidine glycosylase (recognising 8-OH-gua and other damaged purines) and uvrABC (for bulky lesions) are presented. The kinetics of cellular repair after low doses of oxidative damage have been followed with this modified comet assay. Finally, the successful measurement of biomarkers of oxidative damage in human populations establishes the comet assay as a valuable tool in molecular epidemiology.

Cell Nucleus↗

PubMed1996

Comparison of chemically induced DNA breakage in cellular and subcellular systems using the comet assay.

The alkaline comet assay, employing a single cell gel electrophoresis, is a rapid, simple and sensitive technique for visualizing and measuring DNA damage leading to strand breakage in individual mammalian cells. In this report, we describe a modified version of this assay which we used to assess DNA damage as a result of treating lysed cells with genotoxic and antimetabolic agents. By means of this modified assay, DNA is no longer held under the regulation of any metabolic pathway or membrane barrier. Using 3 direct-acting agents, hydrogen peroxide, N-methyl-N-nitrosourea, and bleomycin, we were able to induce increased DNA migration by both the standard and modified comet assays. In contrast, with 4-nitroquinoline 1-oxide, 5-fluorouracil, and methotrexate, which require cellular enzymatic activity to induce DNA damage, we succeeded in inducing increased DNA migration using the standard comet assay conditions only. In some cases, the modified comet assay might be helpful in analyzing chemical and biological characteristics of genotoxic agents when performed in combination with the standard comet assay.

4-Nitroquinoline-1-oxide↗

PubMed1995

Comets in other planetary systems?

Comets in our solar system appear to have provided a bridge between the cold, volatile-rich outer solar system, and the warm, but volatile-poor inner solar system. Excluding tidal and possible extinct radionuclide heating sources, only in the inner solar system are temperatures high enough for liquid water, and therefore life as we know it, to exist for times comparable to the age of the solar system. Comets may have been crucial for providing biogenic volatiles and perhaps organic molecules to this warm environment. It is therefore interesting from an exobiological point of view to ask if comets exist in other planetary systems. Most attempts to detect comets around other stars or in interstellar space have failed. However, there is growing spectroscopic evidence for comet-like bodies orbiting the star Beta Pictoris.

Astronomical Phenomena↗

PubMed1998

Chromosomal aberrations and alkaline comet assay in families with habitual abortion.

Within the pathology of human reproduction, genetic abnormalities play an important role in spontaneous abortions. This paper describes the morphologic, karyotypic features of a consecutive series of singleton spontaneous abortions collected as part of this study and also reports the application of the alkaline comet assay to assess levels of DNA damage in 31 couples comprised of 13 control couples and a patient group of 18 couples with a history of more than one fetal loss. For the cytogenetic analyses, the conventional lymphocyte culture method was applied to all subjects. In this analysis, two women with habitual abortion were determined to carry balanced chromosomal translocation. The alkaline comet assay (single cell gel electrophoresis technique) was applied also to lymphocytes. The comparison of the results of alkaline comet assay in patient and control individuals showed a significant difference in the number of damaged cells. The cells were evaluated according to their grades of damage as: normal (undamaged-no migration), limited migration, (at low damage level) and extensive migration (comet imaged cells-with increasing numbers of breaks, DNA pieces migrate freely into the tail forming a comet image). The frequency of limited migrated and extensive migrated cells in the women in the patient group were higher than in the women in the control group (p<0.001). However, all above parameters were equal for husbands in both the control and patient group (p>0.05).

Abortion, Habitual↗

PubMed1997

Collisional quenching of OH radio emission from comet Hale-Bopp.

Observations of comets in the 18-cm OH transitions offer a means to probe gas production, kinematics, and OH excitation in comets. We present initial results of OH observations of comet Hale-Bopp obtained with the NRAO 43 m antenna located in Greenbank, WV. Maps of the emission provide strong constraints on the amount of quenching of the inversion of the OH ground state A-doublet in the coma. Analysis of the total radio OH flux and maps of its radial brightness distribution indicate a quenched region on the order of approximately 500,000 km during March and April 1997. This large value is generally consistent with previous observations of radio OH quenching in lower production rate comets when the high production rate of comet Hale-Bopp is considered.

Astronomy↗

PubMed1985

Temperatures within comet nuclei.

We have performed a theoretical study of temperatures beneath the surface of a comet's nucleus. We solve the one-dimensional heat conduction equation for the outer portion of the comet. The upper boundary condition of the model is given by energy balance at the surface of the nucleus, including conduction of heat inward, radiation, insolation as modified by the coma, and sublimation. Our coma model assumes single scattering and includes attenuation of direct sunlight by dust grains, scattering of light onto the nucleus, and infrared radiation by dust grains. The lower boundary condition is zero net heat flux around an orbit. The thermal conductivity expression for the nucleus includes direct conduction at grain boundaries, radiative conduction, and Knudsen flow vapor diffusion. The thermal diffusivity of the nucleus and the resultant temperature profiles are shown to be strongly dependent on the physical properties of the material, including porosity, pore size, and compaction. The temperature profiles and the equilibrium temperature deep within the comet also depend on the functional relationship between thermal conductivity and temperature; the highest deep equilibrium temperatures are found for models where the thermal conductivity increases strongly with increasing temperature. The dependence of temperatures on the albedo and thermal emissivity of the nucleus is also calculated, as well as the variation of temperature with latitude for a variety of pole orientations. The effect of a dust mantle on subsurface temperatures is also investigated. All calculations are presented for short-period comets with orbits that make them accessible for exploration by spacecraft rendezvous. In situ measurements of the thermal profile in the upper meter of a comet nucleus can substantially constrain the thermal diffusivity of the material, which in turn can provide significant information about the physical properties of the nucleus.

Cosmic Dust↗

PubMed1999

Identification of two sources of carbon monoxide in comet Hale-Bopp.

The composition of ices in comets may reflect that of the molecular cloud in which the Sun formed, or it may show evidence of chemical processing in the pre-planetary accretion disk around the proto-Sun. As carbon monoxide (CO) is ubiquitous in molecular clouds, its abundance with respect to water could help to determine the degree to which pre-cometary material was processed, although variations in CO abundance may also be influenced by the distance from the Sun at which comets formed. Observations have not hitherto provided an unambiguous measure of CO in the cometary ice (native CO). Evidence for an extended source of CO associated with comet Halley was provided by the Giotto spacecraft, but alternative interpretations exist. Here we report observations of comet Hale-Bopp which show that about half of the CO in the comet comes directly from ice stored in the nucleus. The abundance of this CO with respect to water (12 per cent) is smaller than in quiescent regions of molecular clouds, but is consistent with that measured in proto-stellar envelopes, suggesting that the ices underwent some processing before their inclusion into Hale-Bopp. The remaining CO arises in the coma, probably through thermal destruction of more complex molecules.

Carbon Monoxide↗

PubMed2000

Interception of comet Hyakutake's ion tail at a distance of 500 million kilometres

Remote sensing observations and the direct sampling of material from a few comets have established the characteristic composition of cometary gas. This gas is ionized by solar ultraviolet radiation and the solar wind to form 'pick-up' ions, ions in a low ionization state that retain the same compositional signatures as the original gas. The pick-up ions are carried outward by the solar wind, and they could in principle be detected far from the coma (Sampling of pick-up ions has also been used to study interplanetary dust, Venus' tail and the interstellar medium.) Here we report the serendipitous detection of cometary pick-up ions, most probably associated with the tail of comet Hyakutake, at a distance of 3.4 AU from the nucleus. Previous observations have provided a wealth of physical and chemical information about a small sample of comets, but this detection suggests that remote sampling of comet compositions, and the discovery of otherwise invisible comets, may be possible.

Journal Article↗

PubMed1997

Detection of hypoxic cells in a C3H mouse mammary carcinoma using the comet assay.

The comet assay was used to estimate radiobiological hypoxic fraction across a full range of tumour oxygenations in C3H mammary tumours implanted into the feet of female CDF1 mice. Tumours were either clamped before irradiation or mice were allowed to breath air, 100% oxygen, carbogen or carbon monoxide for 5-35 min before and during exposure to 15 Gy. For the alkaline comet assay, tumours were excised after irradiation and individual tumour cells were analysed for DNA single-strand breaks. Hypoxic cells were defined as those cells with approximately three times fewer single-strand breaks than aerobic cells. Radiobiological hypoxic fraction was calculated by fitting DNA damage histograms to two normal distributions, representing the response of the aerobic and hypoxic populations. The percentage of hypoxic cells estimated using the comet assay was then compared with hypoxic fraction measured using a clamped tumour control assay. Carbogen and oxygen breathing reduced the normal hypoxic fraction from 14% to 2-3% in this tumour, whereas 75-660 p.p.m. carbon monoxide progressively increased the hypoxic fraction from 18% to 82%. The slope of the line comparing the two methods was 1.23 with 95% confidence limits of 1.12-1.33 (r2 = 0.994). In the SCCVII squamous cell carcinoma growing subcutaneously in C3H mice, a similar correlation was observed between hypoxic fraction measured using the comet assay and hypoxic fraction measured in the same tumour cells using the paired survival curve assay (slope = 1.20 with 95% confidence limits of 1.03-1.37). These results confirm the ability of the comet assay to provide an accurate estimate of radiobiological hypoxic fraction over a wide range of tumour oxygenations and between two tumour types.

Animals↗

PubMed2006

Hydrogen cyanide polymers, comets and the origin of life.

Hydrogen cyanide polymers--heterogeneous solids ranging in colour from yellow to orange to brown to black--could be major components of the dark matter observed on many bodies of the outer solar system including asteroids, moons, planets and, especially, comets. The presence on cometary nuclei of frozen volatiles such as methane, ammonia and water subjected to high energy sources makes them attractive sites for the ready formation and condensed-phase polymerization of hydrogen cyanide. This could account for the dark crust observed on Comet Halley in 1986 by the Vega and Giotto missions. Dust emanating from its nucleus would arise partly from HCN polymers as suggested by the Giotto detection of free hydrogen cyanide, CN radicals, solid particles consisting only of H, C and N, or only of H, C, N, O, and nitrogen-containing organic compounds. Further evidence for cometary HCN polymers could be expected from in situ analysis of the ejected material from Comet Tempel 1 after collision with the impactor probe from the two-stage Deep Impact mission on July 4, 2005. Even more revealing will be actual samples of dust collected from the coma of Comet Wild 2 by the Stardust mission, due to return to Earth in January 2006 for analyses which we have predicted will detect these polymers and related compounds. In situ results have already shown that nitriles and polymers of hydrogen cyanide are probable components of the cometary dust that struck the Cometary and Interstellar Dust Analyzer of the Stardust spacecraft as it approached Comet Wild 2 on January 2, 2004. Preliminary evidence (January 2005) obtained by the Huygens probe of the ongoing Cassini-Huygens mission to Saturn and its satellites indicates the presence of nitrogen-containing organic compounds in the refractory organic cores of the aerosols that give rise to the orange haze high in the atmosphere of Titan, Saturn's largest moon. Our continuing investigations suggest that HCN polymers are basically of two types: ladder structures with conjugated -C=N- bonds and polyamidines readily converted by water to polypeptides. Thermochemolysis GC-MS studies show that cleavage products of the polymer include alpha-amino acids, nitrogen heterocycles such as purines and pyrimidines, and provide evidence for peptide linkages. Hydrogen cyanide polymers are a plausible link between cosmochemistry and the origin of informational macromolecules. Implications for prebiotic chemistry are profound. Following persistent bolide bombardment, primitive Earth may have been covered by water and carbonaceous compounds, particularly HCN polymers which would have supplied essential components for establishing protein/nucleic acid life.

Cosmic Dust↗