Comet assay evaluation of six chemicals of known genotoxic potential in rats.

PubMed

Hobbs, Cheryl A; Recio, Leslie; Streicker, Michael; Boyle, Molly H; Tanaka, Jin; Shiga, Atsushi; Witt, Kristine L

2015-07-01

As a part of an international validation of the in vivo rat alkaline comet assay (comet assay) initiated by the Japanese Center for the Validation of Alternative Methods (JaCVAM) we examined six chemicals for potential to induce DNA damage: 2-acetylaminofluorene (2-AAF), N-nitrosodimethylamine (DMN), o-anisidine, 1,2-dimethylhydrazine dihydrochloride (1,2-DMH), sodium chloride, and sodium arsenite. DNA damage was evaluated in the liver and stomach of 7- to 9-week-old male Sprague Dawley rats. Of the five genotoxic carcinogens tested in our laboratory, DMN and 1,2-DMH were positive in the liver and negative in the stomach, 2-AAF and o-anisidine produced an equivocal result in liver and negative results in stomach, and sodium arsenite was negative in both liver and stomach. 1,2-DMH and DMN induced dose-related increases in hedgehogs in the same tissue (liver) that exhibited increased DNA migration. However, no cytotoxicity was indicated by the neutral diffusion assay (assessment of highly fragmented DNA) or histopathology in response to treatment with any of the tested chemicals. Therefore, the increased DNA damage resulting from exposure to DMN and 1,2-DMH was considered to represent a genotoxic response. Sodium chloride, a non-genotoxic non-carcinogen, was negative in both tissues as would be predicted. Although only two (1,2-DMH and DMN) out of five genotoxic carcinogens produced clearly positive results in the comet assay, the results obtained for o-anisidine and sodium arsenite in liver and stomach cells are consistent with the known mode of genotoxicity and tissue specificity exhibited by these carcinogens. In contrast, given the known genotoxic mode-of-action and target organ carcinogenicity of 2-AAF, it is unclear why this chemical failed to convincingly increase DNA migration in the liver. Thus, the results of the comet assay validation studies conducted in our laboratory were considered appropriate for five out of the six test chemicals. Copyright © 2015 Elsevier B.V. All rights reserved.

Comet assay evaluation of six chemicals of known genotoxic potential in rats

PubMed Central

Hobbs, Cheryl A.; Recio, Leslie; Streicker, Michael; Boyle, Molly H.; Tanaka, Jin; Shiga, Atsushi; Witt, Kristine L.

2015-01-01

As a part of an International validation of the in vivo rat alkaline comet assay (comet assay) initiated by the Japanese Center for the Validation of Alternative Methods (JaCVAM) we examined six chemicals for potential to induce DNA damage: 2-acetylaminofluorene (2-AAF), N-nitrosodimethylamine (DMN), o-anisidine, 1,2-dimethylhydrazine dihydrochloride (1,2-DMH), sodium chloride, and sodium arsenite. DNA damage was evaluated in the liver and stomach of 7- to 9-week-old male Sprague Dawley rats. Of the five genotoxic carcinogens tested in our laboratory, DMN and 1,2-DMH were positive in the liver and negative in the stomach, 2-AAF and o-anisidine produced an equivocal result in liver and negative results in stomach, and sodium arsenite was negative in both liver and stomach. 1,2-DMH and DMN induced dose-related increases in hedgehogs in the same tissue (liver) that exhibited increased DNA migration. However, no cytotoxicity was indicated by the neutral diffusion assay (assessment of highly fragmented DNA) or histopathology in response to treatment with any of the tested chemicals. Therefore, the increased DNA damage resulting from exposure to DMN and 1,2-DMH was considered to represent a genotoxic response. Sodium chloride, a non-genotoxic non-carcinogen, was negative in both tissues as would be predicted. Although only two (1,2-DMH and DMN) out of five genotoxic carcinogens produced clearly positive results in the comet assay, the results obtained for o-anisidine and sodium arsenite in liver and stomach cells are consistent with the known mode of genotoxicity and tissue specificity exhibited by these carcinogens. In contrast, given the known genotoxic mode-of-action and target organ carcinogenicity of 2-AAF, it is unclear why this chemical failed to convincingly increase DNA migration in the liver. Thus, the results of the comet assay validation studies conducted in our laboratory were considered appropriate for five out of the six test chemicals. PMID:26212309

Using a medium-throughput comet assay to evaluate the global DNA methylation status of single cells

PubMed Central

Lewies, Angélique; Van Dyk, Etresia; Wentzel, Johannes F.; Pretorius, Pieter J.

2014-01-01

The comet assay is a simple and cost effective technique, commonly used to analyze and quantify DNA damage in individual cells. The versatility of the comet assay allows introduction of various modifications to the basic technique. The difference in the methylation sensitivity of the isoschizomeric restriction enzymes HpaII and MspI are used to demonstrate the ability of the comet assay to measure the global DNA methylation level of individual cells when using cell cultures. In the experiments described here, a medium-throughput comet assay and methylation sensitive comet assay are combined to produce a methylation sensitive medium-throughput comet assay to measure changes in the global DNA methylation pattern in individual cells under various growth conditions. PMID:25071840

A quantitative comet infection assay for influenza virus

PubMed Central

Lindsay, Stephen M.; Timm, Andrea; Yin, John

2011-01-01

Summary The virus comet assay is a cell-based virulence assay used to evaluate an antiviral drug or antibody against a target virus. The comet assay differs from the plaque assay in allowing spontaneous flows in 6-well plates to spread virus. When implemented quantitatively the comet assay has been shown to have an order-of-magnitude greater sensitivity to antivirals than the plaque assay. In this study, a quantitative comet assay for influenza virus is demonstrated, and is shown to have a 13-fold increase in sensitivity to ribavirin. AX4 cells (MDCK cells with increased surface concentration of α2–6 sialic acid, the influenza virus receptor) have reduced the comet size variability relative to MDCK cells, making them a better host cell for use in this assay. Because of enhanced antiviral sensitivity in flow-based assays, less drug is required, which could lead to lower reagent costs, reduced cytotoxicity, and fewer false-negative drug screen results. The comet assay also serves as a readout of flow conditions in the well. Observations from comets formed at varying humidity levels indicate a role for evaporation in the mechanism of spontaneous fluid flow in wells. PMID:22155578

Assessment of the predictive capacity of the optimized in vitro comet assay using HepG2 cells.

PubMed

Hong, Yoon-Hee; Jeon, Hye Lyun; Ko, Kyung Yuk; Kim, Joohwan; Yi, Jung-Sun; Ahn, Ilyoung; Kim, Tae Sung; Lee, Jong Kwon

2018-03-01

Evaluation of DNA damage is critical during the development of new drugs because it is closely associated with genotoxicity and carcinogenicity. The in vivo comet assay to assess DNA damage is globally harmonized as OECD TG 489. However, a comet test guideline that evaluates DNA damage without sacrificing animals does not yet exist. The goal of this study was to select an appropriate cell line for optimization of the in vitro comet assay to assess DNA damage. We then evaluated the predictivity of the in vitro comet assay using the selected cell line. In addition, the effect of adding S9 was evaluated using 12 test chemicals. For cell line selection, HepG2, Chinese hamster lung (CHL/IU), and TK6 cell lines were evaluated. We employed a method for the in vitro comet assay based on that for the in vivo comet assay. The most appropriate cell line was determined by% tail DNA increase after performing in vitro comet assays with 6 test chemicals. The predictivity of the in vitro comet assay using the selected cell line was measured with 10 test chemicals (8 genotoxins and 2 non-genotoxic chemicals). The HepG2 cell line was found to be the most appropriate, and in vitro comet assays using HepG2 cells exhibited a high accuracy of 90% (9/10). This study suggests that HepG2 is an optimal cell line for the in vitro comet assay to assess DNA damage. Copyright © 2018 Elsevier B.V. All rights reserved.

Cytogenetic status and oxidative DNA-damage induced by atorvastatin in human peripheral blood lymphocytes: standard and Fpg-modified comet assay.

PubMed

Gajski, Goran; Garaj-Vrhovac, Vera; Orescanin, Visnja

2008-08-15

To investigate the genotoxic potential of atorvastatin on human lymphocytes in vitro standard comet assay was used in the evaluation of basal DNA damage and to investigate possible oxidative DNA damage produced by reactive oxygen species (ROS) Fpg-modified version of comet assay was also conducted. In addition to these techniques the new criteria for scoring micronucleus test were applied for more complete detection of baseline damage in binuclear lymphocytes exposed to atorvastatin 80 mg/day in different time periods by virtue of measuring the frequency of micronuclei, nucleoplasmic bridges and nuclear buds. All parameters obtained with the standard comet assay and Fpg-modified comet assay were significantly higher in the treated than in control lymphocytes. The Fpg-modified comet assay showed a significantly greater tail length, tail intensity, and tail moment in all treated lymphocytes than did the standard comet assay, which suggests that oxidative stress is likely to be responsible for DNA damage. DNA damage detected by the standard comet assay indicates that some other mechanism is also involved. In addition to the comet assay, a total number of micronuclei, nucleoplasmic bridges and nuclear buds were significantly higher in the exposed than in controlled lymphocytes. Regression analyses showed a positive correlation between the results obtained by the comet (Fpg-modified and standard) and micronucleus assay. Overall, the study demonstrated that atorvastatin in its highest dose is capable of producing damage on the level of DNA molecule and cell.

The next three decades of the comet assay: a report of the 11th International Comet Assay Workshop.

PubMed

Koppen, Gudrun; Azqueta, Amaya; Pourrut, Bertrand; Brunborg, Gunnar; Collins, Andrew R; Langie, Sabine A S

2017-05-01

The International Comet Assay Workshops are a series of scientific conferences dealing with practical and theoretical aspects of the Comet Assay (single-cell gel electrophoresis)-a simple method for detecting DNA strand breaks. The first paper describing such an assay was published over 30 years ago in 1984 by Swedish researchers O. Ostling and K. J. Johanson. Appropriately, the theme for the 2015 meeting was looking to the future: 'The Next 3 Decades of the Comet Assay'. The programme included 25 oral and 43 poster presentations depicting the latest advances in technical developments as well as applications of the comet assay in genotoxicity testing (in vitro and in vivo) and biomonitoring of both humans and the environment. Open discussion sessions based on questions from the participants allowed exchange of practical details on current comet assay protocols. This report summarises technical issues of high importance which were discussed during the sessions. We provide information on ways to improve the assay performance, by testing for cytotoxicity, by using reference samples to reduce or allow for inter-experimental variation, and by standardising quantification of the damage, including replicates and scoring enough comets to ensure statistical validity. After 30 years of experimentation with the comet assay, we are in a position to control the important experimental parameters and make the comet assay a truly reliable method with a wealth of possible applications. © The Author 2017. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Results of the International Validation of the in vivo rodent alkaline comet assay for the detection of genotoxic carcinogens: Individual data for 1,2-dibromoethane, p-anisidine, and o-anthranilic acid in the 2nd step of the 4th phase Validation Study under the JaCVAM initiative.

PubMed

Takasawa, Hironao; Takashima, Rie; Narumi, Kazunori; Kawasako, Kazufumi; Hattori, Akiko; Kawabata, Masayoshi; Hamada, Shuichi

2015-07-01

As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative International Validation Study of an in vivo rat alkaline comet assay, we examined 1,2-dibromoethane (DBE), p-anisidine (ASD), and o-anthranilic acid (ANT) to investigate the effectiveness of the comet assay in detecting genotoxic carcinogens. Each of the three test chemicals was administered to 5 male Sprague-Dawley rats per group by oral gavage at 48, 24, and 3h before specimen preparation. Single cells were collected from the liver and glandular stomach at 3h after the final dosing, and the specimens prepared from these two organs were subjected to electrophoresis under alkaline conditions (pH>13). The percentage of DNA intensity in the comet tail was then assessed using an image analysis system. A micronucleus (MN) assay was also conducted using these three test chemicals with the bone marrow (BM) cells collected from the same animals simultaneously used in the comet assay, i.e., combination study of the comet assay and BM MN assay. A genotoxic (Ames positive) rodent carcinogen, DBE gave a positive result in the comet assay in the present study, while a genotoxic (Ames positive) non-carcinogen, ASD and a non-genotoxic (Ames negative) non-carcinogen, ANT showed negative results in the comet assay. All three chemicals produced negative results in the BM MN assay. While the comet assay findings in the present study were consistent with those obtained from the rodent carcinogenicity studies for the three test chemicals, we consider the positive result in the comet assay for DBE to be particularly meaningful, given that this chemical produced a negative result in the BM MN assay. Therefore, the combination study of the comet assay and BM MN assay is a useful method to detect genotoxic carcinogens that are undetectable with the BM MN assay alone. Copyright © 2015 Elsevier B.V. All rights reserved.

Cytogenetic status and oxidative DNA-damage induced by atorvastatin in human peripheral blood lymphocytes: Standard and Fpg-modified comet assay

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gajski, Goran; Garaj-Vrhovac, Vera; Orescanin, Visnja

2008-08-15

To investigate the genotoxic potential of atorvastatin on human lymphocytes in vitro standard comet assay was used in the evaluation of basal DNA damage and to investigate possible oxidative DNA damage produced by reactive oxygen species (ROS) Fpg-modified version of comet assay was also conducted. In addition to these techniques the new criteria for scoring micronucleus test were applied for more complete detection of baseline damage in binuclear lymphocytes exposed to atorvastatin 80 mg/day in different time periods by virtue of measuring the frequency of micronuclei, nucleoplasmic bridges and nuclear buds. All parameters obtained with the standard comet assay andmore » Fpg-modified comet assay were significantly higher in the treated than in control lymphocytes. The Fpg-modified comet assay showed a significantly greater tail length, tail intensity, and tail moment in all treated lymphocytes than did the standard comet assay, which suggests that oxidative stress is likely to be responsible for DNA damage. DNA damage detected by the standard comet assay indicates that some other mechanism is also involved. In addition to the comet assay, a total number of micronuclei, nucleoplasmic bridges and nuclear buds were significantly higher in the exposed than in controlled lymphocytes. Regression analyses showed a positive correlation between the results obtained by the comet (Fpg-modified and standard) and micronucleus assay. Overall, the study demonstrated that atorvastatin in its highest dose is capable of producing damage on the level of DNA molecule and cell.« less

CometQ: An automated tool for the detection and quantification of DNA damage using comet assay image analysis.

PubMed

Ganapathy, Sreelatha; Muraleedharan, Aparna; Sathidevi, Puthumangalathu Savithri; Chand, Parkash; Rajkumar, Ravi Philip

2016-09-01

DNA damage analysis plays an important role in determining the approaches for treatment and prevention of various diseases like cancer, schizophrenia and other heritable diseases. Comet assay is a sensitive and versatile method for DNA damage analysis. The main objective of this work is to implement a fully automated tool for the detection and quantification of DNA damage by analysing comet assay images. The comet assay image analysis consists of four stages: (1) classifier (2) comet segmentation (3) comet partitioning and (4) comet quantification. Main features of the proposed software are the design and development of four comet segmentation methods, and the automatic routing of the input comet assay image to the most suitable one among these methods depending on the type of the image (silver stained or fluorescent stained) as well as the level of DNA damage (heavily damaged or lightly/moderately damaged). A classifier stage, based on support vector machine (SVM) is designed and implemented at the front end, to categorise the input image into one of the above four groups to ensure proper routing. Comet segmentation is followed by comet partitioning which is implemented using a novel technique coined as modified fuzzy clustering. Comet parameters are calculated in the comet quantification stage and are saved in an excel file. Our dataset consists of 600 silver stained images obtained from 40 Schizophrenia patients with different levels of severity, admitted to a tertiary hospital in South India and 56 fluorescent stained images obtained from different internet sources. The performance of "CometQ", the proposed standalone application for automated analysis of comet assay images, is evaluated by a clinical expert and is also compared with that of a most recent and related software-OpenComet. CometQ gave 90.26% positive predictive value (PPV) and 93.34% sensitivity which are much higher than those of OpenComet, especially in the case of silver stained images. The results are validated using confusion matrix and Jaccard index (JI). Comet assay images obtained after DNA damage repair by incubation in the nutrient medium were also analysed, and CometQ showed a significant change in all the comet parameters in most of the cases. Results show that CometQ is an accurate and efficient tool with good sensitivity and PPV for DNA damage analysis using comet assay images. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Four-fluid MHD simulations of the plasma and neutral gas environment of comet 67P/Churyumov-Gerasimenko near perihelion

NASA Astrophysics Data System (ADS)

Huang, Zhenguang; Tóth, Gábor; Gombosi, Tamas I.; Jia, Xianzhe; Rubin, Martin; Fougere, Nicolas; Tenishev, Valeriy; Combi, Michael R.; Bieler, Andre; Hansen, Kenneth C.; Shou, Yinsi; Altwegg, Kathrin

2016-05-01

The neutral and plasma environment is critical in understanding the interaction of the solar wind and comet 67P/Churyumov-Gerasimenko (CG), the target of the European Space Agency's Rosetta mission. To serve this need and support the Rosetta mission, we have developed a 3-D four-fluid model, which is based on BATS-R-US (Block-Adaptive Tree Solarwind Roe-type Upwind Scheme) within SWMF (Space Weather Modeling Framework) that solves the governing multifluid MHD equations and the Euler equations for the neutral gas fluid. These equations describe the behavior and interactions of the cometary heavy ions, the solar wind protons, the electrons, and the neutrals. This model incorporates different mass loading processes, including photoionization and electron impact ionization, charge exchange, dissociative ion-electron recombination, and collisional interactions between different fluids. We simulated the plasma and neutral gas environment near perihelion in three different cases: an idealized comet with a spherical body and uniform neutral gas outflow, an idealized comet with a spherical body and illumination-driven neutral gas outflow, and comet CG with a realistic shape model and illumination-driven neutral gas outflow. We compared the results of the three cases and showed that the simulations with illumination-driven neutral gas outflow have magnetic reconnection, a magnetic pileup region and nucleus directed plasma flow inside the nightside reconnection region, which have not been reported in the literature.

Use of statistical analysis to validate ecogenotoxicology findings arising from various comet assay components.

PubMed

Hussain, Bilal; Sultana, Tayyaba; Sultana, Salma; Al-Ghanim, Khalid Abdullah; Masoud, Muhammad Shahreef; Mahboob, Shahid

2018-04-01

Cirrhinus mrigala, Labeo rohita, and Catla catla are economically important fish for human consumption in Pakistan, but industrial and sewage pollution has drastically reduced their population in the River Chenab. Statistics are an important tool to analyze and interpret comet assay results. The specific aims of the study were to determine the DNA damage in Cirrhinus mrigala, Labeo rohita, and Catla catla due to chemical pollution and to assess the validity of statistical analyses to determine the viability of the comet assay for a possible use with these freshwater fish species as a good indicator of pollution load and habitat degradation. Comet assay results indicated a significant (P < 0.05) degree of DNA fragmentation in Cirrhinus mrigala followed by Labeo rohita and Catla catla in respect to comet head diameter, comet tail length, and % DNA damage. Regression analysis and correlation matrices conducted among the parameters of the comet assay affirmed the precision and the legitimacy of the results. The present study, therefore, strongly recommends that genotoxicological studies conduct appropriate analysis of the various components of comet assays to offer better interpretation of the assay data.

Can the comet assay be used reliably to detect nanoparticle-induced genotoxicity?

PubMed

Karlsson, Hanna L; Di Bucchianico, Sebastiano; Collins, Andrew R; Dusinska, Maria

2015-03-01

The comet assay is a sensitive method to detect DNA strand breaks as well as oxidatively damaged DNA at the level of single cells. Today the assay is commonly used in nano-genotoxicology. In this review we critically discuss possible interactions between nanoparticles (NPs) and the comet assay. Concerns for such interactions have arisen from the occasional observation of NPs in the "comet head", which implies that NPs may be present while the assay is being performed. This could give rise to false positive or false negative results, depending on the type of comet assay endpoint and NP. For most NPs, an interaction that substantially impacts the comet assay results is unlikely. For photocatalytically active NPs such as TiO2 , on the other hand, exposure to light containing UV can lead to increased DNA damage. Samples should therefore not be exposed to such light. By comparing studies in which both the comet assay and the micronucleus assay have been used, a good consistency between the assays was found in general (69%); consistency was even higher when excluding studies on TiO2 NPs (81%). The strong consistency between the comet and micronucleus assays for a range of different NPs-even though the two tests measure different endpoints-implies that both can be trusted in assessing the genotoxicity of NPs, and that both could be useful in a standard battery of test methods. © 2014 Wiley Periodicals, Inc.

Combining DSMC Simulations and ROSINA/COPS Data of Comet 67P/Churyumov-Gerasimenko to Develop a Realistic Empirical Coma Model and to Determine Accurate Production Rates

NASA Astrophysics Data System (ADS)

Hansen, K. C.; Fougere, N.; Bieler, A. M.; Altwegg, K.; Combi, M. R.; Gombosi, T. I.; Huang, Z.; Rubin, M.; Tenishev, V.; Toth, G.; Tzou, C. Y.

2015-12-01

We have previously published results from the AMPS DSMC (Adaptive Mesh Particle Simulator Direct Simulation Monte Carlo) model and its characterization of the neutral coma of comet 67P/Churyumov-Gerasimenko through detailed comparison with data collected by the ROSINA/COPS (Rosetta Orbiter Spectrometer for Ion and Neutral Analysis/COmet Pressure Sensor) instrument aboard the Rosetta spacecraft [Bieler, 2015]. Results from these DSMC models have been used to create an empirical model of the near comet coma (<200 km) of comet 67P. The empirical model characterizes the neutral coma in a comet centered, sun fixed reference frame as a function of heliocentric distance, radial distance from the comet, local time and declination. The model is a significant improvement over more simple empirical models, such as the Haser model. While the DSMC results are a more accurate representation of the coma at any given time, the advantage of a mean state, empirical model is the ease and speed of use. One use of such an empirical model is in the calculation of a total cometary coma production rate from the ROSINA/COPS data. The COPS data are in situ measurements of gas density and velocity along the ROSETTA spacecraft track. Converting the measured neutral density into a production rate requires knowledge of the neutral gas distribution in the coma. Our empirical model provides this information and therefore allows us to correct for the spacecraft location to calculate a production rate as a function of heliocentric distance. We will present the full empirical model as well as the calculated neutral production rate for the period of August 2014 - August 2015 (perihelion).

A simple and novel modification of comet assay for determination of bacteriophage mediated bacterial cell lysis.

PubMed

Khairnar, Krishna; Sanmukh, Swapnil; Chandekar, Rajshree; Paunikar, Waman

2014-07-01

The comet assay is the widely used method for in vitro toxicity testing which is also an alternative to the use of animal models for in vivo testing. Since, its inception in 1984 by Ostling and Johansson, it is being modified frequently for a wide range of application. In spite of its wide applicability, unfortunately there is no report of its application in bacteriophages research. In this study, a novel application of comet assay for the detection of bacteriophage mediated bacterial cell lysis was described. The conventional methods in bacteriophage research for studying bacterial lysis by bacteriophages are plaque assay method. It is time consuming, laborious and costly. The lytic activity of bacteriophage devours the bacterial cell which results in the release of bacterial genomic material that gets detected by ethidium bromide staining method by the comet assay protocol. The objective of this study was to compare efficacy of comet assay with different assay used to study phage mediated bacterial lysis. The assay was performed on culture isolates (N=80 studies), modified comet assay appear to have relatively higher sensitivity and specificity than other assay. The results of the study showed that the application of comet assay can be an economical, time saving and less laborious alternative to conventional plaque assay for the detection of bacteriophage mediated bacterial cell lysis. Copyright © 2014 Elsevier B.V. All rights reserved.

Performance and data interpretation of the in vivo comet assay in pharmaceutical industry: EFPIA survey results.

PubMed

van der Leede, Bas-Jan; Doherty, Ann; Guérard, Melanie; Howe, Jonathan; O'Donovan, Mike; Plappert-Helbig, Ulla; Thybaud, Véronique

2014-12-01

In genotoxicity testing of pharmaceuticals the rodent alkaline comet assay is being increasingly used as a second in vivo assay in addition to the in vivo micronucleus assay to mitigate in vitro positive results as recommended by the ICH S2(R1) guideline. This paper summarizes a survey suggested by the Safety Working Party of European Medicines Agency (EMA), and conducted by the European Federation of Pharmaceutical Industries and Associations (EFPIA) to investigate the experience among European pharmaceutical companies by conducting the in vivo comet assay for regulatory purpose. A special focus was given on the typology of the obtained results and to identify potential difficulties encountered with the interpretation of study data. The participating companies reported a total of 147 studies (conducted in-house or outsourced) and shared the conclusion on the comet assay response for 136 studies. Most of the studies were negative (118/136). Only about 10% (14/136 studies) of the comet assays showed a positive response. None of the positive comet assay results were clearly associated with organ toxicity indicating that the positive responses are not due to cytotoxic effects of the compound in the tissue examined. The number of comet assays with an equivocal or inconclusive response was rare, respectively <1% (1/147 studies) and 2% (3/147 studies). In case additional information (e.g. repeat assay, organ toxicity, metabolism, tissue exposure) would have been available for evaluation, a final conclusion could most probably have been drawn for most or all of these studies. All (46) negative in vivo comet assays submitted alongside with a negative in vivo micronucleus assay were accepted by the regulatory authorities to mitigate a positive in vitro mammalian cell assay following the current ICH S2 guidance. The survey results demonstrate the robustness of the comet assay and the regulatory acceptance of the current ICH S2 guidance. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

The comet assay: assessment of in vitro and in vivo DNA damage.

PubMed

Bajpayee, Mahima; Kumar, Ashutosh; Dhawan, Alok

2013-01-01

Rapid industrialization and pursuance of a better life have led to an increase in the amount of chemicals in the environment, which are deleterious to human health. Pesticides, automobile exhausts, and new chemical entities all add to air pollution and have an adverse effect on all living organisms including humans. Sensitive test systems are thus required for accurate hazard identification and risk assessment. The Comet assay has been used widely as a simple, rapid, and sensitive tool for assessment of DNA damage in single cells from both in vitro and in vivo sources as well as in humans. Already, the in vivo comet assay has gained importance as the preferred test for assessing DNA damage in animals for some international regulatory guidelines. The advantages of the in vivo comet assay are its ability to detect DNA damage in any tissue, despite having non-proliferating cells, and its sensitivity to detect genotoxicity. The recommendations from the international workshops held for the comet assay have resulted in establishment of guidelines. The in vitro comet assay conducted in cultured cells and cell lines can be used for screening large number of compounds and at very low concentrations. The in vitro assay has also been automated to provide a high-throughput screening method for new chemical entities, as well as environmental samples. This chapter details the in vitro comet assay using the 96-well plate and in vivo comet assay in multiple organs of the mouse.

Recommendations for safety testing with the in vivo comet assay.

PubMed

Vasquez, Marie Z

2012-08-30

While the in vivo comet assay increases its role in regulatory safety testing, deliberations about the interpretation of comet data continue. Concerns can arise regarding comet assay publications with limited data from non-blind testing of positive control compounds and using protocols (e.g. dose concentrations, sample times, and tissues) known to give an expected effect. There may be a tendency towards bias when the validation or interpretation of comet assay data is based on results generated by widely accepted but non-validated assays. The greatest advantages of the comet assay are its sensitivity and its ability to detect genotoxicity in tissues and at sample times that could not previously be evaluated. Guidelines for its use and interpretation in safety testing should take these factors into account. Guidelines should be derived from objective review of data generated by blind testing of unknown compounds dosed at non-toxic concentrations and evaluated in a true safety-testing environment, where the experimental design and conclusions must be defensible. However, positive in vivo comet findings with such compounds are rarely submitted to regulatory agencies and this data is typically unavailable for publication due to its proprietary nature. To enhance the development of guidelines for safety testing with the comet assay, and with the permission of several sponsors, this paper presents and discusses relevant data from multiple GLP comet studies conducted blind, with unknown pharmaceuticals and consumer products. Based on these data and the lessons we have learned through the course of conducting these studies, I suggest significant adjustments to the current conventions, and I provide recommendations for interpreting in vivo comet assay results in situations where risk must be evaluated in the absence of carcinogenicity or clinical data. Copyright © 2012 Elsevier B.V. All rights reserved.

Evaluation of the cytotoxic and genotoxic potential of lecithin/chitosan nanoparticles

NASA Astrophysics Data System (ADS)

Taner, Gökçe; Yeşilöz, Recep; Özkan Vardar, Deniz; Şenyiğit, Taner; Özer, Özgen; Degen, Gisela H.; Başaran, Nurşen

2014-02-01

Nanoparticles-based drug targeting delivery systems have been introduced in the treatment for various diseases because of their effective properties, although there have been conflicting results on the toxicity of nanoparticles. In the present study, the aim was to evaluate the cytotoxicity and the genotoxicity of different concentrations of lecithin/chitosan nanoparticles with and without clobetasol-17-propionate (CP) by neutral red uptake (NRU) cytotoxicity assay and single cell gel electrophoresis (Comet) and cytokinesis-blocked micronucleus assays. The IC50 values of lecithin/chitosan nanoparticles with/without CP were found as 1.9 and 1.8 %, respectively, in the NRU cytotoxicity test. High concentrations of lecithin/chitosan nanoparticles induced DNA damage in human lymphocytes as evaluated by comet assay. The micronucleus frequency was increased by the lecithin/chitosan treatment in a dose-dependent manner. Also at the two highest concentrations, a significant increase in micronucleus formation was observed. Lecithin/chitosan nanoparticles with CP did not increase the frequency of micronucleus and also did not induce additional DNA damage when compared with lecithin/chitosan nanoparticles without CP; therefore, CP itself has not found to be genotoxic at the studied concentration.

The comet assay: ready for 30 more years.

PubMed

Møller, Peter

2018-02-24

During the last 30 years, the comet assay has become widely used for the measurement of DNA damage and repair in cells and tissues. A landmark achievement was reached in 2016 when the Organization for Economic Co-operation and Development adopted a comet assay guideline for in vivo testing of DNA strand breaks in animals. However, the comet assay has much more to offer than being an assay for testing DNA strand breaks in animal organs. The use of repair enzymes increases the range of DNA lesions that can be detected with the assay. It can also be modified to measure DNA repair activity. Still, despite the long-term use of the assay, there is a need for studies that assess the impact of variation in specific steps of the procedure. This is particularly important for the on-going efforts to decrease the variation between experiments and laboratories. The articles in this Special Issue of Mutagenesis cover important technical issues of the comet assay procedure, nanogenotoxicity and ionising radiation sensitivity on plant cells. The included biomonitoring studies have assessed seasonal variation and certain predictors for the basal level of DNA damage in white blood cells. Lastly, the comet assay has been used in studies on genotoxicity of environmental and occupational exposures in human biomonitoring studies and animal models. Overall, the articles in this Special Issue demonstrate the versatility of the comet assay and they hold promise that the assay is ready for the next 30 years.

Experiences with the in vivo and in vitro comet assay in regulatory testing.

PubMed

Frötschl, Roland

2015-01-01

The in vivo comet assay has recently been implemented into regulatory genotoxicity testing of pharmaceuticals with inclusion into the ICH S2R1 guidance. Regulatory genotoxicity testing aims to detect DNA alterations in form of gene mutations, larger scale chromosomal damage and recombination and aneuploidy. The ICH S2R1 guideline offers two options of standard batteries of tests for the detection of these endpoints. Both options start with an AMES assay and option 1 includes an in vitro mammalian cell assay and an in vivo micronucleus assay in rodent, whereas option 2 includes an in vivo micronucleus assay in bone marrow in rodent and a second in vivo assay in a second tissue with a second endpoint. The test recommended as second in vivo test is the comet assay in rat liver. The in vivo comet assay is considered as mature enough to ensure reliable detection of relevant in vivo genotoxicants in combination with the micronucleus test in bone marrow and the AMES assay. Although lots of research papers have been published using the in vitro comet assay, the in vitro version has not been implemented into official regulatory testing guidelines. A survey of the years 1999-2014 revealed 27 in vivo comet assays submitted to BfArM with market authorisation procedures, European and national advice procedures and clinical trial applications. In three procedures, in vitro comet assays had been submitted within the genetic toxicology packages. © The Author 2014. Published by Oxford University Press on behalf of the Mutagenesis Society. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Novel method for the high-throughput processing of slides for the comet assay

PubMed Central

Karbaschi, Mahsa; Cooke, Marcus S.

2014-01-01

Single cell gel electrophoresis (the comet assay), continues to gain popularity as a means of assessing DNA damage. However, the assay's low sample throughput and laborious sample workup procedure are limiting factors to its application. “Scoring”, or individually determining DNA damage levels in 50 cells per treatment, is time-consuming, but with the advent of high-throughput scoring, the limitation is now the ability to process significant numbers of comet slides. We have developed a novel method by which multiple slides may be manipulated, and undergo electrophoresis, in batches of 25 rather than individually and, importantly, retains the use of standard microscope comet slides, which are the assay convention. This decreases assay time by 60%, and benefits from an electrophoresis tank with a substantially smaller footprint, and more uniform orientation of gels during electrophoresis. Our high-throughput variant of the comet assay greatly increases the number of samples analysed, decreases assay time, number of individual slide manipulations, reagent requirements and risk of damage to slides. The compact nature of the electrophoresis tank is of particular benefit to laboratories where bench space is at a premium. This novel approach is a significant advance on the current comet assay procedure. PMID:25425241

Novel method for the high-throughput processing of slides for the comet assay.

PubMed

Karbaschi, Mahsa; Cooke, Marcus S

2014-11-26

Single cell gel electrophoresis (the comet assay), continues to gain popularity as a means of assessing DNA damage. However, the assay's low sample throughput and laborious sample workup procedure are limiting factors to its application. "Scoring", or individually determining DNA damage levels in 50 cells per treatment, is time-consuming, but with the advent of high-throughput scoring, the limitation is now the ability to process significant numbers of comet slides. We have developed a novel method by which multiple slides may be manipulated, and undergo electrophoresis, in batches of 25 rather than individually and, importantly, retains the use of standard microscope comet slides, which are the assay convention. This decreases assay time by 60%, and benefits from an electrophoresis tank with a substantially smaller footprint, and more uniform orientation of gels during electrophoresis. Our high-throughput variant of the comet assay greatly increases the number of samples analysed, decreases assay time, number of individual slide manipulations, reagent requirements and risk of damage to slides. The compact nature of the electrophoresis tank is of particular benefit to laboratories where bench space is at a premium. This novel approach is a significant advance on the current comet assay procedure.

Further characterization of benzo[a]pyrene diol-epoxide (BPDE)-induced comet assay effects.

PubMed

Bausinger, Julia; Schütz, Petra; Piberger, Ann Liza; Speit, Günter

2016-03-01

The present study aims to further characterize benzo[a]pyrene diol-epoxide (BPDE)-induced comet assay effects. Therefore, we measured DNA effects by the comet assay and adduct levels by high-performance liquid chromatography (HPLC) in human lymphocytes and A549 cells exposed to (±)-anti-benzo[a]pyrene-7,8-diol 9,10-epoxide [(±)-anti-BPDE] or (+)-anti-benzo[a]pyrene-7,8-diol 9,10-epoxide [(+)-anti-BPDE]. Both, the racemic form and (+)-anti-BPDE, which is the most relevant metabolite with regard to mutagenicity and carcinogenicity, induced DNA migration in cultured lymphocytes in the same range of concentrations to a similar extent in the alkaline comet assay after exposure for 2h. Nevertheless, (+)-anti-BPDE induced significantly enhanced DNA migration after 16 and 18h post-cultivation which was not seen in response to (±)-anti-BPDE. Combination of the comet assay with the Fpg (formamidopyrimidine-DNA glycosylase) protein did not enhance BPDE-induced effects and thus indicated the absence of Fpg-sensitive sites (oxidized purines, N7-guanine adducts, AP-sites). The aphidicolin (APC)-modified comet assay suggested significant excision repair activity of cultured lymphocytes during the first 18h of culture after a 2 h-exposure to BPDE. In contrast to these repair-related effects measured by the comet assay, HPLC analysis of stable adducts did not reveal any significant removal of (+)-anti-BPDE-induced adducts from lymphocytes during the first 22h of culture. On the other hand, HPLC measurements indicated that A549 cells repaired about 70% of (+)-anti-BPDE-induced DNA-adducts within 22h of release. However, various experiments with the APC-modified comet assay did not indicate significant repair activity during this period in A549 cells. The conflicting results obtained with the comet assay and the HPLC-based adduct analysis question the real cause for BPDE-induced DNA migration in the comet assay and the reliability of the APC-modified comet assay for the determination of DNA excision repair activity in response to BPDE in different cell types. © The Author 2015. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Micropatterned comet assay enables high throughput and sensitive DNA damage quantification

PubMed Central

Ge, Jing; Chow, Danielle N.; Fessler, Jessica L.; Weingeist, David M.; Wood, David K.; Engelward, Bevin P.

2015-01-01

The single cell gel electrophoresis assay, also known as the comet assay, is a versatile method for measuring many classes of DNA damage, including base damage, abasic sites, single strand breaks and double strand breaks. However, limited throughput and difficulties with reproducibility have limited its utility, particularly for clinical and epidemiological studies. To address these limitations, we created a microarray comet assay. The use of a micrometer scale array of cells increases the number of analysable comets per square centimetre and enables automated imaging and analysis. In addition, the platform is compatible with standard 24- and 96-well plate formats. Here, we have assessed the consistency and sensitivity of the microarray comet assay. We showed that the linear detection range for H2O2-induced DNA damage in human lymphoblastoid cells is between 30 and 100 μM, and that within this range, inter-sample coefficient of variance was between 5 and 10%. Importantly, only 20 comets were required to detect a statistically significant induction of DNA damage for doses within the linear range. We also evaluated sample-to-sample and experiment-to-experiment variation and found that for both conditions, the coefficient of variation was lower than what has been reported for the traditional comet assay. Finally, we also show that the assay can be performed using a 4× objective (rather than the standard 10× objective for the traditional assay). This adjustment combined with the microarray format makes it possible to capture more than 50 analysable comets in a single image, which can then be automatically analysed using in-house software. Overall, throughput is increased more than 100-fold compared to the traditional assay. Together, the results presented here demonstrate key advances in comet assay technology that improve the throughput, sensitivity, and robustness, thus enabling larger scale clinical and epidemiological studies. PMID:25527723

Micropatterned comet assay enables high throughput and sensitive DNA damage quantification.

PubMed

Ge, Jing; Chow, Danielle N; Fessler, Jessica L; Weingeist, David M; Wood, David K; Engelward, Bevin P

2015-01-01

The single cell gel electrophoresis assay, also known as the comet assay, is a versatile method for measuring many classes of DNA damage, including base damage, abasic sites, single strand breaks and double strand breaks. However, limited throughput and difficulties with reproducibility have limited its utility, particularly for clinical and epidemiological studies. To address these limitations, we created a microarray comet assay. The use of a micrometer scale array of cells increases the number of analysable comets per square centimetre and enables automated imaging and analysis. In addition, the platform is compatible with standard 24- and 96-well plate formats. Here, we have assessed the consistency and sensitivity of the microarray comet assay. We showed that the linear detection range for H2O2-induced DNA damage in human lymphoblastoid cells is between 30 and 100 μM, and that within this range, inter-sample coefficient of variance was between 5 and 10%. Importantly, only 20 comets were required to detect a statistically significant induction of DNA damage for doses within the linear range. We also evaluated sample-to-sample and experiment-to-experiment variation and found that for both conditions, the coefficient of variation was lower than what has been reported for the traditional comet assay. Finally, we also show that the assay can be performed using a 4× objective (rather than the standard 10× objective for the traditional assay). This adjustment combined with the microarray format makes it possible to capture more than 50 analysable comets in a single image, which can then be automatically analysed using in-house software. Overall, throughput is increased more than 100-fold compared to the traditional assay. Together, the results presented here demonstrate key advances in comet assay technology that improve the throughput, sensitivity, and robustness, thus enabling larger scale clinical and epidemiological studies. © The Author 2014. Published by Oxford University Press on behalf of the Mutagenesis Society. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Neutral Na in comets tails: a chemical story

NASA Astrophysics Data System (ADS)

Ellinger, Y.; Pauzat, F.; Mousis, O.; Guilbert-Lepoutre, A.; Leblanc, F.; Ali-Dib, M.; Doronin, M.; Zicler, E.; Doressoundiram, A.

2015-10-01

The origin of the neutral sodium comet tail discovered in comet Hale-Bopp in 1997 is still a matter of discussion. Here we propose a scenario which is based on chemical grounds. The starting point is the chemical trapping of the Na+ ion in the refractory material during the condensation phase of the protosolar nebula, followed by its incorporation in the building blocks of the comets parent bodies. In the next step, the Na+ ions are washed out of the refractory material by the water formed by the melting of the ice due to the heat released in the radioactive decay of short period elements. When the water freezes again, the Na+ ion looses its positive charge to evolve progressively toward a neutral atom when approaching the surface of the ice. As shown by high-level numerical simulations based on first principle periodic density functional theory (DFT) to describe the solid structure of the ice, it is a neutral Na that is ejected with the sublimation of the ice top layer.

In Situ Space Gas Dynamic Measurements by the ROSINA Comet Pressure Sensor COPS on the Rosetta Spacecraft

NASA Astrophysics Data System (ADS)

Tzou, C. Y.; Altwegg, K.; Fiethe, B.; Gasc, S.; Rubin, M.

2014-12-01

Rosetta is part of the cornerstone missions executed by the European Space Agency. It is the first space mission to orbit and also land on a comet. Starting in August 2014 Rosetta will be able to carry out a close study of comet 67P/Churyumov-Gerasimenko. The Rosetta Orbiter Spectrometer for Ion and Neutral Analysis (ROSINA) is one of the core payloads on board of the Rosetta spacecraft [Balsiger et al, 2007]. ROSINA's main objective is to determine the major atmospheric and ionospheric composition in the coma and to investigate the gas dynamics around the comet. ROSINA consists of two mass spectrometers and a pressure sensor. The Comet Pressure Sensor (COPS) includes two gauges: the "nude gauge" measures total neutral density in the coma and the "ram gauge" measures the dynamic pressure of the cometary gas flux to obtain the bulk velocity of the neutral gas. The combination of these two gauges makes COPS capable to derive the gas dynamics at the location of the spacecraft. We performed laboratory gas dynamic measurements with the identical flight-spare instrument of COPS. Using the Calibration System for The Mass Spectrometer Instrument ROSINA (CASYMIR) we produce neutral gas beams to model cometary gas jets with velocities from thermal up to 2 km/s. We expect that COPS will be able to detect the faint and expanding atmosphere of comet 67P/Churyumov-Gerasimenko as early as August 2014 when the comet is still farther than 3 AU from the Sun. We will present the first ROSINA COPS observations of the gas dynamics around the comet together with the corresponding laboratory measurements required for the interpretation of these data. Reference: Balsiger, H. et al.: ROSINA-Rosetta Orbiter Spectrometer for Ion and Neutral Analysis, Space Science Reviews, Vol. 128, 745-801, 2007.

DNA comet Giemsa staining for conventional bright-field microscopy.

PubMed

Osipov, Andreyan; Arkhangelskaya, Ekaterina; Vinokurov, Alexei; Smetaninа, Nadezhda; Zhavoronkov, Alex; Klokov, Dmitry

2014-04-10

This study was undertaken to evaluate the compatibility of Giemsa staining protocol with the comet assay. We showed, for the first time, that DNA comets can be visualized and analyzed using Giemsa staining. We generated DNA damage dose response curves for human peripheral blood lymphocytes exposed to X-ray radiation using the comet assay with either SybrGreen I or Giemsa stain. The dose response curves were fitted by linear regressions (R2>0.977). The SybrGreen I results showed only ~1.2-fold higher slope coefficient (method sensitivity) compared to the Giemsa results. The unexpectedly high sensitivity of Giemsa staining for the comet assay is due to the Romanowsky-Giemsa effect, the stain photo-stability and the higher resolution of bright-field imaging compared to fluorescence imaging. Our results demonstrate that Giemsa staining can effectively be used for measuring DNA damage by the comet assay. The low cost and availability of Giemsa stain makes this method affordable for any low budget research and will facilitate new applications of the comet assay in biology and medicine.

Automated segmentation of comet assay images using Gaussian filtering and fuzzy clustering.

PubMed

Sansone, Mario; Zeni, Olga; Esposito, Giovanni

2012-05-01

Comet assay is one of the most popular tests for the detection of DNA damage at single cell level. In this study, an algorithm for comet assay analysis has been proposed, aiming to minimize user interaction and providing reproducible measurements. The algorithm comprises two-steps: (a) comet identification via Gaussian pre-filtering and morphological operators; (b) comet segmentation via fuzzy clustering. The algorithm has been evaluated using comet images from human leukocytes treated with a commonly used DNA damaging agent. A comparison of the proposed approach with a commercial system has been performed. Results show that fuzzy segmentation can increase overall sensitivity, giving benefits in bio-monitoring studies where weak genotoxic effects are expected.

Assessment of genotoxic effects of flumorph by the comet assay in mice organs.

PubMed

Zhang, T; Zhao, Q; Zhang, Y; Ning, J

2014-03-01

The present study investigated the genotoxic effects of flumorph in various organs (brain, liver, spleen, kidney and sperm) of mice. The DNA damage, measured as comet tail length (µm), was determined using the alkaline comet assay. The comet assay is a sensitive assay for the detection of genotoxicity caused by flumorph using mice as a model. Statistically significant increases in comet assay for both dose-dependent and duration-dependent DNA damage were observed in all the organs assessed. The organs exhibited the maximum DNA damage in 96 h at 54 mg/kg body weight. Brain showed maximum DNA damage followed by spleen > kidney > liver > sperm. Our data demonstrated that flumorph had induced systemic genotoxicity in mammals as it caused DNA damage in all tested vital organs, especially in brain and spleen.

HT-COMET: a novel automated approach for high throughput assessment of human sperm chromatin quality.

PubMed

Albert, Océane; Reintsch, Wolfgang E; Chan, Peter; Robaire, Bernard

2016-05-01

Can we make the comet assay (single-cell gel electrophoresis) for human sperm a more accurate and informative high throughput assay? We developed a standardized automated high throughput comet (HT-COMET) assay for human sperm that improves its accuracy and efficiency, and could be of prognostic value to patients in the fertility clinic. The comet assay involves the collection of data on sperm DNA damage at the level of the single cell, allowing the use of samples from severe oligozoospermic patients. However, this makes comet scoring a low throughput procedure that renders large cohort analyses tedious. Furthermore, the comet assay comes with an inherent vulnerability to variability. Our objective is to develop an automated high throughput comet assay for human sperm that will increase both its accuracy and efficiency. The study comprised two distinct components: a HT-COMET technical optimization section based on control versus DNAse treatment analyses ( ITALIC! n = 3-5), and a cross-sectional study on 123 men presenting to a reproductive center with sperm concentrations categorized as severe oligozoospermia, oligozoospermia or normozoospermia. Sperm chromatin quality was measured using the comet assay: on classic 2-well slides for software comparison; on 96-well slides for HT-COMET optimization; after exposure to various concentrations of a damage-inducing agent, DNAse, using HT-COMET; on 123 subjects with different sperm concentrations using HT-COMET. Data from the 123 subjects were correlated to classic semen quality parameters and plotted as single-cell data in individual DNA damage profiles. We have developed a standard automated HT-COMET procedure for human sperm. It includes automated scoring of comets by a fully integrated high content screening setup that compares well with the most commonly used semi-manual analysis software. Using this method, a cross-sectional study on 123 men showed no significant correlation between sperm concentration and sperm DNA damage, confirming the existence of hidden chromatin damage in men with apparently normal semen characteristics, and a significant correlation between percentage DNA in the tail and percentage of progressively motile spermatozoa. Finally, the use of DNA damage profiles helped to distinguish subjects between and within sperm concentration categories, and allowed a determination of the proportion of highly damaged cells. The main limitations of the HT-COMET are the high, yet indispensable, investment in an automated liquid handling system and heating block to ensure accuracy, and the availability of an automated plate reading microscope and analysis software. This standardized HT-COMET assay offers many advantages, including higher accuracy and evenness due to automation of sensitive steps, a 14.4-fold increase in sample analysis capacity, and an imaging and scoring time of 1 min/well. Overall, HT-COMET offers a decrease in total experimental time of more than 90%. Hence, this assay constitutes a more efficient option to assess sperm chromatin quality, paves the way to using this assay to screen large cohorts, and holds prognostic value for infertile patients. Funded by the CIHR Institute of Human Development, Child and Youth Health (IHDCYH; RHF 100625). O.A. is a fellow supported by the Fonds de la Recherche du Québec - Santé (FRQS) and the CIHR Training Program in Reproduction, Early Development, and the Impact on Health (REDIH). B.R. is a James McGill Professor. The authors declare no conflicts of interest. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

The application of the comet assay to assess the genotoxicity of environmental pollutants in the nematode Caenorhabditis elegans.

PubMed

Imanikia, Soudabeh; Galea, Francesca; Nagy, Eszter; Phillips, David H; Stürzenbaum, Stephen R; Arlt, Volker M

2016-07-01

This study aimed to establish a protocol for cell dissociation from the nematode Caenorhabditis elegans (C. elegans) to assess the genotoxicity of the environmental pollutant benzo[a]pyrene (BaP) using the alkaline version of the single cell electrophoresis assay (comet assay). BaP genotoxicity was assessed in C. elegans (wild-type [WT]; N2, Bristol) after 48h exposure (0-40μM). Induction of comets by BaP was concentration-dependent up to 20μM; comet% tail DNA was ∼30% at 20μM BaP and ∼10% in controls. Similarly, BaP-induced DNA damage was evaluated in C. elegans mutant strains deficient in DNA repair. In xpa-1 and apn-1 mutants BaP-induced comet formation was diminished to WT background levels suggesting that the damage formed by BaP that is detected in the comet assay is not recognised in cells deficient in nucleotide and base excision repair, respectively. In summary, our study provides a protocol to evaluate DNA damage of environmental pollutants in whole nematodes using the comet assay. Copyright © 2016 The Author(s). Published by Elsevier B.V. All rights reserved.

Alkaline Comet Assay for Assessing DNA Damage in Individual Cells.

PubMed

Pu, Xinzhu; Wang, Zemin; Klaunig, James E

2015-08-06

Single-cell gel electrophoresis, commonly called a comet assay, is a simple and sensitive method for assessing DNA damage at the single-cell level. It is an important technique in genetic toxicological studies. The comet assay performed under alkaline conditions (pH >13) is considered the optimal version for identifying agents with genotoxic activity. The alkaline comet assay is capable of detecting DNA double-strand breaks, single-strand breaks, alkali-labile sites, DNA-DNA/DNA-protein cross-linking, and incomplete excision repair sites. The inclusion of digestion of lesion-specific DNA repair enzymes in the procedure allows the detection of various DNA base alterations, such as oxidative base damage. This unit describes alkaline comet assay procedures for assessing DNA strand breaks and oxidative base alterations. These methods can be applied in a variety of cells from in vitro and in vivo experiments, as well as human studies. Copyright © 2015 John Wiley & Sons, Inc.

Dose-Response Assessment of Four Genotoxic Chemicals in a Combined Mouse and Rat Micronucleus and Comet Assay Protocol

PubMed Central

Recio, Leslie; Hobbs, Cheryl; Caspary, William; Witt, Kristine L.

2012-01-01

The in vivo micronucleus (MN) assay has proven to be an effective measure of genotoxicity potential. However, sampling a single tissue (bone marrow) for a single indicator of genetic damage using the MN assay provides a limited genotoxicity profile. The in vivo alkaline (pH>13) Comet assay, which detects a broad spectrum of DNA damage, can be applied to a variety of rodent tissues following administration of test agents. To determine if the Comet assay is a useful supplement to the in vivo MN assay, a combined test protocol (MN/Comet assay) was conducted in male B6C3F1 mice and F344/N rats using four model genotoxicants: ethyl methanesulfonate (EMS), acrylamide (ACM), cyclophosphamide (CP), and vincristine sulfate (VS). Test compounds were administered on 4 consecutive days at 24-hour intervals (VS was administered to rats for 3 days); animals were euthanized 4 hours after the last administration. All compounds induced significant increases in micronucleated reticulocytes (MN-RET) in the peripheral blood of mice, and all but ACM induced MN-RET in rats. EMS and ACM induced significant increases in DNA damage, measured by the Comet assay, in multiple tissues of mice and rats. CP-induced DNA damage was detected in leukocytes and duodenum cells. VS, a spindle fiber disrupting agent, was negative in the Comet assay. Based on these results, the MN/Comet assay holds promise for providing more comprehensive assessments of potential genotoxicants, and the National Toxicology Program is presently using this combined protocol in its overall evaluation of the genotoxicity of substances of public health concern. PMID:20371966

Study of the coma of comet 67P/Churyumov-Gerasimenko based on the ROSINA/RTOF instrument onboard Rosetta

NASA Astrophysics Data System (ADS)

Hoang, M.; Garnier, P.; Rème, H.; Altwegg, K.; Balsiger, H.; Calmonte, U.; Fiethe, B.; Galli, A.; Gasc, S.; Jäckel, A.; Mall, U.; Le Roy, L.; Rubin, M.; Tzou, C.-Y.; Waite, J. H.; Wurz, P.

2015-10-01

The Rosetta ESA mission investigates the environment of the comet 67P / Churyumov- Gerasimenko since August 2014. Among the experiments onboard the satellite, the ROSINA experiment (Rosetta Orbiter Spectrometer for Ion and Neutral Analysis) includes two mass spectrometers (DFMS and RTOF) to analyze the composition of neutrals and ions, and an instrument (COPS) to monitor the density and velocity of neutrals in the coma [1]. We will here analyze and discuss the data of the ROSINA/RTOF instrument during the comet escort phase. A detailed description of the main volatiles (H2O, CO2, CO) dynamics and of the heterogeneities of the coma will be provided.

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

PubMed Central

Ding, Wei; Bishop, Michelle E.; Lyn-Cook, Lascelles E.; Davis, Kelly J.; Manjanatha, Mugimane G.

2016-01-01

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents is important for protecting public health. The in vivo alkaline comet assay, which detects DNA damage as strand breaks, is especially relevant for assessing the genotoxic hazards of xenobiotics, as its responses reflect the in vivo absorption, tissue distribution, metabolism and excretion (ADME) of chemicals, as well as DNA repair process. Compared to other in vivo DNA damage assays, the assay is rapid, sensitive, visual and inexpensive, and, by converting oxidative DNA damage into strand breaks using specific repair enzymes, the assay can measure oxidative DNA damage in an efficient and relatively artifact-free manner. Measurement of DNA damage with the comet assay can be performed using both acute and subchronic toxicology study designs, and by integrating the comet assay with other toxicological assessments, the assay addresses animal welfare requirements by making maximum use of animal resources. Another major advantage of the assays is that they only require a small amount of cells, and the cells do not have to be derived from proliferating cell populations. The assays also can be performed with a variety of human samples obtained from clinically or occupationally exposed individuals. PMID:27166647

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver.

PubMed

Ding, Wei; Bishop, Michelle E; Lyn-Cook, Lascelles E; Davis, Kelly J; Manjanatha, Mugimane G

2016-05-04

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents is important for protecting public health. The in vivo alkaline comet assay, which detects DNA damage as strand breaks, is especially relevant for assessing the genotoxic hazards of xenobiotics, as its responses reflect the in vivo absorption, tissue distribution, metabolism and excretion (ADME) of chemicals, as well as DNA repair process. Compared to other in vivo DNA damage assays, the assay is rapid, sensitive, visual and inexpensive, and, by converting oxidative DNA damage into strand breaks using specific repair enzymes, the assay can measure oxidative DNA damage in an efficient and relatively artifact-free manner. Measurement of DNA damage with the comet assay can be performed using both acute and subchronic toxicology study designs, and by integrating the comet assay with other toxicological assessments, the assay addresses animal welfare requirements by making maximum use of animal resources. Another major advantage of the assays is that they only require a small amount of cells, and the cells do not have to be derived from proliferating cell populations. The assays also can be performed with a variety of human samples obtained from clinically or occupationally exposed individuals.

Seasonal variations as predictive factors of the comet assay parameters: a retrospective study.

PubMed

Geric, Marko; Gajski, Goran; Orešcanin, Višnja; Garaj-Vrhovac, Vera

2018-02-24

Since there are several predicting factors associated with the comet assay parameters, we have decided to assess the impact of seasonal variations on the comet assay results. A total of 162 volunteers were retrospectively studied, based on the date when blood donations were made. The groups (winter, spring, summer and autumn) were matched in terms of age, gender, smoking status, body mass index and medical diagnostic exposure in order to minimise the impact of other possible predictors. Means and medians of the comet assay parameters were higher when blood was sampled in the warmer period of the year, the values of parameters being the highest during summer. Correlation of meteorological data (air temperature, sun radiation and sun insolation) was observed when data were presented as the median per person. Using multivariate analysis, sampling season and exposure to medical radiation were proved to be the most influential predictors for the comet assay parameters. Taken together, seasonal variation is another variable that needs to be accounted for when conducting a cohort study. Further studies are needed in order to improve the statistical power of the results related to the impact of sun radiation, air temperature and sun insolation on the comet assay parameters.

The effect of different methods and image analyzers on the results of the in vivo comet assay.

PubMed

Kyoya, Takahiro; Iwamoto, Rika; Shimanura, Yuko; Terada, Megumi; Masuda, Shuichi

2018-01-01

The in vivo comet assay is a widely used genotoxicity test that can detect DNA damage in a range of organs. It is included in the Organisation for Economic Co-operation and Development Guidelines for the Testing of Chemicals. However, various protocols are still used for this assay, and several different image analyzers are used routinely to evaluate the results. Here, we verified a protocol that largely contributes to the equivalence of results, and we assessed the effect on the results when slides made from the same sample were analyzed using two different image analyzers (Comet Assay IV vs Comet Analyzer). Standardizing the agarose concentrations and DNA unwinding and electrophoresis times had a large impact on the equivalence of the results between the different methods used for the in vivo comet assay. In addition, there was some variation in the sensitivity of the two different image analyzers tested; however this variation was considered to be minor and became negligible when the test conditions were standardized between the two different methods. By standardizing the concentrations of low melting agarose and DNA unwinding and electrophoresis times between both methods used in the current study, the sensitivity to detect the genotoxicity of a positive control substance in the in vivo comet assay became generally comparable, independently of the image analyzer used. However, there may still be the possibility that other conditions, except for the three described here, could affect the reproducibility of the in vivo comet assay.

Slight hypercalcemia is not associated with positive responses in the Comet Assay in male rat liver.

PubMed

Thiel, Anette; Hamel, Annie; Schaefer, Katrien; Cardoso, Renato; Beilstein, Paul

2017-08-01

Maintenance of physiological levels of intracellular and extracellular calcium is essential for life. Increased intracellular calcium levels are involved in cell death (apoptosis and necrosis) and are associated with positive responses in the Comet assay in vitro. In addition, high calcium and vitamin D intakes were reported to induce apoptosis in adipose tissue in obese mice and to increase DNA-migration in the Comet assay. To investigate increased serum concentration of calcium as a potential confounding factor in the regulatory Comet assay in vivo, we induced mild hypercalcemia in male Wistar rats by 3-day continuous intravenous infusion of calcium gluconate and performed the Comet assay in the liver in line with regulatory guidelines. The results of the study showed that mild increases in serum calcium concentration (up to 1.4 times above the concurrent control) and increased urinary calcium concentration (up to 27.8 times above the concurrent control) results in clinical signs like mild tremor, faster respiration rate and decreased activity in a few animals. However, under the conditions of the study, no increase in the %Tail DNA in the Comet assay and no indication of liver damage as determined by histopathological means were observed. Thus, mild increases in plasma calcium did not lead to positive results in a genotoxicity assessment by the Comet assay in the rat liver. This result is important as it confirms the reliability of this assay for regulatory evaluation of safety. Copyright © 2017 DSM Nutritional Products AG. Published by Elsevier B.V. All rights reserved.

Epithelial cells as alternative human biomatrices for comet assay.

PubMed

Rojas, Emilio; Lorenzo, Yolanda; Haug, Kristiane; Nicolaissen, Bjørn; Valverde, Mahara

2014-01-01

The comet assay is a valuable experimental tool aimed at mapping DNA damage in human cells in vivo for environmental and occupational monitoring, as well as for therapeutic purposes, such as storage prior to transplant, during tissue engineering, and in experimental ex vivo assays. Furthermore, due to its great versatility, the comet assay allows to explore the use of alternative cell types to assess DNA damage, such as epithelial cells. Epithelial cells, as specialized components of many organs, have the potential to serve as biomatrices that can be used to evaluate genotoxicity and may also serve as early effect biomarkers. Furthermore, 80% of solid cancers are of epithelial origin, which points to the importance of studying DNA damage in these tissues. Indeed, studies including comet assay in epithelial cells have either clear clinical applications (lens and corneal epithelial cells) or examine genotoxicity within human biomonitoring and in vitro studies. We here review improvements in determining DNA damage using the comet assay by employing lens, corneal, tear duct, buccal, and nasal epithelial cells. For some of these tissues invasive sampling procedures are needed. Desquamated epithelial cells must be obtained and dissociated prior to examination using the comet assay, and such procedures may induce varying amounts of DNA damage. Buccal epithelial cells require lysis enriched with proteinase K to obtain free nucleosomes. Over a 30 year period, the comet assay in epithelial cells has been little employed, however its use indicates that it could be an extraordinary tool not only for risk assessment, but also for diagnosis, prognosis of treatments and diseases.

Epithelial cells as alternative human biomatrices for comet assay

PubMed Central

Rojas, Emilio; Lorenzo, Yolanda; Haug, Kristiane; Nicolaissen, Bjørn; Valverde, Mahara

2014-01-01

The comet assay is a valuable experimental tool aimed at mapping DNA damage in human cells in vivo for environmental and occupational monitoring, as well as for therapeutic purposes, such as storage prior to transplant, during tissue engineering, and in experimental ex vivo assays. Furthermore, due to its great versatility, the comet assay allows to explore the use of alternative cell types to assess DNA damage, such as epithelial cells. Epithelial cells, as specialized components of many organs, have the potential to serve as biomatrices that can be used to evaluate genotoxicity and may also serve as early effect biomarkers. Furthermore, 80% of solid cancers are of epithelial origin, which points to the importance of studying DNA damage in these tissues. Indeed, studies including comet assay in epithelial cells have either clear clinical applications (lens and corneal epithelial cells) or examine genotoxicity within human biomonitoring and in vitro studies. We here review improvements in determining DNA damage using the comet assay by employing lens, corneal, tear duct, buccal, and nasal epithelial cells. For some of these tissues invasive sampling procedures are needed. Desquamated epithelial cells must be obtained and dissociated prior to examination using the comet assay, and such procedures may induce varying amounts of DNA damage. Buccal epithelial cells require lysis enriched with proteinase K to obtain free nucleosomes. Over a 30 year period, the comet assay in epithelial cells has been little employed, however its use indicates that it could be an extraordinary tool not only for risk assessment, but also for diagnosis, prognosis of treatments and diseases. PMID:25506353

Alkaline comet assay in liver and stomach, and micronucleus assay in bone marrow, from rats treated with 2-acetylaminofluorene, azidothymidine, cisplatin, or isobutyraldehyde.

PubMed

Kraynak, A R; Barnum, J E; Cunningham, C L; Ng, A; Ykoruk, B A; Bennet, B; Stoffregen, D; Merschman, M; Freeland, E; Galloway, S M

2015-07-01

As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM) initiative international validation study of the in vivo rat alkaline comet assay (comet assay), we examined the ability of the assay to determine the genotoxicity of 2-acetylaminofluorene (AAF), azidothymidine (AZT), cisplatin (CPN), and isobutyraldehyde (IBA) in liver and glandular stomach of male Sprague-Dawley rats. Rats were given oral doses of test compound or control once daily for three days. High dose levels were approximately maximum tolerated doses and were based on preliminary range-finding studies. Tissues were harvested 3h after the final dose (48h after the initial dose). A bone marrow micronucleus assay (MN) was also conducted on the rats treated with AZT, CPN, and IBA. Acute toxic effects of treatment were determined primarily through histomorphologic analysis of liver and stomach but also by body weight and serum liver enzyme changes. The comet assay was conducted on fresh tissue preparations but frozen samples from two studies were also assayed. Statistically significant dose-related differences in comet % DNA in tail were found in liver and stomach for the genotoxin AZT and in liver for the genotoxin CPN, but not in liver or stomach for the non-genotoxin IBA. Statistically significant differences in % DNA in tail were measured in liver for the low and mid dose of the genotoxin AAF, but not the high dose. The comet assays of frozen liver suspensions from CPN- and AAF-treated rats yielded comparable results to the assays of fresh preparations. There were no indications of significant toxicity induced by any treatment. The micronucleus assay was positive for CPN and AZT and negative for IBA. In conclusion, the in vivo comet assay is capable of detecting genotoxic effects of a variety of chemicals and may fill an important role in the genotoxicity test battery. Copyright © 2015 Elsevier B.V. All rights reserved.

Interpreting sperm DNA damage in a diverse range of mammalian sperm by means of the two-tailed comet assay

PubMed Central

Cortés-Gutiérrez, Elva I.; López-Fernández, Carmen; Fernández, José Luis; Dávila-Rodríguez, Martha I.; Johnston, Stephen D.; Gosálvez, Jaime

2014-01-01

Key Concepts The two-dimensional Two-Tailed Comet assay (TT-comet) protocol is a valuable technique to differentiate between single-stranded (SSBs) and double-stranded DNA breaks (DSBs) on the same sperm cell.Protein lysis inherent with the TT-comet protocol accounts for differences in sperm protamine composition at a species-specific level to produce reliable visualization of sperm DNA damage.Alkaline treatment may break the sugar–phosphate backbone in abasic sites or at sites with deoxyribose damage, transforming these lesions into DNA breaks that are also converted into ssDNA. These lesions are known as Alkali Labile Sites “ALSs.”DBD–FISH permits the in situ visualization of DNA breaks, abasic sites or alkaline-sensitive DNA regions.The alkaline comet single assay reveals that all mammalian species display constitutive ALS related with the requirement of the sperm to undergo transient changes in DNA structure linked with chromatin packing.Sperm DNA damage is associated with fertilization failure, impaired pre-and post- embryo implantation and poor pregnancy outcome.The TT is a valuable tool for identifying SSBs or DSBs in sperm cells with DNA fragmentation and can be therefore used for the purposes of fertility assessment. Sperm DNA damage is associated with fertilization failure, impaired pre-and post- embryo implantation and poor pregnancy outcome. A series of methodologies to assess DNA damage in spermatozoa have been developed but most are unable to differentiate between single-stranded DNA breaks (SSBs) and double-stranded DNA breaks (DSBs) on the same sperm cell. The two-dimensional Two-Tailed Comet assay (TT-comet) protocol highlighted in this review overcomes this limitation and emphasizes the importance in accounting for the difference in sperm protamine composition at a species-specific level for the appropriate preparation of the assay. The TT-comet is a modification of the original comet assay that uses a two dimensional electrophoresis to allow for the simultaneous evaluation of DSBs and SSBs in mammalian spermatozoa. Here we have compiled a retrospective overview of how the TT-comet assay has been used to investigate the structure and function of sperm DNA across a diverse range of mammalian species (eutheria, metatheria, and prototheria). When conducted as part of the TT-comet assay, we illustrate (a) how the alkaline comet single assay has been used to help understand the constitutive and transient changes in DNA structure associated with chromatin packing, (b) the capacity of the TT-comet to differentiate between the presence of SSBs and DSBs (c) and the possible implications of SSBs or DSBs for the assessment of infertility. PMID:25505901

Comparative evaluation of genotoxicity by micronucleus assay in the buccal mucosa over comet assay in peripheral blood in oral precancer and cancer patients.

PubMed

Katarkar, Atul; Mukherjee, Sanjit; Khan, Masood H; Ray, Jay G; Chaudhuri, Keya

2014-09-01

Early detection and quantification of DNA damage in oral premalignancy or malignancy may help in management of the disease and improve survival rates. The comet assay has been successfully utilised to detect DNA damage in oral premalignant or malignancy. However, due to the invasive nature of collecting blood, it may be painful for many unwilling patients. This study compares the micronucleus (MN) assay in oral buccal mucosa cells with the comet assay in peripheral blood cells in a subset of oral habit-induced precancer and cancer patients. For this, MN assay of exfoliated epithelial cells was compared with comet assay of peripheral blood leucocytes among 260 participants, including those with oral lichen planus (OLP; n = 52), leukoplakia (LPK; n = 51), oral submucous fibrosis (OSF; n = 51), oral squamous cell carcinoma (OSCC; n = 54) and normal volunteers (n = 52). Among the precancer groups, LPK patients showed significantly higher levels of DNA damage as reflected by both comet tail length (P < 0.0001) and micronuclei (MNi) frequency (P = 0.0009). The DNA damage pattern in precancer and cancer patients was OLP < OSF < LPK < OSCC, and with respective oral habits, it was multiple habits > cigarette + khaini > cigarette smokers > areca + khaini > areca. There was no significant difference in the comet length and MNi frequency between males and females who had oral chewing habits. An overall significant correlation was observed between MNi frequency and comet tail length with r = 0.844 and P < 0.0001. Thus, the extent of DNA damage evaluation by the comet assay in peripheral blood cells is perfectly reflected by the MN assay on oral exfoliated epithelial cells, and MNi frequency can be used with the same effectiveness and greater efficiency in early detection of oral premalignant conditions. © The Author 2014. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Cryopreservation of human blood for alkaline and Fpg-modified comet assay.

PubMed

Pu, Xinzhu; Wang, Zemin; Klaunig, James E

2016-01-01

The Comet assay is a reproducible and sensitive assay for the detection of DNA damage in eukaryotic cells and tissues. Incorporation of lesion specific, oxidative DNA damage repair enzymes (for example, Fpg, OGG1 and EndoIII) in the standard alkaline Comet assay procedure allows for the detection and measurement of oxidative DNA damage. The Comet assay using white blood cells (WBC) has proven useful in monitoring DNA damage from environmental agents in humans. However, it is often impractical to performance Comet assay immediately after blood sampling. Thus, storage of blood sample is required. In this study, we developed and tested a simple storage method for very small amount of whole blood for standard and Fpg-modified modified Comet assay. Whole blood was stored in RPMI 1640 media containing 10% FBS, 10% DMSO and 1 mM deferoxamine at a sample to media ratio of 1:50. Samples were stored at -20 °C and -80 °C for 1, 7, 14 and 28 days. Isolated lymphocytes from the same subjects were also stored under the same conditions for comparison. Direct DNA strand breakage and oxidative DNA damage in WBC and lymphocytes were analyzed using standard and Fpg-modified alkaline Comet assay and compared with freshly analyzed samples. No significant changes in either direct DNA strand breakage or oxidative DNA damage was seen in WBC and lymphocytes stored at -20 °C for 1 and 7 days compared to fresh samples. However, significant increases in both direct and oxidative DNA damage were seen in samples stored at -20 °C for 14 and 28 days. No changes in direct and oxidative DNA damage were observed in WBC and lymphocytes stored at -80 °C for up to 28 days. These results identified the proper storage conditions for storing whole blood or isolated lymphocytes to evaluate direct and oxidative DNA damage using standard and Fpg-modified alkaline Comet assay.

Evaluation of genotoxic activity of maleic hydrazide, ethyl methane sulfonate, and N-nitroso diethylamine in Tradescantia.

PubMed

Alvarez-Moya, C; Santerre-Lucas, A; Zúñiga-González, G; Torres-Bugarín, O; Padilla-Camberos, E; Feria-Velasco, A

2001-01-01

To assess the genotoxic activity of N-nitroso diethylamine (NDEA), maleic hydrazide (MH), and ethyl methane sulfonate (EMS) using two systems: the comet assay on nuclei from Tradescantia, and the pink mutation test on Tradescantia staminal hairs (clone 4430). Tradescantia cups was obtained from Laboratorio de Citogenética y Mutagénesis del Centro de Ciencias de la Atmósfera de la Universidad Nacional Autónoma de México and treated with: N-nitroso diethylamine (NDEA) at 1, 5, 10 mM, maleic hydrazide (MH) at 1, 5, 10 mM and ethyl methane sulfonate (EMS) at 15, 30 and 45 mM; and used in both pink mutation assay and comet assay using cellular nuclei from Tradescantia staminal hairs. The observation of staminal hair was realized along eight days (6-14) after treatment), flowers produced day 14 after treatment were utilized done according to Underbrink. In previous reports on plants, were comet assay was used, breaking cellular wall and separating by centrifugation gradient are necessary. Here, nuclei from staminal hairs were obtained by squashing the cells (is not necessary to utilize to break special procedure cellular wall), collected using a nylon mesh of 80 Mm and next the comet assay was applied. Student's T test was the statistical test used for analyzing the comet assay data. Both assays showed a great sensitivity to the studied mutagens. A relationship between the dose-pink event and the dose-tail length was evident. Even though the Tradescantia mutation assay is a sensitive test with MH and EMS, low doses of NDEA were not able to induce a significant increase in the pink event frequencies; however, the comet assay was able to detect the mutagenic effect of NDEA at the same dose. Thus, it is clear that the comet assay is highly sensitive to the lowest dose of chemical mutagens. The comet assay on nuclei from Tradescantia staminal hairs is a useful tool to monitor genotoxic agents; it is simple, highly sensitive, and faster than the pink mutation test.

Random, double- and single-strand DNA breaks can be differentiated in the method of Comet assay by the shape of the comet image.

PubMed

Georgieva, Milena; Zagorchev, Plamen; Miloshev, George

2015-10-01

Comet assay is an invaluable tool in DNA research. It is widely used to detect DNA damage as an indicator of exposure to genotoxic stress. A canonical set of parameters and specialized software programs exist for Comet assay data quantification and analysis. None of them so far has proven its potential to employ a computer-based algorithm for assessment of the shape of the comet as an indicator of the exact mechanism by which the studied genotoxins cut in the molecule of DNA. Here, we present 14 unique measurements of the comet image based on the comet morphology. Their mathematical derivation and statistical analysis allowed precise description of the shape of the comet image which in turn discriminated the cause of genotoxic stress. This algorithm led to the development of the "CometShape" software which allowed easy discrimination among different genotoxins depending on the type of DNA damage they induce. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Four-fluid MHD Simulations of the Plasma and Neutral Gas Environment of Comet 67P/Churyumov-Gerasimenko Near Perihelion

NASA Astrophysics Data System (ADS)

Huang, Zhenguang; Toth, Gabor; Gombosi, Tamas; Jia, Xianzhe; Rubin, Martin; Fougere, Nicolas; Tenishev, Valeriy; Combi, Michael; Bieler, Andre; Hansen, Kenneth; Shou, Yinsi; Altwegg, Kathrin

2016-04-01

The neutral and plasma environment is critical in understanding the interaction of the solar wind and comet 67P/Churyumov-Gerasimenko (CG), the target of the European Space Agency's Rosetta mission. In this study, we have developed a 3-D four-fluid model, which is based on BATS-R-US (Block-Adaptive Tree Solarwind Roe-type Upwind Scheme) within SWMF (Space Weather Modeling Framework) that solves the governing multi-fluid MHD equations and the Euler equations for the neutral gas fluid. These equations describe the behavior and interactions of the cometary heavy ions, the solar wind protons, the electrons, and the neutrals. We simulated the plasma and neutral gas environment of comet CG with SHAP5 model near perihelion and we showed that the plasma environment in the inner coma region have some new features: magnetic reconnection in the tail region, a magnetic pile-up region on the nightside, and nucleus directed plasma flow inside the nightside reconnection region.

[Use of comet assay for the risk assessment of oil- and chemical-industry workers].

PubMed

Megyesi, János; Biró, Anna; Wigmond, László; Major, Jenő; Tompa, Anna

2014-11-23

The comet assay is a fluorescent microscopic method that is able to detect DNA strand-breaks even in non-proliferative cells in samples with low cell counts. The aim of the authors was to measure genotoxic DNA damage and assess oxidative DNA damage caused by occupational exposure in groups exposed to benzene, polycyclic aromatic carbohydrates and styrene at the workplace in order to clarify whether the comet assay can be used as an effect marker tool in genotoxicology monitoring. In addition to the basic steps of the comet assay, one sample was treated with formamido-pirimidine-DNA-glycolase restriction-enzyme that measures oxidative DNA damage. An increase was observed in tail moments in each group of untreated and Fpg-treated samples compared to the control. It can be concluded that occupational exposure can be detected with the method. The comet assay may prove to be an excellent effect marker and a supplementary technique for monitoring the presence or absence of genotoxic effects.

Comet assay: an essential tool in toxicological research.

PubMed

Glei, M; Schneider, T; Schlörmann, W

2016-10-01

The comet assay is a versatile, reliable, cost-efficient, and fast technique for detecting DNA damage and repair in any tissue. It is useable in almost any cell type and applicable to both eukaryotic and prokaryotic organisms. Instead of highlighting one of the numerous specific aspects of the comet assay, the present review aims at giving an overview about the evolution of this widely applicable method from the first description by Ostling and Johanson to the OECD Guideline 489 for the in vivo mammalian comet assay. In addition, methodical aspects and the influence of critical steps of the assay as well as the evaluation of results and improvements of the method are reviewed. Methodical aspects regarding oxidative DNA damage and repair are also addressed. An overview about the most recent works and relevant cutting-edge reviews based on the comet assay with special regard to, e.g., clinical applications, nanoparticles or environmental risk assessment concludes this review. Taken together, the presented overview raises expectations to further decades of successful applications and enhancements of this excellent method.

The comet assay as a tool for human biomonitoring studies: the ComNet project.

PubMed

Collins, Andrew; Koppen, Gudrun; Valdiglesias, Vanessa; Dusinska, Maria; Kruszewski, Marcin; Møller, Peter; Rojas, Emilio; Dhawan, Alok; Benzie, Iris; Coskun, Erdem; Moretti, Massimo; Speit, Günter; Bonassi, Stefano

2014-01-01

The comet assay is widely used in human biomonitoring to measure DNA damage as a marker of exposure to genotoxic agents or to investigate genoprotective effects. Studies often involve small numbers of subjects, and design may be sub-optimal in other respects. In addition, comet assay protocols in use in different laboratories vary significantly. In spite of these difficulties, it is appropriate to carry out a pooled analysis of all available comet assay biomonitoring data, in order to establish baseline parameters of DNA damage, and to investigate associations between comet assay measurements and factors such as sex, age, smoking status, nutrition, lifestyle, etc. With this as its major objective, the ComNet project has recruited almost 100 research groups willing to share datasets. Here we provide a background to this project, discussing the history of the comet assay and practical issues that can critically affect its performance. We survey its diverse applications in biomonitoring studies, including environmental and occupational exposure to genotoxic agents, genoprotection by dietary and other factors, DNA damage associated with various diseases, and intrinsic factors that affect DNA damage levels in humans. We examine in depth the quality of data from a random selection of studies, from an epidemiological and statistical point of view. Copyright © 2013 Elsevier B.V. All rights reserved.

Modified in vivo comet assay detects the genotoxic potential of 14-hydroxycodeinone, an α,β-unsaturated ketone in oxycodone.

PubMed

Pant, Kamala; Roden, Nicholas; Zhang, Charles; Bruce, Shannon; Wood, Craig; Pendino, Kimberly

2015-12-01

14-Hydroxycodeinone (14-HC) is an α,β-unsaturated ketone impurity found in oxycodone drug substance and has a structural alert for genotoxicity. 14-HC was tested in a combined Modified and Standard Comet Assay to determine if the slight decrease in % Tail DNA noted in a previously conducted Standard Comet Assay with 14-HC could be magnified to clarify if the response was due to cross-linking activity. One limitation of the Standard Comet Assay is that DNA cross-links cannot be reliably detected. However, under certain modified testing conditions, DNA cross-links and chemical moieties that elicit such cross-links can be elucidated. One such modification involves the induction of additional breakages of DNA strands by gamma or X-ray irradiation. To determine if 14-HC is a DNA crosslinker in vivo, a Modified Comet Assay was conducted using X-ray irradiation as the modification to visualize crosslinking activity. In this assay, 14-HC was administered orally to mice up to 320 mg/kg/day. Results showed a statistically significant reduction in percent tail DNA in duodenal cells at 320 mg/kg/day, with a nonstatistically significant but dose-related reduction in percent tail DNA also observed at the mid dose of 160 mg/kg/day. Similar decreases were not observed in cells from the liver or stomach, and no increases in percent tail DNA were noted for any tissue in the concomitantly conducted Standard Comet Assay. Taken together, 14-HC was identified as a cross-linking agent in the duodenum in the Modified Comet Assay. © 2015 Wiley Periodicals, Inc.

Recent Advances in In Vivo Genotoxicity Testing: Prediction of Carcinogenic Potential Using Comet and Micronucleus Assay in Animal Models

PubMed Central

Kang, Seung Hun; Kwon, Jee Young; Lee, Jong Kwon; Seo, Young Rok

2013-01-01

Genotoxic events have been known as crucial step in the initiation of cancer. To assess the risk of cancer, genotoxicity assays, including comet, micronucleus (MN), chromosomal aberration, bacterial reverse, and sister chromatid exchange assay, can be performed. Compared with in vitro genotoxicity assay, in vivo genotoxicity assay has been used to verify in vitro assay result and definitely provide biological significance for certain organs or cell types. The comet assay can detect DNA strand breaks as markers of genotoxicity. Methods of the in vivo comet assay have been established by Japanese Center for the Validation of Alternative Methods (JaCVAM) validation studies depending on tissue and sample types. The MN can be initiated by segregation error and lagging acentric chromosome fragment. Methods of the in vivo MN assay have been established by Organization for Economic Co-operation and Development (OECD) test guidelines and many studies. Combining the in vivo comet and MN assay has been regarded as useful methodology for evaluating genetic damage, and it has been used in the assessment of potential carcinogenicity by complementarily presenting two distinct endpoints of the in vivo genotoxicity individual test. Few studies have investigated the quantitative relation between in vivo genotoxicity results and carcinogenicity. Extensive studies emphasizes that positive correlation is detectable. This review summarizes the results of the in vivo comet and MN assays that have investigated the genotoxicity of carcinogens as classified by the International Agency for Research on Cancer (IARC) carcinogenicity database. As a result, these genotoxicity data may provide meaningful information for the assessment of potential carcinogenicity and for implementation in the prevention of cancer. PMID:25337557

Inter-laboratory comparison of the in vivo comet assay including three image analysis systems.

PubMed

Plappert-Helbig, Ulla; Guérard, Melanie

2015-12-01

To compare the extent of potential inter-laboratory variability and the influence of different comet image analysis systems, in vivo comet experiments were conducted using the genotoxicants ethyl methanesulfonate and methyl methanesulfonate. Tissue samples from the same animals were processed and analyzed-including independent slide evaluation by image analysis-in two laboratories with extensive experience in performing the comet assay. The analysis revealed low inter-laboratory experimental variability. Neither the use of different image analysis systems, nor the staining procedure of DNA (propidium iodide vs. SYBR® Gold), considerably impacted the results or sensitivity of the assay. In addition, relatively high stability of the staining intensity of propidium iodide-stained slides was found in slides that were refrigerated for over 3 months. In conclusion, following a thoroughly defined protocol and standardized routine procedures ensures that the comet assay is robust and generates comparable results between different laboratories. © 2015 Wiley Periodicals, Inc.

HT-COMET: a novel automated approach for high throughput assessment of human sperm chromatin quality

PubMed Central

Albert, Océane; Reintsch, Wolfgang E.; Chan, Peter; Robaire, Bernard

2016-01-01

STUDY QUESTION Can we make the comet assay (single-cell gel electrophoresis) for human sperm a more accurate and informative high throughput assay? SUMMARY ANSWER We developed a standardized automated high throughput comet (HT-COMET) assay for human sperm that improves its accuracy and efficiency, and could be of prognostic value to patients in the fertility clinic. WHAT IS KNOWN ALREADY The comet assay involves the collection of data on sperm DNA damage at the level of the single cell, allowing the use of samples from severe oligozoospermic patients. However, this makes comet scoring a low throughput procedure that renders large cohort analyses tedious. Furthermore, the comet assay comes with an inherent vulnerability to variability. Our objective is to develop an automated high throughput comet assay for human sperm that will increase both its accuracy and efficiency. STUDY DESIGN, SIZE, DURATION The study comprised two distinct components: a HT-COMET technical optimization section based on control versus DNAse treatment analyses (n = 3–5), and a cross-sectional study on 123 men presenting to a reproductive center with sperm concentrations categorized as severe oligozoospermia, oligozoospermia or normozoospermia. PARTICIPANTS/MATERIALS, SETTING, METHODS Sperm chromatin quality was measured using the comet assay: on classic 2-well slides for software comparison; on 96-well slides for HT-COMET optimization; after exposure to various concentrations of a damage-inducing agent, DNAse, using HT-COMET; on 123 subjects with different sperm concentrations using HT-COMET. Data from the 123 subjects were correlated to classic semen quality parameters and plotted as single-cell data in individual DNA damage profiles. MAIN RESULTS AND THE ROLE OF CHANCE We have developed a standard automated HT-COMET procedure for human sperm. It includes automated scoring of comets by a fully integrated high content screening setup that compares well with the most commonly used semi-manual analysis software. Using this method, a cross-sectional study on 123 men showed no significant correlation between sperm concentration and sperm DNA damage, confirming the existence of hidden chromatin damage in men with apparently normal semen characteristics, and a significant correlation between percentage DNA in the tail and percentage of progressively motile spermatozoa. Finally, the use of DNA damage profiles helped to distinguish subjects between and within sperm concentration categories, and allowed a determination of the proportion of highly damaged cells. LIMITATIONS, REASONS FOR CAUTION The main limitations of the HT-COMET are the high, yet indispensable, investment in an automated liquid handling system and heating block to ensure accuracy, and the availability of an automated plate reading microscope and analysis software. WIDER IMPLICATIONS OF THE FINDINGS This standardized HT-COMET assay offers many advantages, including higher accuracy and evenness due to automation of sensitive steps, a 14.4-fold increase in sample analysis capacity, and an imaging and scoring time of 1 min/well. Overall, HT-COMET offers a decrease in total experimental time of more than 90%. Hence, this assay constitutes a more efficient option to assess sperm chromatin quality, paves the way to using this assay to screen large cohorts, and holds prognostic value for infertile patients. STUDY FUNDING/COMPETING INTEREST(S) Funded by the CIHR Institute of Human Development, Child and Youth Health (IHDCYH; RHF 100625). O.A. is a fellow supported by the Fonds de la Recherche du Québec - Santé (FRQS) and the CIHR Training Program in Reproduction, Early Development, and the Impact on Health (REDIH). B.R. is a James McGill Professor. The authors declare no conflicts of interest. PMID:26975326

Pre- and Post-equinox ROSINA production rates calculated using a realistic empirical coma model derived from AMPS-DSMC simulations of comet 67P/Churyumov-Gerasimenko

NASA Astrophysics Data System (ADS)

Hansen, Kenneth; Altwegg, Kathrin; Berthelier, Jean-Jacques; Bieler, Andre; Calmonte, Ursina; Combi, Michael; De Keyser, Johan; Fiethe, Björn; Fougere, Nicolas; Fuselier, Stephen; Gombosi, Tamas; Hässig, Myrtha; Huang, Zhenguang; Le Roy, Lena; Rubin, Martin; Tenishev, Valeriy; Toth, Gabor; Tzou, Chia-Yu

2016-04-01

We have previously used results from the AMPS DSMC (Adaptive Mesh Particle Simulator Direct Simulation Monte Carlo) model to create an empirical model of the near comet coma (<400 km) of comet 67P for the pre-equinox orbit of comet 67P/Churyumov-Gerasimenko. In this work we extend the empirical model to the post-equinox, post-perihelion time period. In addition, we extend the coma model to significantly further from the comet (~100,000-1,000,000 km). The empirical model characterizes the neutral coma in a comet centered, sun fixed reference frame as a function of heliocentric distance, radial distance from the comet, local time and declination. Furthermore, we have generalized the model beyond application to 67P by replacing the heliocentric distance parameterizations and mapping them to production rates. Using this method, the model become significantly more general and can be applied to any comet. The model is a significant improvement over simpler empirical models, such as the Haser model. For 67P, the DSMC results are, of course, a more accurate representation of the coma at any given time, but the advantage of a mean state, empirical model is the ease and speed of use. One application of the empirical model is to de-trend the spacecraft motion from the ROSINA COPS and DFMS data (Rosetta Orbiter Spectrometer for Ion and Neutral Analysis, Comet Pressure Sensor, Double Focusing Mass Spectrometer). The ROSINA instrument measures the neutral coma density at a single point and the measured value is influenced by the location of the spacecraft relative to the comet and the comet-sun line. Using the empirical coma model we can correct for the position of the spacecraft and compute a total production rate based on the single point measurement. In this presentation we will present the coma production rate as a function of heliocentric distance both pre- and post-equinox and perihelion.

Validation of the 3D Skin Comet assay using full thickness skin models: Transferability and reproducibility.

PubMed

Reisinger, Kerstin; Blatz, Veronika; Brinkmann, Joep; Downs, Thomas R; Fischer, Anja; Henkler, Frank; Hoffmann, Sebastian; Krul, Cyrille; Liebsch, Manfred; Luch, Andreas; Pirow, Ralph; Reus, Astrid A; Schulz, Markus; Pfuhler, Stefan

2018-03-01

Recently revised OECD Testing Guidelines highlight the importance of considering the first site-of-contact when investigating the genotoxic hazard. Thus far, only in vivo approaches are available to address the dermal route of exposure. The 3D Skin Comet and Reconstructed Skin Micronucleus (RSMN) assays intend to close this gap in the in vitro genotoxicity toolbox by investigating DNA damage after topical application. This represents the most relevant route of exposure for a variety of compounds found in household products, cosmetics, and industrial chemicals. The comet assay methodology is able to detect both chromosomal damage and DNA lesions that may give rise to gene mutations, thereby complementing the RSMN which detects only chromosomal damage. Here, the comet assay was adapted to two reconstructed full thickness human skin models: the EpiDerm™- and Phenion ® Full-Thickness Skin Models. First, tissue-specific protocols for the isolation of single cells and the general comet assay were transferred to European and US-American laboratories. After establishment of the assay, the protocol was then further optimized with appropriate cytotoxicity measurements and the use of aphidicolin, a DNA repair inhibitor, to improve the assay's sensitivity. In the first phase of an ongoing validation study eight chemicals were tested in three laboratories each using the Phenion ® Full-Thickness Skin Model, informing several validation modules. Ultimately, the 3D Skin Comet assay demonstrated a high predictive capacity and good intra- and inter-laboratory reproducibility with four laboratories reaching a 100% predictivity and the fifth yielding 70%. The data are intended to demonstrate the use of the 3D Skin Comet assay as a new in vitro tool for following up on positive findings from the standard in vitro genotoxicity test battery for dermally applied chemicals, ultimately helping to drive the regulatory acceptance of the assay. To expand the database, the validation will continue by testing an additional 22 chemicals. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Assessment of gamma ray-induced DNA damage in Lasioderma serricorne using the comet assay

NASA Astrophysics Data System (ADS)

Kameya, Hiromi; Miyanoshita, Akihiro; Imamura, Taro; Todoriki, Setsuko

2012-03-01

We attempted a DNA comet assay under alkaline conditions to verify the irradiation treatment of pests. Lasioderma serricorne (Fabricius) were chosen as test insects and irradiated with gamma rays from a 60Co source at 1 kGy. We conducted the comet assay immediately after irradiation and over time for 7 day. Severe DNA fragmentation in L. serricorne cells was observed just after irradiation and the damage was repaired during the post-irradiation period in a time-dependent manner. The parameters of the comet image analysis were calculated, and the degree of DNA damage and repair were evaluated. Values for the Ratio (a percentage determined by fluorescence in the damaged area to overall luminance, including intact DNA and the damaged area of a comet image) of individual cells showed that no cells in the irradiated group were included in the Ratio<0.1 category, the lowest grade. This finding was observed consistently throughout the 7-day post-irradiation period. We suggest that the Ratio values of individual cells can be used as an index of irradiation history and conclude that the DNA comet assay under alkaline conditions, combined with comet image analysis, can be used to identify irradiation history.

Quantification of applied dose in irradiated citrus fruits by DNA Comet Assay together with image analysis.

PubMed

Cetinkaya, Nurcan; Ercin, Demet; Özvatan, Sümer; Erel, Yakup

2016-02-01

The experiments were conducted for quantification of applied dose for quarantine control in irradiated citrus fruits. Citrus fruits exposed to doses of 0.1 to 1.5 kGy and analyzed by DNA Comet Assay. Observed comets were evaluated by image analysis. The tail length, tail moment and tail DNA% of comets were used for the interpretation of comets. Irradiated citrus fruits showed the separated tails from the head of the comet by increasing applied doses from 0.1 to 1.5 kGy. The mean tail length and mean tail moment% levels of irradiated citrus fruits at all doses are significantly different (p < 0.01) from control even for the lowest dose at 0.1 kGy. Thus, DNA Comet Assay may be a practical quarantine control method for irradiated citrus fruits since it has been possible to estimate the applied low doses as small as 0.1 kGy when it is combined with image analysis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Electronic cigarettes induce DNA strand breaks and cell death independently of nicotine in cell lines.

PubMed

Yu, Vicky; Rahimy, Mehran; Korrapati, Avinaash; Xuan, Yinan; Zou, Angela E; Krishnan, Aswini R; Tsui, Tzuhan; Aguilera, Joseph A; Advani, Sunil; Crotty Alexander, Laura E; Brumund, Kevin T; Wang-Rodriguez, Jessica; Ongkeko, Weg M

2016-01-01

Evaluate the cytotoxicity and genotoxicity of short- and long-term e-cigarette vapor exposure on a panel of normal epithelial and head and neck squamous cell carcinoma (HNSCC) cell lines. HaCaT, UMSCC10B, and HN30 were treated with nicotine-containing and nicotine-free vapor extract from two popular e-cigarette brands for periods ranging from 48 h to 8 weeks. Cytotoxicity was assessed using Annexin V flow cytometric analysis, trypan blue exclusion, and clonogenic assays. Genotoxicity in the form of DNA strand breaks was quantified using the neutral comet assay and γ-H2AX immunostaining. E-cigarette-exposed cells showed significantly reduced cell viability and clonogenic survival, along with increased rates of apoptosis and necrosis, regardless of e-cigarette vapor nicotine content. They also exhibited significantly increased comet tail length and accumulation of γ-H2AX foci, demonstrating increased DNA strand breaks. E-cigarette vapor, both with and without nicotine, is cytotoxic to epithelial cell lines and is a DNA strand break-inducing agent. Further assessment of the potential carcinogenic effects of e-cigarette vapor is urgently needed. Copyright © 2015 Elsevier Ltd. All rights reserved.

Electronic cigarettes induce DNA strand breaks and cell death independently of nicotine in cell lines

PubMed Central

Yu, Vicky; Rahimy, Mehran; Korrapati, Avinaash; Xuan, Yinan; Zou, Angela E.; Krishnan, Aswini R.; Tsui, Tzuhan; Aguilera, Joseph A.; Advani, Sunil; Crotty Alexander, Laura E.; Brumund, Kevin T.; Wang-Rodriguez, Jessica

2016-01-01

Objectives Evaluate the cytotoxicity and genotoxicity of short- and long-term e-cigarette vapor exposure on a panel of normal epithelial and head and neck squamous cell carcinoma (HNSCC) cell lines. Materials and Methods HaCaT, UMSCC10B, and HN30 were treated with nicotine-containing and nicotine-free vapor extract from two popular e-cigarette brands for periods ranging from 48 hours to 8 weeks. Cytotoxicity was assessed using Annexin V flow cytometric analysis, trypan blue exclusion, and clonogenic assays. Genotoxicity in the form of DNA strand breaks was quantified using the neutral comet assay and γ-H2AX immunostaining. Results E-cigarette-exposed cells showed significantly reduced cell viability and clonogenic survival, along with increased rates of apoptosis and necrosis, regardless of e-cigarette vapor nicotine content. They also exhibited significantly increased comet tail length and accumulation of γ-H2AX foci, demonstrating increased DNA strand breaks. Conclusion E-cigarette vapor, both with and without nicotine, is cytotoxic to epithelial cell lines and is a DNA strand break-inducing agent. Further assessment of the potential carcinogenic effects of e-cigarette vapor is urgently needed. PMID:26547127

The Comet assay in insects--Status, prospects and benefits for science.

PubMed

Augustyniak, Maria; Gladysz, Marcin; Dziewięcka, Marta

2016-01-01

The Comet assay has been recently adapted to investigate DNA damage in insects. The first reports of its use in Drosophila melanogaster appeared in 2002. Since then, the interest in the application of the Comet assay to studies of insects has been rapidly increasing. Many authors see substantial potential in the use of the Comet assay in D. melanogaster for medical toxicology studies. This application could allow the testing of drugs and result in an understanding of the mechanisms of action of toxins, which could significantly influence the limited research that has been performed on vertebrates. The possible perspectives and benefits for science are considered in this review. In the last decade, the use of the Comet assay has been described in insects other than D. melanogaster. Specifically, methods to prepare a cell suspension from insect tissues, which is a difficult task, were analyzed and compared in detail. Furthermore, attention was paid to any differences and modifications in the research protocols, such as the buffer composition and electrophoresis conditions. Various scientific fields in addition to toxicological and ecotoxicological research were considered. We expect the Comet assay to be used in environmental risk assessments and to improve our understanding of many important phenomena of insect life, such as metamorphosis, molting, diapause and quiescence. The use of this method to study species that are of key importance to humans, such as pests and beneficial insects, appears to be highly probable and very promising. The use of the Comet assay for DNA stability testing in insects will most likely rapidly increase in the future. Copyright © 2015 Elsevier B.V. All rights reserved.

Different sensitivities of cultured mammalian cells towards aphidicolin-enhanced DNA effects in the comet assay.

PubMed

Speit, Günter; Schütz, Petra; Bausinger, Julia

2016-06-01

The comet assay in combination with the polymerase inhibitor aphidicolin (APC) has been used to measure DNA excision repair activity, DNA repair kinetics and individual DNA repair capacity. Since APC can enhance genotoxic effects of mutagens measured by the comet assay, this approach has been proposed for increasing the sensitivity of the comet assay in human biomonitoring. The APC-modified comet assay has mainly been performed with human blood and it was shown that it not only enhances the detection of DNA damage repaired by nucleotide excision repair (NER) but also damage typically repaired by base excision repair (BER). Recently, we reported that in contrast to blood leukocytes, A549 cells (a human lung adenocarcinoma cell line) seem to be insensitive towards the repair-inhibiting action of APC. To further elucidate the general usefulness of the APC-modified comet assay for studying repair in cultured mammalian cells, we comparatively investigated further cell lines (HeLa, TK6, V79). DNA damage was induced by BPDE (benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide) and MMS (methyl methanesulfonate) in the absence and presence of APC (3 or 15μM). APC was either added for 2h together with the mutagen or cells were pre-incubated for 30min with APC before the mutagen was added. The results indicate that the cell lines tested differ fundamentally with regard to their sensitivity and specificity towards the repair-inhibiting effect of APC. The actual cause for these differences is still unclear but potential molecular explanations are discussed. Irrespective of the underlying mechanism(s), our study revealed practical limitations of the use of the APC-modified comet assay. Copyright © 2016 Elsevier B.V. All rights reserved.

DNA Damage Analysis in Children with Non-syndromic Developmental Delay by Comet Assay.

PubMed

Susai, Surraj; Chand, Parkash; Ballambattu, Vishnu Bhat; Hanumanthappa, Nandeesha; Veeramani, Raveendranath

2016-05-01

Majority of the developmental delays in children are non-syndromic and they are believed to have an underlying DNA damage, though not well substantiated. Hence the present study was carried out to find out if there is any increased DNA damage in children with non-syndromic developmental delay by using the comet assay. The present case-control study was undertaken to assess the level of DNA damage in children with non syndromic developmental delay and compare the same with that of age and sex matched controls using submarine gel electrophoresis (Comet Assay). The blood from clinically diagnosed children with non syndromic developmental delay and controls were subjected for alkaline version of comet assay - Single cell gel electrophoresis using lymphocytes isolated from the peripheral blood. The comets were observed under a bright field microscope; photocaptured and scored using the Image J image quantification software. Comet parameters were compared between the cases and controls and statistical analysis and interpretation of results was done using the statistical software SPSS version 20. The mean comet tail length in cases and control was 20.77+7.659μm and 08.97+4.398μm respectively which was statistically significant (p<0.001). Other comet parameters like total comet length and % DNA in tail also showed a statistically significant difference (p < 0.001) between cases and controls. The current investigation unraveled increased levels of DNA damage in children with non syndromic developmental delay when compared to the controls.

The use of comet assay in plant toxicology: recent advances

PubMed Central

Santos, Conceição L. V.; Pourrut, Bertrand; Ferreira de Oliveira, José M. P.

2015-01-01

The systematic study of genotoxicity in plants induced by contaminants and other stress agents has been hindered to date by the lack of reliable and robust biomarkers. The comet assay is a versatile and sensitive method for the evaluation of DNA damages and DNA repair capacity at single-cell level. Due to its simplicity and sensitivity, and the small number of cells required to obtain robust results, the use of plant comet assay has drastically increased in the last decade. For years its use was restricted to a few model species, e.g., Allium cepa, Nicotiana tabacum, Vicia faba, or Arabidopsis thaliana but this number largely increased in the last years. Plant comet assay has been used to study the genotoxic impact of radiation, chemicals including pesticides, phytocompounds, heavy metals, nanoparticles or contaminated complex matrices. Here we will review the most recent data on the use of this technique as a standard approach for studying the genotoxic effects of different stress conditions on plants. Also, we will discuss the integration of information provided by the comet assay with other DNA-damage indicators, and with cellular responses including oxidative stress, cell division or cell death. Finally, we will focus on putative relations between transcripts related with DNA damage pathways, DNA replication and repair, oxidative stress and cell cycle progression that have been identified in plant cells with comet assays demonstrating DNA damage. PMID:26175750

Laboratory Gas Dynamic Measurements of the Comet Pressure Sensor COPS on the Rosetta Spacecraft

NASA Astrophysics Data System (ADS)

Tzou, Chia-Yu; Altwegg, Kathrin; Gasc, Sébastien; Rubin, Martin

2014-05-01

Rosetta is part of the cornerstone missions executed by the European Space Agency (ESA). It is the first space mission to orbit and also land on a comet. By the end of July 2014 Rosetta will be able to carry out a close study of comet 67P/Churyumov-Gerasimenko. The Rosetta Orbiter Spectrometer for Ion and Neutral Analysis (ROSINA) is one of the core payloads on board of the Rosetta spacecraft [Balsiger et al, 2007]. ROSINA's main objective is to determine the major atmospheric and ionospheric composition in the coma and to investigate the gas dynamics around the comet. ROSINA consists of two mass spectrometers and a pressure sensor. The Comet Pressure Sensor (COPS) is not only a pressure sensor but also plays the role of a safety instrument for Rosetta by providing high-density alerts to the other payload instruments. It includes two gauges: the "nude gauge" measures total neutral density in the coma and the "ram gauge" measures the dynamic pressure of the cometary gas flux to obtain the bulk velocity of the neutral gas. The combination of these two gauges makes COPS capable to derive the gas dynamics in the coma. We recently performed laboratory gas dynamic measurements with the identical flight-spare instrument of COPS. Using the Calibration System for The Mass Spectrometer Instrument ROSINA (CASYMIR) we produce neutral gas beams to model cometary gas jets with velocities from thermal to 2 km/s. For COPS calibration we measure gas beams with different incident angles to derive the velocity and the temperature of the gas using different mixtures expected at the comet. We demonstrate that COPS will be ready for the prime mission and it will be fascinating to compare COPS measurements with numerous observation results and computer models starting in summer 2014 to gain new insights into the gas dynamics around a comet. Reference: Balsiger, H. et al.: ROSINA-Rosetta Orbiter Spectrometer for Ion and Neutral Analysis, Space Science Reviews, Vol. 128, 745-801, 2007.

In Situ Space Gas Dynamic Measurements by the ROSINA Comet Pressure Sensor COPS Onboard Rosetta Spacecraft

NASA Astrophysics Data System (ADS)

Tzou, Chia-Yu; Altwegg, Kathrin; Fiethe, Björn; Gasc, Sébastien; Rubin, Martin

2015-04-01

Rosetta is part of the cornerstone missions executed by the European Space Agency. It is the first space mission to orbit and also land on a comet. The Rosetta Orbiter Spectrometer for Ion and Neutral Analysis (ROSINA) is one of the core payloads on board of the Rosetta spacecraft [Balsiger et al, 2007]. ROSINA's main objective is to determine the major atmospheric and ionospheric composition in the coma and to investigate the gas dynamics around the comet. ROSINA consists of two mass spectrometers and a pressure sensor. The COmet Pressure Sensor (COPS) includes two gauges: the "nude gauge" measures total neutral density in the coma and the "ram gauge" measures the dynamic pressure of the cometary gas flux. The combination of these two gauges makes COPS capable to derive the gas dynamics (velocity and temperature) at the location of the spacecraft. Over several months Rosetta has been carrying out a close study of comet 67P/Churyumov-Gerasimenko. In early August 2014 COPS detected the faint and expanding atmosphere of the comet while it was still outside of 3.5 AU from the Sun. We will present ROSINA COPS observations of the evolution and gas dynamics of the cometary coma following these first observations until spring 2015. Reference: Balsiger, H. et al.: ROSINA-Rosetta Orbiter Spectrometer for Ion and Neutral Analysis, Space Science Reviews, Vol. 128, 745-801, 2007.

Monitoring the Genotoxic and Cytotoxic Potential and the Presence of Pesticides and Hydrocarbons in Water of the Sinos River Basin, Southern Brazil.

PubMed

Bianchi, Eloisa; Lessing, Gustavo; Brina, Karisa Roxo; Angeli, Larissa; Andriguetti, Natália Bordin; Peruzzo, Jaqueline Regina Soares; do Nascimento, Carlos Augusto; Spilki, Fernando Rosado; Ziulkoski, Ana Luiza; da Silva, Luciano Basso

2017-04-01

The Sinos River is one of the most polluted rivers in Brazil. The purpose of this work was to monitor the presence of some pesticides and hydrocarbons as well as the genotoxic and cytotoxic potential on HEp-2 cells from water samples collected at seven sites in the Sinos River Basin (SRB), southern Brazil. Nine samples were taken from the three main rivers in the SRB and used as a solution to dilute the HEp-2 cell culture medium after microfiltration. Twenty-four pesticides and 19 hydrocarbons were measured. Cytotoxicity was assessed by methyl thiazolyl tetrazolium (MTT) and neutral red (NR) assays, in which cells were exposed to different concentrations of the water samples for 24 h. Genotoxicity of the microfiltrated raw water samples was assessed by comet assay after 6 and 24 h of exposure. Among the chemicals analyzed, only the 2,4-D, dichloromethane, tetrachloroethene, chloroform, bromodichloromethane, styrene, and toluene were detected, but they were all lower than the limit established by Brazilian regulations. Twenty samples from a total of 60 had a cytotoxic effect in the MTT assay and 30 in the NR assay. The comet assay indicated the presence of genotoxic substances in the water at the seven locations monitored. Temporal and spatial variation was observed in the cytotoxicity and genotoxicity assays. Results indicated that the water in all stretches of the SRB is contaminated and it can cause harmful effects to humans and to the aquatic biota. This HEp-2 cell-line approach can be an additional tool for environmental monitoring.

Applicability of the comet assay in evaluation of DNA damage in healthcare providers' working with antineoplastic drugs: a systematic review and meta-analysis.

PubMed

Zare Sakhvidi, Mohammad Javad; Hajaghazadeh, Mohammad; Mostaghaci, Mehrdad; Mehrparvar, Amir Houshang; Zare Sakhvidi, Fariba; Naghshineh, Elham

2016-01-01

Unintended occupational exposure to antineoplastic drugs (ANDs) may occur in medical personnel. Some ANDs are known human carcinogens and exposure can be monitored by genotoxic biomarkers. To evaluate the obstacles to obtaining conclusive results from a comet assay test to determine DNA damage among AND exposed healthcare workers. We systematically reviewed studies that used alkaline comet assay to determine the magnitude and significance of DNA damage among health care workers with potential AND exposure. Fifteen studies were eligible for review and 14 studies were used in the meta-analysis. Under random effect assumption, the estimated standardized mean difference (SMD) in the DNA damage of health care workers was 1.93 (95% CI: 1.15-2.71, p < 0.0001). The resulting SMD was reduced to 1.756 (95% CI: 0.992-2.52, p < 0.0001) when the analysis only included nurses. In subgroup analyses based on gender and smoking, heterogeneity was observed. Only for studies reporting comet moment, I2 test results, as a measure of heterogeneity, dropped to zero. Heterogeneity analysis showed that date of study publication was a possible source of heterogeneity (B = -0.14; p < 0.0001). A mixture of personal parameters, comet assay methodological variables, and exposure characteristics may be responsible for heterogenic data from comet assay studies and interfere with obtaining conclusive results. Lack of quantitative environmental exposure measures and variation in comet assay protocols across studies are important obstacles in generalization of results.

Semen quality parameters as fertility predictors of water buffalo bull spermatozoa during low-breeding season.

PubMed

Ahmed, Hussain; Andrabi, S Murtaza Hassan; Jahan, Sarwat

2016-10-01

The present study was carried out to assess various postthaw semen quality parameters for the prediction of fertility in buffalo bull during low-breeding season. Semen (30 ejaculates) was collected from five adult buffalo bulls with artificial vagina (42 °C). Sperm motility parameters, velocity distribution, motion kinematics, and subpopulations were analyzed by computer-aided sperm motion analyzer (CASA). Moreover, sperm visual motility, supravital plasma membrane integrity, viability/acrosome integrity, viability/mitochondrial transmembrane potential, DNA fragmentation/integrity, and morphology were analyzed by phase-contrast microscope, supravital hypoosmotic swelling test, Trypan blue/Giemsa staining, propidium iodide/"5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl carbocyanine iodide" (JC-1) fluorochromes, neutral comet assay/acridine orange assay and wet mount technique, respectively. Outcome of 528 inseminations was analyzed for in vivo fertility. Pearson's correlation coefficients revealed that sperm progressive motility (%), rapid velocity (%), average path velocity (μm/s), straight line velocity (μm/s), subpopulation one (most rapid, and progressive) of motile spermatozoa (%), supravital plasma membrane integrity (%), and viable spermatozoa with intact acrosome (%) were significantly correlated with in vivo fertility (r = 0.64, P < 0.01; r = 0.57, P < 0.01; r = 0.52, P < 0.01; r = 0.56, P < 0.01; r = 0.73, P < 0.001; r = 0.74, P < 0.001; r = 0.88, P < 0.001); whereas nonviable spermatozoa with damaged acrosome or low-mitochondrial transmembrane potential and comet length (μm) of neutral comet assay were negatively associated with in vivo fertility (r = -0.79, r = -0.75, P < 0.001, and r = -0.60, P < 0.05, respectively). Multiple regression analysis reported that combination of semen quality parameters as predictor of fertility were better (R(2) adjusted = 81.30%, P < 0.001) as compared with single parameter (R(2) adjusted = 50.20%, P < 0.007). It is concluded that assessment of CASA parameters and some other sperm structural and functional parameters, that is, integrity of plasma membrane and acrosome, and transmembrane potential of mitochondria were able to predict the in vivo fertility of water buffalo bull during low-breeding season. Copyright © 2016 Elsevier Inc. All rights reserved.

Evaluating In Vitro DNA Damage Using Comet Assay.

PubMed

Lu, Yanxin; Liu, Yang; Yang, Chunzhang

2017-10-11

DNA damage is a common phenomenon for each cell during its lifespan, and is defined as an alteration of the chemical structure of genomic DNA. Cancer therapies, such as radio- and chemotherapy, introduce enormous amount of additional DNA damage, leading to cell cycle arrest and apoptosis to limit cancer progression. Quantitative assessment of DNA damage during experimental cancer therapy is a key step to justify the effectiveness of a genotoxic agent. In this study, we focus on a single cell electrophoresis assay, also known as the comet assay, which can quantify single and double-strand DNA breaks in vitro. The comet assay is a DNA damage quantification method that is efficient and easy to perform, and has low time/budget demands and high reproducibility. Here, we highlight the utility of the comet assay for a preclinical study by evaluating the genotoxic effect of olaparib/temozolomide combination therapy to U251 glioma cells.

Estimation of serum malondialdehyde and assessment of DNA damage using comet assay in patients with oral submucous fibrosis.

PubMed

Paulose, Swetha; Rangdhol, Vishwanath; Ramesh, Ramasamy; Jeelani, Siccandar Ali; Brooklyin, Sivakumar

2016-08-01

To quantify the level of serum malondialdehyde and extent of DNA damage using comet assay in patients with oral submucous fibrosis (SMF) in comparison to normal individuals and to correlate the extent of DNA damage with MDA levels. Study included 30 cases of SMF (n = 30) and equal number of healthy volunteers. Serum malondialdehyde was measured using the thiobarbituric-trichloroacetitic acid (TBA-TCA) method. Comet assay was used to assess the DNA damage. Association between the extent of DNA damage and serum MDA levels was analyzed in SMF statistically. Comet assay results showed that there was an increase in tail length, percentage of tail DNA and tail moment among SMF subjects (P < 0.05). Serum MDA levels were elevated in SMF patients compared with healthy subjects. A significant positive correlation was observed between serum MDA levels and comet tail length in SMF group (r = 0.56; P < 0.05). Patients with SMF have increased DNA damage and elevated levels of lipid peroxidation compared with healthy controls. Evaluation of MDA levels as an oxidative biomarker along with comet assay analysis will serve as a diagnostic tool to identify patients with high risk of malignant potential in SMF. © 2015 Wiley Publishing Asia Pty Ltd.

New Application of the Comet Assay

PubMed Central

Cortés-Gutiérrez, Elva I.; Dávila-Rodríguez, Martha I.; Fernández, José Luís; López-Fernández, Carmen; Gosálbez, Altea; Gosálvez, Jaime

2011-01-01

The comet assay is a well-established, simple, versatile, visual, rapid, and sensitive tool used extensively to assess DNA damage and DNA repair quantitatively and qualitatively in single cells. The comet assay is most frequently used to analyze white blood cells or lymphocytes in human biomonitoring studies, although other cell types have been examined, including buccal, nasal, epithelial, and placental cells and even spermatozoa. This study was conducted to design a protocol that can be used to generate comets in subnuclear units, such as chromosomes. The new technique is based on the chromosome isolation protocols currently used for whole chromosome mounting in electron microscopy, coupled to the alkaline variant of the comet assay, to detect DNA damage. The results show that migrant DNA fragments can be visualized in whole nuclei and isolated chromosomes and that they exhibit patterns of DNA migration that depend on the level of DNA damage produced. This protocol has great potential for the highly reproducible study of DNA damage and repair in specific chromosomal domains. PMID:21540337

First application of comet assay in blood cells of Mediterranean loggerhead sea turtle (Caretta caretta).

PubMed

Caliani, Ilaria; Campani, Tommaso; Giannetti, Matteo; Marsili, Letizia; Casini, Silvia; Fossi, Maria Cristina

2014-05-01

The aim of this study was to validate the comet assay in erythrocytes of Caretta caretta, a species never investigated for genotoxicity. We studied 31 loggerhead sea turtles from three Italian marine rescue centres. Peripheral blood samples were collected from all the animals and the comet assay applied. All comet cells were analysed using two methods: visual scoring and computer image analysis. The % DNA in tail mean value ± SD and Damage Index were 21.56 ± 15.41 and 134.83 ± 94.12, respectively. A strong and statistically significant statistically correlation between the two analytical methods was observed (r = 0.95; p < 0.05). These results demonstrate that the comet assay is a useful method to detect the possible effects of genotoxic agents in loggerhead sea turtle and to increase the knowledge about the ecotoxicological health status of this threatened species. Copyright © 2013 Elsevier Ltd. All rights reserved.

The comet assay in Environmental Risk Assessment of marine pollutants: applications, assets and handicaps of surveying genotoxicity in non-model organisms.

PubMed

Martins, Marta; Costa, Pedro M

2015-01-01

Determining the genotoxic effects of pollutants has long been a priority in Environmental Risk Assessment (ERA) for coastal ecosystems, especially of complex areas such as estuaries and other confined waterbodies. The acknowledged link between DNA damage, mutagenicity and carcinogenicity to the exposure to certain toxicants has been responsible to the growing interest in determining the genotoxic effects of xenobiotics to wildlife as a measure of environmental risk. The comet assay, although widely employed in in vivo and in vitro toxicology, still holds many constraints in ERA, in large part owing to difficulties in obtaining conclusive cause-effect relationships from complex environments. Nevertheless, these challenges do not hinder the attempts to apply the alkaline comet assay on sentinel organisms, wild or subjected to bioassays in or ex situ (from fish to molluscs) as well to standardise protocols and establish general guidelines to the interpretation of findings. Fish have been regarded as an appealing subject due to the ease of performing the comet assay in whole blood. However, the application of the comet assay is becoming increasingly common in invertebrates (e.g. in molluscan haemocytes and solid tissues such as gills). Virtually all sorts of results have been obtained from the application of the comet assay in ERA (null, positive and inconclusive). However, it has become clear that interpreting DNA damage data from wild organisms is particularly challenging due to their ability to adapt to continuous environmental stressors, including toxicants. Also, the comet assay in non-model organisms for the purpose of ERA implies different constraints, assumptions and interpretation of findings, compared with the in vitro procedures from which most guidelines have been derived. This paper critically reviews the application of the comet assay in ERA, focusing on target organisms and tissues; protocol developments, case studies plus data handling and interpretation. © The Author 2014. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Worldwide interest in the comet assay: a bibliometric study.

PubMed

Neri, Monica; Milazzo, Daniele; Ugolini, Donatella; Milic, Mirta; Campolongo, Alessandra; Pasqualetti, Patrizio; Bonassi, Stefano

2015-01-01

The comet assay is a rapid, sensitive and relatively simple method for measuring DNA damage. A bibliometric study was performed to evaluate temporal and geographical trends, research quality and main areas of interest in scientific production in this field. A PubMed search strategy was developed and 7674 citations were retrieved in the period 1990-2013. Notably, the MeSH (Medical Subject Headings) term 'comet assay', officially introduced in 2000, is used by indexers only in two thirds of papers retrieved. Articles on the comet assay were published in 78 countries, spread over the 5 continents. The EU contributed the greatest output, producing >2900 articles with IF (42.0%) and totalling almost 10000 IF points, and was followed by USA. In the new millennium, research with this assay reached a plateau or slow decline in the most industrialised areas (USA, Germany, UK, Italy), while its use has boomed in emerging countries, with increases of 5- to 7-fold in the last 10 years in China, India and Brazil, for instance. This transition resulted in a slow decrease of scientific production quality, as the countries that increased their relative weight typically had lower mIFs. The most common MeSH terms used in papers using the comet assay referred to wide areas of interest, such as DNA damage and repair, cell survival and apoptosis, cancer and oxidative stress, occupational and environmental health. Keywords related to humans, rodents and cell culture were also frequently used. The top journal for the comet assay articles was found to be Mutation Research, followed by Mutagenesis. Most papers using the comet assay as a biomarker were published in genetic and toxicology journals, with a stress on environmental and occupational disciplines. © The Author 2014. Published by Oxford University Press on behalf of the Mutagenesis Society. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Structure and dynamics of the umagnetized plasma around comet 67P/CG

NASA Astrophysics Data System (ADS)

Henri, P.; Vallières, X.; Gilet, N.; Hajra, R.; Moré, J.; Goetz, C.; Richter, I.; Glassmeier, K. H.; Galand, M. F.; Heritier, K. L.; Eriksson, A. I.; Nemeth, Z.; Tsurutani, B.; Rubin, M.; Altwegg, K.

2016-12-01

At distances close enough to the Sun, when comets are characterised by a significant outgassing, the cometary neutral density may become large enough for both the cometary plasma and the cometary gas to be coupled, through ion-neutral and electron-neutral collisions. This coupling enables the formation of an unmagnetised expanding cometary ionosphere around the comet nucleus, also called diamagnetic cavity, within which the solar wind magnetic field cannot penetrate. The instruments of the Rosetta Plasma Consortium (RPC), onboard the Rosetta Orbiter, enable us to better constrain the structure, dynamics and stability of the plasma around comet 67P/CG. Recently, magnetic field measurements (RPC-MAG) have shown the existence of such a diamagnetic region around comet 67P/CG [Götz et al., 2016]. Contrary to a single, large scale, diamagnetic cavity such as what was observed around comet Halley, Rosetta have crossed several diamagnetic structures along its trajectory around comet 67P/CG. Using electron density measurements from the Mutual Impedance Probe (RPC-MIP) during the different diamagnetic cavity crossings, identified by the flux gate magnetometer (RPC-MAG), we map the unmagnetised plasma density around comet 67P/CG. Our aims is to better constrain the structure, dynamics and stability of this inner cometary plasma layer characterised by cold electrons (as witnessed by the Langmuir Probes RPC-LAP). The ionisation ratio in these unmagnetised region(s) is computed from the measured electron (RPC-MIP) and neutral gas (ROSINA/COPS) densities. In order to assess the importance of solar EUV radiation as a source of ionisation, the observed electron density will be compared to a the density expected from an ionospheric model taking into account solar radiation absorption. The crossings of diamagnetic region(s) by Rosetta show that the unmagnetised cometary plasma is particularly homogeneous, compared to the highly dynamical magnetised plasma observed in adjacent magnetised regions. Moreover, during the crossings of multiple, successive diamagnetic region(s) over time scales of tens of minutes or hours, the plasma density is almost identical in the different unmagnetised regions, suggesting that these unmagnetised regions may be a single diamagnetic structure crossed several times by Rosetta.

Comparative study on toxicity of methylmercury chloride and methylmercury hydroxide to the human neuroblastoma cell line SH-SY5Y.

PubMed

Patnaik, Rajashree; Padhy, Rabindra N

2018-05-11

Toxicities of methylmercury chloride (CH 3 HgCl) and methylmercury hydroxide (CH 3 HgOH) to cultured neuroblastoma cell line SH-SY5Y in vitro are evaluated. This is the comparative study between two methylmercury compounds to find out the extent of toxicity of these compounds are toxic to SH-SY5Y cell line. Both cytotoxicity and genotoxicity experiments were carried out to find out the more toxic compound. For cytotoxicity study, four staining assay methods independently with trypan blue (TB), acridine orange/ethidium bromide (AO/EB), 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide (MTT), and neutral red (NR) were used and the comet assay method was done for genotoxicity study. The obtained toxicity data were used for probit analysis. In cytotoxicity, CH 3 HgCl had minimum inhibitory concentration (MIC) value in each assay method as 3 mg/L invariably; LC 25 values were in the range 7.41 to 10.23 mg/L, and LC 50 values were 14.79 to 15.48 mg/L; while LC 75 values were 20.89 to 26.91 mg/L. Moreover, LC 100 value was 30 mg/L, known from comet assay experiments for CH 3 HgCl. Similarly for CH 3 HgOH, the MIC value in each assay method was invariably 3 mg/L, the LC 25 values were in the range 12.58 to 16.59 mg/L, and LC 50 values were 19.49 to 23.44 mg/L; LC 75 values were 27.54 to 30.90 mg/L and LC 100 value was 42 mg/L in each assay done for cytotoxicity and genotoxicity studies. Computed DNA fragmentation indices in comet assays were 98.6 ± 0.57 30 mg/L with CH 3 HgCl and 76 ± 5.29 30 mg/L with CH 3 HgOH. This study clearly indicated that methylmercury chloride is more toxic than methylmercury hydroxide to SH-SY5Y cell line. Toxicity of Hg had been quantified with in vitro cultured human neuroblastoma cell line; since it has neurotoxic effects, its neural evaluation has implications in environmental health issues.

Identification of low level gamma-irradiation of meats by high sensitivity comet assay

NASA Astrophysics Data System (ADS)

Miyahara, Makoto; Saito, Akiko; Ito, Hitoshi; Toyoda, Masatake

2002-03-01

The detection of low levels of irradiation in meats (pork, beef, and chicken) using the new comet assay was investigated in order to assess the capability of the procedure. The new assay includes a process that improves its sensitivity to irradiation and a novel evaluation system for each slide (influence score and comet-type distribution). Samples used were purchased at retailers and were irradiated at 0.5 and 2kGy at 0°C. The samples were processed to obtain comets. Slides were evaluated by typing comets, calculating the influence score and analyzing the comet-type distribution chart of shown on the slide. Influence scores of beef, pork, and chicken at 0kGy were 287(SD=8.0), 305 (SD=12.9), and 320 (SD=21.0), respectively. Those at 500Gy, were 305 (SD=5.3), 347 (SD=10.6), and 364 (12.6), respectively. Irradiation levels in food were successfully determined. Sensitivity to irradiation differed among samples (chicken>pork>beef).

Applicability of the comet assay in evaluation of DNA damage in healthcare providers’ working with antineoplastic drugs: a systematic review and meta-analysis

PubMed Central

Zare Sakhvidi, Mohammad Javad; Hajaghazadeh, Mohammad; Mostaghaci, Mehrdad; Mehrparvar, Amir houshang; Zare Sakhvidi, Fariba; Naghshineh, Elham

2016-01-01

Background Unintended occupational exposure to antineoplastic drugs (ANDs) may occur in medical personnel. Some ANDs are known human carcinogens and exposure can be monitored by genotoxic biomarkers. Objective To evaluate the obstacles to obtaining conclusive results from a comet assay test to determine DNA damage among AND exposed healthcare workers. Methods We systematically reviewed studies that used alkaline comet assay to determine the magnitude and significance of DNA damage among health care workers with potential AND exposure. Fifteen studies were eligible for review and 14 studies were used in the meta-analysis. Results Under random effect assumption, the estimated standardized mean difference (SMD) in the DNA damage of health care workers was 1.93 (95% CI: 1.15–2.71, p < 0.0001). The resulting SMD was reduced to 1.756 (95% CI: 0.992–2.52, p < 0.0001) when the analysis only included nurses. In subgroup analyses based on gender and smoking, heterogeneity was observed. Only for studies reporting comet moment, I2 test results, as a measure of heterogeneity, dropped to zero. Heterogeneity analysis showed that date of study publication was a possible source of heterogeneity (B = −0.14; p < 0.0001). Conclusions A mixture of personal parameters, comet assay methodological variables, and exposure characteristics may be responsible for heterogenic data from comet assay studies and interfere with obtaining conclusive results. Lack of quantitative environmental exposure measures and variation in comet assay protocols across studies are important obstacles in generalization of results. PMID:27110842

Human sperm sex chromosome disomy and sperm DNA damage assessed by the neutral comet assay.

PubMed

McAuliffe, M E; Williams, P L; Korrick, S A; Dadd, R; Marchetti, F; Martenies, S E; Perry, M J

2014-10-10

Is there an association between human sperm sex chromosome disomy and sperm DNA damage? An increase in human sperm XY disomy was associated with higher comet extent; however, there was no other consistent association of sex chromosome disomies with DNA damage. There is limited published research on the association between sex chromosome disomy and sperm DNA damage and the findings are not consistent across studies. We conducted a cross-sectional study of 190 men (25% ever smoker, 75% never smoker) from subfertile couples presenting at the Massachusetts General Hospital Fertility Clinic from January 2000 to May 2003. Multiprobe fluorescence in situ hybridization for chromosomes X, Y and 18 was used to determine XX, YY, XY and total sex chromosome disomy in sperm nuclei using an automated scoring method. The neutral comet assay was used to measure sperm DNA damage, as reflected by comet extent, percentage DNA in the comet tail, and tail distributed moment. Univariate and multiple linear regression models were constructed with sex chromosome disomy (separate models for each of the four disomic conditions) as the independent variable, and DNA damage parameters (separate models for each measure of DNA damage) as the dependent variable. Men with current or past smoking history had significantly greater comet extent (µm: regression coefficients with 95% CI) [XX18: 15.17 (1.98, 28.36); YY18: 14.68 (1.50, 27.86); XY18: 15.41 (2.37, 28.45); Total Sex Chromosome Disomy: 15.23 (2.09, 28.38)], and tail distributed moment [XX18: 3.01 (0.30, 5.72); YY18: 2.95 (0.24, 5.67); XY18: 3.04 (0.36, 5.72); Total Sex Chromosome Disomy: 3.10 (0.31, 5.71)] than men who had never smoked. In regression models adjusted for age and smoking, there was a positive association between XY disomy and comet extent. For an increase in XY disomy from 0.56 to 1.47% (representing the 25th to 75th percentile), there was a mean increase of 5.08 µm in comet extent. No other statistically significant findings were observed. A potential limitation of this study is that it is cross-sectional. Cross-sectional analyses by nature do not lend themselves to inference about directionality for any observed associations; therefore we cannot determine which variable is the cause and which one is the effect. A small sample size may be a further limitation. Comparison of these findings to other studies is limited due to methodological differences. Although consistent associations across sex chromosome disomies or DNA damage measures were not observed, this study highlights the need to explore etiologies of sperm DNA damage and sex chromosome disomy to better understand the potential mechanistic overlaps between the two. This work was supported by NIOSH Grant T42 OH008416, and NIH/NIEHS Grants ES 009718, ES 000002, and R01 ES017457. During the study M.E.M. was affiliated with the Department of Environmental Health at the Harvard School of Public Health. N/A. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Human sperm sex chromosome disomy and sperm DNA damage assessed by the neutral comet assay

PubMed Central

McAuliffe, M.E.; Williams, P.L.; Korrick, S.A.; Dadd, R.; Marchetti, F.; Martenies, S.E.; Perry, M.J.

2014-01-01

STUDY QUESTION Is there an association between human sperm sex chromosome disomy and sperm DNA damage? SUMMARY ANSWER An increase in human sperm XY disomy was associated with higher comet extent; however, there was no other consistent association of sex chromosome disomies with DNA damage. WHAT IS KNOWN ALREADY There is limited published research on the association between sex chromosome disomy and sperm DNA damage and the findings are not consistent across studies. STUDY DESIGN, SIZE, AND DURATION We conducted a cross-sectional study of 190 men (25% ever smoker, 75% never smoker) from subfertile couples presenting at the Massachusetts General Hospital Fertility Clinic from January 2000 to May 2003. PARTICIPANTS/MATERIALS, SETTING, METHODS Multiprobe fluorescence in situ hybridization for chromosomes X, Y and 18 was used to determine XX, YY, XY and total sex chromosome disomy in sperm nuclei using an automated scoring method. The neutral comet assay was used to measure sperm DNA damage, as reflected by comet extent, percentage DNA in the comet tail, and tail distributed moment. Univariate and multiple linear regression models were constructed with sex chromosome disomy (separate models for each of the four disomic conditions) as the independent variable, and DNA damage parameters (separate models for each measure of DNA damage) as the dependent variable. MAIN RESULTS AND THE ROLE OF CHANCE Men with current or past smoking history had significantly greater comet extent (µm: regression coefficients with 95% CI) [XX18: 15.17 (1.98, 28.36); YY18: 14.68 (1.50, 27.86); XY18: 15.41 (2.37, 28.45); Total Sex Chromosome Disomy: 15.23 (2.09, 28.38)], and tail distributed moment [XX18: 3.01 (0.30, 5.72); YY18: 2.95 (0.24, 5.67); XY18: 3.04 (0.36, 5.72); Total Sex Chromosome Disomy: 3.10 (0.31, 5.71)] than men who had never smoked. In regression models adjusted for age and smoking, there was a positive association between XY disomy and comet extent. For an increase in XY disomy from 0.56 to 1.47% (representing the 25th to 75th percentile), there was a mean increase of 5.08 µm in comet extent. No other statistically significant findings were observed. LIMITATIONS, REASONS FOR CAUTION A potential limitation of this study is that it is cross-sectional. Cross-sectional analyses by nature do not lend themselves to inference about directionality for any observed associations; therefore we cannot determine which variable is the cause and which one is the effect. A small sample size may be a further limitation. Comparison of these findings to other studies is limited due to methodological differences. WIDER IMPLICATIONS OF THE FINDINGS Although consistent associations across sex chromosome disomies or DNA damage measures were not observed, this study highlights the need to explore etiologies of sperm DNA damage and sex chromosome disomy to better understand the potential mechanistic overlaps between the two. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by NIOSH Grant T42 OH008416, and NIH/NIEHS Grants ES 009718, ES 000002, and R01 ES017457. During the study M.E.M. was affiliated with the Department of Environmental Health at the Harvard School of Public Health. TRIAL REGISTRATION NUMBER N/A. PMID:25069502

Genotoxic effects of the water-soluble fraction of heavy oil in the brackish/freshwater amphipod Quadrivisio aff. lutzi (Gammaridea) as assessed using the comet assay.

PubMed

Weber, Laura; Carvalho, Ligia; Sá, Natália; Silva, Viviane; Beraldini, Nathalia; Souza, Valderes; Conceição, Moisés

2013-05-01

Amphipod crustaceans have been widely used as invertebrate models in ecotoxicology due to their importance in the food chain. However, few studies have evaluated the genotoxic effects of pollutants in this model using the comet assay. The main obstacle to using amphipods in the comet assay is the difficulty in obtaining enough blood cells from a single individual. In this study, we evaluated the genotoxic effects of the water-soluble fraction (WSF) of heavy oil on the brackish/freshwater amphipod Quadrivisio aff. lutzi, which is common in the coastal lagoons of southeastern Brazil, using hemocytes obtained from single amphipods (without pooling) after optimizing hemolymph extraction. The comet assay revealed significantly higher DNA damage levels (2- to 6-fold higher) in treated amphipods compared to untreated ones with a sublethal concentration of 17.6 % of the WSF within 72 h of treatment. Two independent experiments confirmed an "up and down" pattern of DNA damage, measured as the % of DNA contained in the tail of the comets. Elevations in DNA damage levels were observed at the 6 and 48 h time points, while very low levels of DNA damage were observed at the 24 and 72 h time points. Furthermore, the comet assay revealed gender variability in the levels of DNA damage after short-term exposure.

Composition/Structure/Dynamics of comet and planetary satellite atmospheres

NASA Technical Reports Server (NTRS)

Combi, Michael R. (Principal Investigator)

1995-01-01

This research program addresses two cases of tenuous planetary atmospheres: comets and Io. The comet atmospheric research seeks to analyze a set of spatial profiles of CN in comet Halley taken in a 7.4-day period in April 1986; to apply a new dust coma model to various observations; and to analyze observations of the inner hydrogen coma, which can be optically thick to the resonance scattering of Lyman-alpha radiation, with the newly developed approach that combines a spherical radiative transfer model with our Monte Carlo H coma model. The Io research seeks to understand the atmospheric escape from Io with a hybrid-kinetic model for neutral gases and plasma given methods and algorithms developed for the study of neutral gas cometary atmospheres and the earth's polar wind and plasmasphere. Progress is reported on cometary Hydrogen Lyman-alpha studies; time-series analysis of cometary spatial profiles; model analysis of the dust comae of comets; and a global kinetic atmospheric model of Io.

In vitro genotoxicity of neutral red after photo-activation and metabolic activation in the Ames test, the micronucleus test and the comet assay.

PubMed

Guérard, Melanie; Zeller, Andreas; Singer, Thomas; Gocke, Elmar

2012-07-04

Neutral red (Nr) is relatively non-toxic and is widely used as indicator dye in many biological test systems. It absorbs visible light and is known to act as a photosensitizer, involving the generation of reactive oxygen species (type-I reaction) and singlet oxygen (type-II reaction). The mutagenicity of Nr was determined in the Ames test (with Salmonella typhimurium strains TA1535, TA97, TA98, TA98NR, TA100, and TA102) with and without metabolic activation, and with and without photo-activation on agar plates. Similarly to the situation following metabolic activation, photo-mutagenicity of Nr was seen with all Salmonella strains tested, albeit with different effects between these strains. To our knowledge, Nr is the only photo-mutagen showing such a broad action. Since the effects are also observed in strains not known to be responsive to ROS, this indicates that ROS production is not the sole mode of action that leads to photo-genotoxicity. The reactive species produced by irradiation are short-lived as pre-irradiation of an Nr solution did not produce mutagenic effects when added to the bacteria. In addition, mutagenicity in TA98 following irradiation was stronger than in the nitroreductase-deficient strain TA98NR, indicating that nitro derivatives that are transformed by bacterial nitroreductase to hydroxylamines appear to play a role in the photo-mutagenicity of Nr. Photo-genotoxicity of Nr was further investigated in the comet assay and micronucleus test in L5178Y cells. Concentration-dependent increases in primary DNA damage and in the frequency of micronuclei were observed after irradiation. Copyright © 2012 Elsevier B.V. All rights reserved.

Critical issues with the in vivo comet assay: A report of the comet assay working group in the 6th International Workshop on Genotoxicity Testing (IWGT).

PubMed

Speit, Günter; Kojima, Hajime; Burlinson, Brian; Collins, Andrew R; Kasper, Peter; Plappert-Helbig, Ulla; Uno, Yoshifumi; Vasquez, Marie; Beevers, Carol; De Boeck, Marlies; Escobar, Patricia A; Kitamoto, Sachiko; Pant, Kamala; Pfuhler, Stefan; Tanaka, Jin; Levy, Dan D

2015-05-01

As a part of the 6th IWGT, an expert working group on the comet assay evaluated critical topics related to the use of the in vivo comet assay in regulatory genotoxicity testing. The areas covered were: identification of the domain of applicability and regulatory acceptance, identification of critical parameters of the protocol and attempts to standardize the assay, experience with combination and integration with other in vivo studies, demonstration of laboratory proficiency, sensitivity and power of the protocol used, use of different tissues, freezing of samples, and choice of appropriate measures of cytotoxicity. The standard protocol detects various types of DNA lesions but it does not detect all types of DNA damage. Modifications of the standard protocol may be used to detect additional types of specific DNA damage (e.g., cross-links, bulky adducts, oxidized bases). In addition, the working group identified critical parameters that should be carefully controlled and described in detail in every published study protocol. In vivo comet assay results are more reliable if they were obtained in laboratories that have demonstrated proficiency. This includes demonstration of adequate response to vehicle controls and an adequate response to a positive control for each tissue being examined. There was a general agreement that freezing of samples is an option but more data are needed in order to establish generally accepted protocols. With regard to tissue toxicity, the working group concluded that cytotoxicity could be a confounder of comet results. It is recommended to look at multiple parameters such as histopathological observations, organ-specific clinical chemistry as well as indicators of tissue inflammation to decide whether compound-specific toxicity might influence the result. The expert working group concluded that the alkaline in vivo comet assay is a mature test for the evaluation of genotoxicity and can be recommended to regulatory agencies for use. Copyright © 2014 Elsevier B.V. All rights reserved.

Induction and repair of DNA damage measured by the comet assay in human T lymphocytes separated by immunomagnetic cell sorting.

PubMed

Bausinger, Julia; Speit, Günter

2014-11-01

The comet assay is widely used in human biomonitoring to measure DNA damage in whole blood or isolated peripheral blood mononuclear cells (PBMC) as a marker of exposure to genotoxic agents. Cytogenetic assays with phytohemagglutinin (PHA)-stimulated cultured T lymphocytes are also frequently performed in human biomonitoring. Cytogenetic effects (micronuclei, chromosome aberrations, sister chromatid exchanges) may be induced in vivo but also occur ex vivo during the cultivation of lymphocytes as a consequence of DNA damage present in lymphocytes at the time of sampling. To better understand whether DNA damage measured by the comet assay in PBMC is representative for DNA damage in T cells, we comparatively investigated DNA damage and its repair in PBMC and T cells obtained by immunomagnetic cell sorting. PBMC cultures and T cell cultures were exposed to mutagens with different modes of genotoxic action and DNA damage was measured by the comet assay after the end of a 2h exposure and after 18h post-incubation. The mutagens tested were methyl methanesulfonate (MMS), (±)-anti-B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE), 4-nitroquinoline-1-oxide (4NQO), styrene oxide and potassium bromate. MMS and potassium bromate were also tested by the modified comet assay with formamido pyrimidine glycosylase (FPG) protein. The results indicate that the mutagens tested induce DNA damage in PBMC and T cells in the same range of concentrations and removal of induced DNA lesions occurs to a comparable extent. Based on these results, we conclude that the comet assay with PBMC is suited to predict DNA damage and its removal in T cells. Copyright © 2014 Elsevier B.V. All rights reserved.

Cytogenetic status of healthy children assessed with the alkaline comet assay and the cytokinesis-block micronucleus cytome assay.

PubMed

Gajski, Goran; Gerić, Marko; Oreščanin, Višnja; Garaj-Vrhovac, Vera

2013-01-20

In the present study the alkaline comet assay and the cytokinesis-block micronucleus cytome (CBMN Cyt) assay were used to evaluate the baseline frequency of cytogenetic damage in peripheral blood lymphocytes (PBLs) of 50 healthy children from the general population in Croatia (age, 11.62±1.81 years). Mean values of tail length, tail intensity and tail moment, as comet assay parameters, were 12.92±0.10, 0.73±0.06 and 0.08±0.01, respectively. The mean frequency of micronuclei (MN) for all subjects was 2.32±0.28 per 1000 bi-nucleated cells, while the mean frequency of nucleoplasmic bridges (NPBs) was 1.72±0.24 and of nuclear buds (NBUDs) 1.44±0.19. The mean nuclear division index (NDI) was 1.70±0.05. When comet-assay parameters were considered, higher mean values for all three were found for the female population. According to the Mann-Whitney U test applied on the results of the comet assay, the only statistically significant difference between the male and female populations was found for tail length. Similar to the results obtained by the comet assay, girls showed higher mean values of all three measured parameters of the CBMN Cyt assay. This difference was statistically significant for total number of NPBs only. In the case of the NDI, a higher mean value was also obtained in girls, but this difference was not statistically significant. The results obtained present background data that could be considered as normal values for healthy children living in urban areas, and can later on serve as baseline values for further toxicological monitoring. Additionally, the usefulness of both techniques in measuring cytogenetic damage during bio-monitoring of children is confirmed. Copyright © 2012 Elsevier B.V. All rights reserved.

Evaluation of genome damage in subjects occupationally exposed to possible carcinogens.

PubMed

Zeljezic, Davor; Mladinic, Marin; Kopjar, Nevenka; Radulovic, Azra Hursidic

2016-09-01

In occupational exposures, populations are simultaneously exposed to a mixture of chemicals. We aimed to evaluate DNA damage due to possible carcinogen exposure (phenylhydrazine, ethylene oxide, dichloromethane, and 1,2-dichloroethane) in lymphocytes of pharmaceutical industry workers from the same production line. Population comprised 16 subjects (9 females and 7 males) who were exposed to multiple chemicals for 8 months. Genome damage was assessed using alkaline comet assay, micronucleus assay, and comet assay coupled with fluorescent in situ hybridization (comet-FISH). After 8 months of exposure, the issue of irregular use of all available personal protective equipment (PPE) came into light. To decrease the risk of exposure, strict use of PPE was enforced. After 8 months of strict PPE use, micronuclei frequency and comet assay parameters in lymphocytes of pharmaceutical workers significantly decreased compared with prior period of irregular PPE use. Comet-FISH results indicated a significant shift in distribution of signals for the TP 53 gene toward a more frequent occurrence in the comet tail. Prolonged exposure to possible carcinogens may hinder DNA repair mechanisms and affect structural integrity of TP 53 Two indicators of loss of TP 53 gene integrity have risen, namely, TP 53 fragmentation rate in lymphocytes with persistently elevated primary damage and incidence of TP 53 deletions in undamaged lymphocytes. © The Author(s) 2015.

On the global nature of the solar wind interaction with Comet Halley

NASA Technical Reports Server (NTRS)

Mendis, D. A.; Flammer, K. R.; Reme, H.; Sauvaud, J. A.; D'Uston, C.

1989-01-01

Data obtained by two instruments of the RPA-Copernic experiment aboard Giotto during its encounter with Comet Halley are used to determine the positions of several sharp boundaries delineating transitions from one flow state to another. Production rates of the neutrals are obtained, along with ion-neutral drag coefficients. It is suggested that the cometopause observed between the shock and the ionopause coincides with the expected position of a previously proposed collisionopause.

Statistical analysis of dust signals observed by ROSINA/COPS onboard of the Rosetta spacecraft at comet 67P/Churyumov-Gerasimenko

NASA Astrophysics Data System (ADS)

Tzou, Chia-Yu; altwegg, kathrin; Bieler, Andre; Calmonte, Ursina; Gasc, Sébastien; Le Roy, Léna; Rubin, Martin

2016-10-01

ROSINA is the in situ Rosetta Orbiter Spectrometer for Ion and Neutral Analysis on board of Rosetta, one of the corner stone missions of the European Space Agency (ESA) to land and orbit the Jupiter family comet 67P/Churyumov-Gerasimenko (67P). ROSINA consists of two mass spectrometers and a pressure sensor. The Reflectron Time of Flight Spectrometer (RTOF) and the Double Focusing Mass Spectrometer (DFMS) complement each other in mass and time resolution.The Comet Pressure Sensor (COPS) provides density measurements of the neutral molecules in the cometary coma of 67P. COPS has two gauges, a nude gauge that measures the total neutral density and a ram gauge that measures the dynamic pressure from the comet. Combining the two COPS is also capable of providing gas dynamic information such as gas velocity and gas temperature of the coma.While Rosetta started orbiting around 67P in August 2014, COPS observed diurnal and seasonal variations of the neutral gas density in the coma. Surprisingly, additional to these major density variation patterns, COPS occasionally observed small spikes in the density that are associated with dust. These dust signals can be interpreted as a result of cometary dust releasing volatiles while heated up near COPS. A statistical analysis of dust signals detected by COPS will be presented.

An Integrated Modeling Study for Coordinated Observations of H, O, OH, and H2O(+) Emissions in the Coma and Ion Tail of the Comet Hale-Bopp

NASA Technical Reports Server (NTRS)

Smyth, William H.

2001-01-01

This project has two overall objectives. One objective is to advance our general understanding of both the comet neutral atmosphere and the cometary plasma in the atmosphere and ion tall. The other objective is to obtain specific key information about comet Hale-Bopp that is generally important for Hale-Bopp studies. The primary emphasis in this project is to analyze, in a self-consistent manner, excellent quality high resolution image and line profile observations obtained by the University of Wisconsin for H, O, OH, and H2O+ emissions from the inner coma, outer coma, and ion tail of Hale-Bopp. The information on the spatial and velocity distributions of H2O neutral and ionized photo-products in the inner coma, outer coma, and in the H2O+ ion tail is of substantial and direct importance in the development of an integrated understanding of the complex structure and dynamics of the neutral and plasma species in the atmosphere of Hale-Bopp in particular and comets in general. The H2O production rate of Hale-Bopp is determined and, together with the other information related to the structure and dynamics of the neutral and plasma atmospheres obtained in this study, provide critical information important for a wide variety of research conducted by other groups.

The contribution of electron collisions to rotational excitations of cometary water

NASA Technical Reports Server (NTRS)

Xie, Xingfa; Mumma, Michael J.

1992-01-01

The e-H2O collisional rate for exciting rotational transitions in cometary water is evaluated for conditions found in comet Halley during the Giotto spacecraft encounter. In the case of the O(sub 00) yields 1(sub 11) rotational transition, the e-H2O collisional rate exceeds that for excitation by neutral-neutral collisions at distances exceeding 3000 km from the cometary nucleus. Thus, the rotational temperature of the water molecule in the intermediate coma may be controlled by collisions with electrons rather than with neutral collisions, and the rotational temperature retrieved from high resolution infrared spectra of water in comet Halley may reflect electron temperatures rather than neutral gas temperature in the intermediate coma.

Evaluation of Cytotoxicity and Genotoxicity of Acacia aroma Leaf Extracts

PubMed Central

Mattana, C. M.; Cangiano, M. A.; Alcaráz, L. E.; Sosa, A.; Escobar, F.; Sabini, C.; Sabini, L.; Laciar, A. L.

2014-01-01

Acacia aroma, native plant from San Luis, Argentina, is commonly used as antiseptic and for healing of wounds. The present study was conducted to investigate the in vitro cytotoxicity and genotoxicity of hot aqueous extract (HAE) and ethanolic extract (EE) of A. aroma. The cytotoxic activity was assayed by neutral red uptake assay on Vero cell. Cell treatment with a range from 100 to 5000 μg/mL of HAE and EE showed that 500 μg/mL and 100 μg/mL were the maximum noncytotoxic concentrations, respectively. The CC50 was 658 μg/mL for EE and 1020 μg/mL for HAE. The genotoxicity was tested by the single-cell gel electrophoresis comet assay. The results obtained in the evaluation of DNA cellular damage exposed to varied concentrations of the HAE showed no significant genotoxic effect at range of 1–20 mg/mL. The EE at 20 mg/mL showed moderate genotoxic effect related to the increase of the DNA percentage contained in tail of the comet; DNA was classified in category 2. At concentrations below 5 mg/mL, the results of cytotoxicity and genotoxicity of aqueous and ethanolic extracts of Acacia aroma guarantee the safety at cell and genomic level. However further studies are needed for longer periods including animal models to confirm the findings. PMID:25530999

Vehicle and positive control values from the in vivo rodent comet assay and biomonitoring studies using human lymphocytes: historical database and influence of technical aspects.

PubMed

Pant, Kamala; Springer, S; Bruce, S; Lawlor, T; Hewitt, N; Aardema, M J

2014-10-01

There is increased interest in the in vivo comet assay in rodents as a follow-up approach for determining the biological relevance of chemicals that are genotoxic in in vitro assays. This is partly because, unlike other assays, DNA damage can be assessed in this assay in virtually any tissue. Since background levels of DNA damage can vary with the species, tissue, and cell processing method, a robust historical control database covering multiple tissues is essential. We describe extensive vehicle and positive control data for multiple tissues from rats and mice. In addition, we report historical data from control and genotoxin-treated human blood. Technical issues impacting comet results are described, including the method of cell preparation and freezing. Cell preparation by scraping (stomach and other GI tract organs) resulted in higher % tail DNA than mincing (liver, spleen, kidney etc) or direct collection (blood or bone marrow). Treatment with the positive control genotoxicant, ethyl methanesulfonate (EMS) in rats and methyl methanesulfonate in mice, resulted in statistically significant increases in % tail DNA. Background DNA damage was not markedly increased when cell suspensions were stored frozen prior to preparing slides, and the outcome of the assay was unchanged (EMS was always positive). In conclusion, historical data from our laboratory for the in vivo comet assay for multiple tissues from rats and mice, as well as human blood show very good reproducibility. These data and recommendations provided are aimed at contributing to the design and proper interpretation of results from comet assays. © 2014 Wiley Periodicals, Inc.

Effective ion speeds at ˜200-250 km from comet 67P/Churyumov-Gerasimenko near perihelion

NASA Astrophysics Data System (ADS)

Vigren, E.; André, M.; Edberg, N. J. T.; Engelhardt, I. A. D.; Eriksson, A. I.; Galand, M.; Goetz, C.; Henri, P.; Heritier, K.; Johansson, F. L.; Nilsson, H.; Odelstad, E.; Rubin, M.; Stenberg-Wieser, G.; Tzou, C.-Y.; Vallières, X.

2017-07-01

In 2015 August, comet 67P/Churyumov-Gerasimenko, the target comet of the ESA Rosetta mission, reached its perihelion at ˜1.24 au. Here, we estimate for a three-day period near perihelion, effective ion speeds at distances ˜200-250 km from the nucleus. We utilize two different methods combining measurements from the Rosetta Plasma Consortium (RPC)/Mutual Impedance Probe with measurements either from the RPC/Langmuir Probe or from the Rosetta Orbiter Spectrometer for Ion and Neutral Analysis (ROSINA)/Comet Pressure Sensor (COPS) (the latter method can only be applied to estimate the effective ion drift speed). The obtained ion speeds, typically in the range 2-8 km s-1, are markedly higher than the expected neutral outflow velocity of ˜1 km s-1. This indicates that the ions were de-coupled from the neutrals before reaching the spacecraft location and that they had undergone acceleration along electric fields, not necessarily limited to acceleration along ambipolar electric fields in the radial direction. For the limited time period studied, we see indications that at increasing distances from the nucleus, the fraction of the ions' kinetic energy associated with radial drift motion is decreasing.

Optical observations of the AMPTE artificial comet and magnetotail barium releases

NASA Technical Reports Server (NTRS)

Hallinan, T. J.; Stenbaek-Nielsen, H.; Brown, N.

1985-01-01

The first AMPTE artificial comet was observed with a low light level television camera operated aboard the NASA CV990 flying out of Moffett Field, California. The comet head, neutral cloud, and comet tail were all observed for four minutes with an unifiltered camera. Brief observations at T + 4 minutes through a 4554A Ba(+) filter confirmed the identification of the structures. The ion cloud expanded along with the neutral cloud at a rate of 2.3 km/sec (diameter) until it reached a final diameter of approx. 170 km at approx. T + 90 s. It also drifted with the neutral cloud until approx. 165 s. By T + 190 s it had reached a steady state velocity of 5.4 km/sec southward. A barium release in the magnetotail was observed from the CV990 in California, Eagle, Alaska, and Fairbanks, Alaska. Over a twenty-five minute period, the center of the barium streak drifted southward (approx. 500 m/sec), upward (24 km/sec) and eastward (approx 1 km/sec) in a nonrotating reference frame. An all-sky TV at Eagle showed a single auroral arc in the far North during this period.

THE PLASMA ENVIRONMENT IN COMETS OVER A WIDE RANGE OF HELIOCENTRIC DISTANCES: APPLICATION TO COMET C/2006 P1 (MCNAUGHT)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shou, Y.; Combi, M.; Gombosi, T.

2015-08-20

On 2007 January 12, comet C/2006 P1 (McNaught) passed its perihelion at 0.17 AU. Abundant remote observations offer plenty of information on the neutral composition and neutral velocities within 1 million kilometers of the comet nucleus. In early February, the Ulysses spacecraft made an in situ measurement of the ion composition, plasma velocity, and magnetic field when passing through the distant ion tail and the ambient solar wind. The measurement by Ulysses was made when the comet was at around 0.8 AU. With the constraints provided by remote and in situ observations, we simulated the plasma environment of Comet C/2006more » P1 (McNaught) using a multi-species comet MHD model over a wide range of heliocentric distances from 0.17 to 1.75 AU. The solar wind interaction of the comet at various locations is characterized and typical subsolar standoff distances of the bow shock and contact surface are presented and compared to analytic solutions. We find the variation in the bow shock standoff distances at different heliocentric distances is smaller than the contact surface. In addition, we modified the multi-species model for the case when the comet was at 0.7 AU and achieved comparable water group ion abundances, proton densities, plasma velocities, and plasma temperatures to the Ulysses/SWICS and SWOOPS observations. We discuss the dominating chemical reactions throughout the comet-solar wind interaction region and demonstrate the link between the ion composition near the comet and in the distant tail as measured by Ulysses.« less

[Endonuclease modified comet assay for oxidative DNA damage induced by detection of genetic toxicants].

PubMed

Zhao, Jian; Li, Hongli; Zhai, Qingfeng; Qiu, Yugang; Niu, Yong; Dai, Yufei; Zheng, Yuxin; Duan, Huawei

2014-03-01

The aim of this study was to investigate the use of the lesion-specific endonucleases-modified comet assay for analysis of DNA oxidation in cell lines. DNA breaks and oxidative damage were evaluated by normal alkaline and formamidopyrimidine-DNA-glycosylase (FPG) modified comet assays. Cytotoxicity were assessed by MTT method. The human bronchial epithelial cell (16HBE) were treated with benzo (a) pyrene (B(a)P), methyl methanesulfonate (MMS), colchicine (COL) and vincristine (VCR) respectively, and the dose is 20 µmol/L, 25 mg/ml, 5 mg/L and 0.5 mg/L for 24 h, respectively. Oxidative damage was also detected by levels of reactive oxygen species in treated cells. Four genotoxicants give higher cytotoxicity and no significant changes on parameters of comet assay treated by enzyme buffer. Cell survival rate were (59.69 ± 2.60) %, (54.33 ± 2.81) %, (53.11 ± 4.00) %, (51.43 ± 3.92) % in four groups, respectively. There was the direct DNA damage induced by test genotoxicants presented by tail length, Olive tail moment (TM) and tail DNA (%) in the comet assay. The presence of FPG in the assays increased DNA migration in treated groups when compared to those without it, and the difference was statistically significant which indicated that the clastogen and aneugen could induce oxidative damage in DNA strand. In the three parameters, the Olive TM was changed most obviously after genotoxicants treatment. In the contrast group, the Olive TM of B(a) P,MMS, COL,VCR in the contrast groups were 22.99 ± 17.33, 31.65 ± 18.86, 19.86 ± 9.56 and 17.02 ± 9.39, respectively, after dealing with the FPG, the Olive TM were 34.50 ± 17.29, 43.80 ± 10.06, 33.10 ± 12.38, 28.60 ± 10.53, increased by 58.94%, 38.48%, 66.86% and 68.21%, respectively (t value was 3.91, 3.89, 6.66 and 3.87, respectively, and all P < 0.05), and the correlation between Olive TM and reactive oxygen species was better than other parameters (r = 0.77, P < 0.05). This study indicates that FPG-comet assay appears more specific for detecting oxidative DNA damage induced by genotoxicants exposure, and the application of comet assay will be expanded. The endonuclease modified comet assay will be used widely in the toxicology and molecular epidemiology study.

Study of the coma of comet 67P/Churyumov-Gerasimenko based on the ROSINA/RTOF instrument onboard Rosetta

NASA Astrophysics Data System (ADS)

Hoang, M.; Garnier, P.; Lasue, J.; Reme, H.; Altwegg, K.; Balsiger, H. R.; Bieler, A. M.; Calmonte, U.; Fiethe, B.; Galli, A.; Gasc, S.; Gombosi, T. I.; Jäckel, A.; Mall, U.; Le Roy, L.; Rubin, M.; Tzou, C. Y.; Waite, J. H., Jr.; Wurz, P.

2015-12-01

The ROSETTA spacecraft of ESA is in the environment of comet 67P/Churyumov-Gerasimenko since August 2014. Among the experiments onboard the spacecraft, the ROSINA experiment (Rosetta Orbiter Spectrometer for Ion and Neutral Analysis) includes two mass spectrometers (DFMS and RTOF) to analyze the composition of neutrals and ions, and a pressure sensor (COPS) to monitor the density and velocity of neutrals in the coma [1]. We will here analyze and discuss the data of the ROSINA/RTOF instrument during the comet escort phase. The Reflectron-type Time-Of-Flight (RTOF) mass spectrometer possesses a wide mass range and a high temporal resolution [1,2]. It was designed to measure cometary neutral gas as well as cometary ions. A detailed description of the main volatiles (H2O, CO2, CO) dynamics and of the heterogeneities of the coma will then be provided. The influence of various parameters on the coma measurements is investigated on a statistical basis, with the parameters being distance to the comet, heliocentric distance, longitude and latitude of nadir point. Our analysis of the northern hemisphere summer season shows the presence of water vapor mostly in the illuminated northern hemisphere near the neck region with cyclic diurnal variations whereas CO2 was confined to the cold southern hemisphere with a more spatially homogeneous composition, in agreement with previous observations of 67P [2] or Hartley 2 [3]. A comparison will also be provided with the COPS total density and DFMS abundance measurements. [1] Balsiger et al., "ROSINA - Rosetta Orbiter Spectrometer for Ion and Neutral Analysis", Space Sci. Rev., 2007. [2] Scherer et al., "A novel principle for an ion mirror design in time-of-flight mass spectrometry," Int. Jou. Mass Spectr., 2006. [3] Hässig et al., "Time variability and heterogeneity in the coma of 67P/Churyumov-Gerasimenko", Science, 2015. [4] A'Hearn et al., "EPOXI at comet Hartley 2", Science, 2011.

Modeling the neutral gas and dust coma of Comet 1P/Halley

NASA Astrophysics Data System (ADS)

Rubin, Martin; Tenishev, Valeriy M.; Combi, Michael R.; Hansen, Kenneth C.; Gombosi, Tamas I.; Altwegg, Kathrin; Balsiger, Hans

2010-05-01

The neutral gas environment of a comet is largely influenced by dissociation of parent molecules created at the surface of the comet and collisions of all the involved species. We compare the results from a kinetic model of the neutral cometary environment with measurements from the Neutral Mass Spectrometer (NMS) and the Dust Impact Detection System (DIDSY) onboard the Giotto spacecraft which flew-by at comet 1P/Halley in 1986. We further show that our model is in good agreement to measurements obtained by the International Ultraviolet Explorer (IUE), sounding rocket experiments, and the International Halley Watch (IHW). The model solves the Boltzmann equation with a Direct Simulation Monte Carlo technique [Tenishev et al. (2008, Astrophys. J., 685, 659-677)] by tracking trajectories of gas molecules and dust grains under the influence of the comet's weak gravity field with momentum exchange among particles modeled in a probabilistic manner. The cometary nucleus is considered to be the source of dust and the parent species (in our model: H2O, CO, H2CO, CO2, CH3OH, C2H6, C2H4, C2H2, HCN, NH3, and CH4) in the coma. Subsequently our model also tracks the corresponding dissociation products (H, H2, O, OH, C, CH, CH2, CH3, N, NH, NH2, C2, C2H, C2H5, CN, and HCO). This work has been supported by JPL subcontract 1266313 under NASA grant NMO710889, NASA planetary atmospheres program grant NNX09AB59G, grant AST-0707283 from the NSF Planetary Astronomy program, and the Swiss National Science Foundation.

Protective effect of grape seed extracts on human lymphocytes: a preliminary study.

PubMed

Szeto, Yim Tong; Lee, Kit Yee; Kalle, Wouter; Pak, Sok Cheon

2013-03-01

Grape seed extracts (GSEs) possess a broad spectrum of antioxidative properties that protects various cells from free radicals and oxidative stress. In this study, the genoprotective effect of GSE on human lymphocytic DNA was studied using standard and lysed cell comet assays. Lymphocytes from 5 healthy subjects were pretreated with GSE in different concentrations. The standard and lysed cell comet assays were performed on treated, untreated, challenged, and unchallenged cells in parallel. Cells were then subjected to an oxidant challenge induced with 5-min exposures to hydrogen peroxide. In the standard comet assay, GSE significantly diminished hydrogen-peroxide-induced DNA damage in a dose-dependent manner. In the lysed cell assay, however, the antioxidant effect was diminished at a higher GSE concentration. Data indicate that the cell membrane might play a role in limiting cellular access to antioxidants, which directly affects the genoprotective or potential pro-oxidant effect of antioxidants on human DNA. Using both standard and lysed cell comet assays in parallel could be a useful way to elucidate the mechanism of protection or damage by antioxidants.

JaCVAM-organized international validation study of the in vivo rodent alkaline comet assay for the detection of genotoxic carcinogens: I. Summary of pre-validation study results.

PubMed

Uno, Yoshifumi; Kojima, Hajime; Omori, Takashi; Corvi, Raffaella; Honma, Masamistu; Schechtman, Leonard M; Tice, Raymond R; Burlinson, Brian; Escobar, Patricia A; Kraynak, Andrew R; Nakagawa, Yuzuki; Nakajima, Madoka; Pant, Kamala; Asano, Norihide; Lovell, David; Morita, Takeshi; Ohno, Yasuo; Hayashi, Makoto

2015-07-01

The in vivo rodent alkaline comet assay (comet assay) is used internationally to investigate the in vivo genotoxic potential of test chemicals. This assay, however, has not previously been formally validated. The Japanese Center for the Validation of Alternative Methods (JaCVAM), with the cooperation of the U.S. NTP Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM)/the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), the European Centre for the Validation of Alternative Methods (ECVAM), and the Japanese Environmental Mutagen Society/Mammalian Mutagenesis Study Group (JEMS/MMS), organized an international validation study to evaluate the reliability and relevance of the assay for identifying genotoxic carcinogens, using liver and stomach as target organs. The ultimate goal of this validation effort was to establish an Organisation for Economic Co-operation and Development (OECD) test guideline. The purpose of the pre-validation studies (i.e., Phase 1 through 3), conducted in four or five laboratories with extensive comet assay experience, was to optimize the protocol to be used during the definitive validation study. Copyright © 2015 Elsevier B.V. All rights reserved.

The effect of gamma radiation on the Common carp (Cyprinus carpio): In vivo genotoxicity assessment with the micronucleus and comet assays.

PubMed

M K, Praveen Kumar; Soorambail K, Shyama; Bhagatsingh Harisingh, Sonaye; D'costa, Avelyno; Ramesh Chandra, Chaubey

2015-10-01

Radioactive wastes may be leached into freshwater, either accidentally or in industrial effluents. We have studied gamma radiation-induced DNA damage in the freshwater fish Cyprinus carpio. Fish were irradiated with 2-10Gy gamma radiation and genotoxic effects in blood cells were studied with the micronucleus (MN) and comet assays. Micronuclei and a dose-dependent increase in comet-tail DNA were seen in dose- and time-dependent studies. The highest % tail DNA was observed at 24h, declining until 72h, which may indicate the repair of radiation-induced DNA single-strand breaks after gamma radiation. However, double-stranded DNA damage may not have been repaired, as indicated by increased micronuclei at later periods. A positive correlation was observed between the comet and micronucleus assay results. This study confirms the mutagenic/genotoxic potential of gamma radiation in the Common carp, as well as the possible combined use of the micronucleus and comet assays for in vivo laboratory studies with fresh-water fish for screening the genotoxic potential of radioactive pollution. Copyright © 2015 Elsevier B.V. All rights reserved.

Two-Tailed Comet Assay (2T-Comet): Simultaneous Detection of DNA Single and Double Strand Breaks.

PubMed

Cortés-Gutiérrez, Elva I; Fernández, José Luis; Dávila-Rodríguez, Martha I; López-Fernández, Carmen; Gosálvez, Jaime

2017-01-01

A modification of the original comet assay was developed for the simultaneous evaluation of DNA single strand breaks (SSBs) and double strand breaks (DSBs) in human spermatozoa. The two-dimensional perpendicular tail comet assay (2T-comet) combines non-denaturing and denaturant conditions to the same sperm nucleoid. In this case, the species-specific deproteinized sperm is first subjected to an electrophoretic field under non-denaturing conditions to mobilize isolated free discrete DNA fragments produced from DSBs; this is then followed by a second electrophoresis running perpendicular to the first one but under alkaline conditions to produce DNA denaturation, exposing SSBs on the same linear DNA chain or DNA fragments flanked by DSBs. This procedure results in a two dimensional comet tail emerging from the core where two types of original DNA affected molecule can be simultaneously discriminated. The 2T-comet is a fast, sensitive, and reliable procedure to distinguish between single and double strand DNA damage within the same cell. It is an innovative method for assessing sperm DNA integrity, which has important implications for human fertility and andrological pathology. This technique may be adapted to assess different DNA break types in other species and other cell types.

Estimates of DNA damage by the comet assay in the direct-developing frog Eleutherodactylus johnstonei (Anura, Eleutherodactylidae)

PubMed Central

Valencia, Laura Carolina; García, Adriana; Ramírez-Pinilla, Martha Patricia; Fuentes, Jorge Luis

2011-01-01

The aim of this study was to use the Comet assay to assess genetic damage in the direct-developing frog Eleutherodactylus johnstonei. A DNA diffusion assay was used to evaluate the effectiveness of alkaline, enzymatic and alkaline/enzymatic treatments for lysing E. johnstonei blood cells and to determine the amount of DNA strand breakage associated with apoptosis and necrosis. Cell sensitivity to the mutagens bleomycin (BLM) and 4-nitro-quinoline-1-oxide (4NQO) was also assessed using the Comet assay, as was the assay reproducibility. Alkaline treatment did not lyse the cytoplasmic and nuclear membranes of E. johnstonei blood cells, whereas enzymatic digestion with proteinase K (40 μg/mL) yielded naked nuclei. The contribution of apoptosis and necrosis (assessed by the DNA diffusion assay) to DNA damage was estimated to range from 0% to 8%. BLM and 4NQO induced DNA damage in E. johnstonei blood cells at different concentrations and exposure times. Dose-effect curves with both mutagens were highly reproducible and showed consistently low coefficients of variation (CV ≤ 10%). The results are discussed with regard to the potential use of the modified Comet assay for assessing the exposure of E. johnstonei to herbicides in ecotoxicological studies. PMID:22215974

Estimates of DNA damage by the comet assay in the direct-developing frog Eleutherodactylus johnstonei (Anura, Eleutherodactylidae).

PubMed

Valencia, Laura Carolina; García, Adriana; Ramírez-Pinilla, Martha Patricia; Fuentes, Jorge Luis

2011-10-01

The aim of this study was to use the Comet assay to assess genetic damage in the direct-developing frog Eleutherodactylus johnstonei. A DNA diffusion assay was used to evaluate the effectiveness of alkaline, enzymatic and alkaline/enzymatic treatments for lysing E. johnstonei blood cells and to determine the amount of DNA strand breakage associated with apoptosis and necrosis. Cell sensitivity to the mutagens bleomycin (BLM) and 4-nitro-quinoline-1-oxide (4NQO) was also assessed using the Comet assay, as was the assay reproducibility. Alkaline treatment did not lyse the cytoplasmic and nuclear membranes of E. johnstonei blood cells, whereas enzymatic digestion with proteinase K (40 μg/mL) yielded naked nuclei. The contribution of apoptosis and necrosis (assessed by the DNA diffusion assay) to DNA damage was estimated to range from 0% to 8%. BLM and 4NQO induced DNA damage in E. johnstonei blood cells at different concentrations and exposure times. Dose-effect curves with both mutagens were highly reproducible and showed consistently low coefficients of variation (CV ≤ 10%). The results are discussed with regard to the potential use of the modified Comet assay for assessing the exposure of E. johnstonei to herbicides in ecotoxicological studies.

Assessment of the in vivo genotoxicity of cadmium chloride, chloroform, and D,L-menthol as coded test chemicals using the alkaline comet assay.

PubMed

Wada, Kunio; Fukuyama, Tomoki; Nakashima, Nobuaki; Matsumoto, Kyomu

2015-07-01

As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM) international validation study of in vivo rat alkaline comet assays, we examined cadmium chloride, chloroform, and D,L-menthol under blind conditions as coded chemicals in the liver and stomach of Sprague-Dawley rats after 3 days of administration. Cadmium chloride showed equivocal responses in the liver and stomach, supporting previous reports of its poor mutagenic potential and non-carcinogenic effects in these organs. Treatment with chloroform, which is a non-genotoxic carcinogen, did not induce DNA damage in the liver or stomach. Some histopathological changes, such as necrosis and degeneration, were observed in the liver; however, they did not affect the comet assay results. D,L-Menthol, a non-genotoxic non-carcinogen, did not induce liver or stomach DNA damage. These results indicate that the comet assay can reflect genotoxic properties under blind conditions. Copyright © 2015 Elsevier B.V. All rights reserved.

Lymphocyte DNA damage in Turkish asphalt workers detected by the comet assay.

PubMed

Bacaksiz, Aysegul; Kayaalti, Zeliha; Soylemez, Esma; Tutkun, Engin; Soylemezoglu, Tulin

2014-01-01

Asphalt has a highly complex structure and it contains several organic compounds including polycyclic aromatic hydrocarbons and heterocyclic compounds. In this study, comet assay was used to detect the DNA damage in blood lymphocytes of 30 workers exposed to asphalt fumes and 30 nonexposed controls. This is the first report on Turkish asphalt workers' investigated DNA damage using the alkaline single cell gel electrophoresis (SCGE). The DNA damage was evaluated by the percentage of DNA in the comet tail (% tail DNA) for each cell. According to our results, workers exposed to asphalt fumes had higher DNA damage than the control group (p < 0.01). The present study showed that asphalt fumes caused a significant increase in DNA damage and the comet assay is a suitable method for determining DNA damage in asphalt workers.

Weak silica nanomaterial-induced genotoxicity can be explained by indirect DNA damage as shown by the OGG1-modified comet assay and genomic analysis.

PubMed

Pfuhler, Stefan; Downs, Thomas R; Allemang, Ashley J; Shan, Yuching; Crosby, Meredith E

2017-01-01

In a previous study, 15-nm silica nanoparticles (NPs) caused small increases in DNA damage in liver as measured in the in vivo comet and micronucleus assays after intravenous administration to rats at their maximum tolerated dose, a worst-case exposure scenario. Histopathological examination supported a particle-induced, tissue damage-mediated inflammatory response. This study used a targeted approach to provide insight into the mode of action (MoA) by examining transcriptional regulation of genes in liver in a time and dose-dependent manner at 1, 2, 4, 8 and 24 h after intravenous administration of 15-nm silica NPs. DNA damage was assessed using the standard comet assay and hOGG1 glycosylase-modified comet assay that also measures oxidative DNA damage. Potassium bromate, an IARC Class 2B carcinogen that specifically operates via an oxidative stress MoA, was used as a positive control for the hOGG1 comet assay and gave a strong signal in its main target organ, the kidney, while showing less activity in liver. Treatment of rats with silica NPs at 50 mg/kg body weight (bw) caused small, statistically insignificant increases in DNA damage in liver measured by the standard comet assay, while a statistically significant increase was observed at 4 h with the hOGG1 comet assay, consistent with a MoA involving reactive oxygen species. Histopathology showed liver damage and neutrophil involvement while genomic analysis and response pattern of key genes involved in inflammation and oxidative stress supported a tissue damage-mediated inflammatory response involving the complement system for removing/phagocytising damaged cells. No changes were observed for histopathology or gene array for the low-dose (5 mg/kg bw) silica NPs. The results of this study confirm our hypothesis that the weak DNA damage observed by silica NPs occurs secondary to inflammation/immune response, indicating that a threshold can be applied in the risk assessment of these materials. © The Author 2016. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

The effect of obstructive sleep apnea on DNA damage and oxidative stress.

PubMed

Kang, Il Gyu; Jung, Joo Hyun; Kim, Seon Tae

2013-06-01

Obstructive sleep apnea syndrome (OSAS) is associated with repeated hypoxia and re-oxygenation. This characteristic of OSAS may cause oxidative stress and DNA damage. However, the link of OSAS with oxidative stress and DNA damage is still controversial. In the current study, we investigated whether OSAS causes DNA damage using alkaline single-cell gel electrophoresis (comet assay) and measuring oxidative stress by monitoring serum malondialdehyde (MDA) levels. From March 2009 to August 2010, 51 patients who underwent polysomnography (PSG) during the night were enrolled in this study. We obtained serum from the patients at 6 AM. DNA damage and oxidative stress were evaluated using a comet assay and measuring serum MDA, respectively. We divided the patients into two groups according to the existence of comets appearing in the comet assay. Group 1 included 44 patients with negative assay results and group 2 consisted of seven patients with positive comet assay findings. We compared the age, gender proportion, PSG data (respiratory disturbance index [RDI], lowest O2 saturation level, and arousal index [AI]), time of disease onset, smoking habits, and serum MDA levels between the two groups. The average age and gender proportion of the two groups were not statistically different (P>0.05). The average of RDI for group 1 was 30.4±18.4 and 8.0±7.7 (P<0.01) for group 2. The average of lowest O2 saturation level for group 1 was 81.2±7.2 and 87.4±6.5 (P<0.05) for group 2. The average AI for group 1 was 32.8±15.1 and 20.8±7.7 (P<0.05) for group 2. Similarly, serum MDA levels of the two groups were not statistically different (P>0.05). No relationship between positive comet assay results and OSAS severity was identified. Results of the current study showed that OSAS was not associated with DNA damage as measured by comet assays or oxidative stress according to serum MDA levels.

Measuring oxidative damage to DNA and its repair with the comet assay.

PubMed

Collins, Andrew R

2014-02-01

Single cell gel electrophoresis, or the comet assay, was devised as a sensitive method for detecting DNA strand breaks, at the level of individual cells. A simple modification, incorporating a digestion of DNA with a lesion-specific endonuclease, makes it possible to measure oxidised bases. With the inclusion of formamidopyrimidine DNA glycosylase to recognise oxidised purines, or Nth (endonuclease III) to detect oxidised pyrimidines, the comet assay has been used extensively in human biomonitoring to monitor oxidative stress, usually in peripheral blood mononuclear cells. There is evidence to suggest that the enzymic approach is more accurate than chromatographic methods, when applied to low background levels of base oxidation. However, there are potential problems of over-estimation (because the enzymes are not completely specific) or under-estimation (failure to detect lesions that are close together). Attempts have been made to improve the inter-laboratory reproducibility of the comet assay. In addition to measuring DNA damage, the assay can be used to monitor the cellular or in vitro repair of strand breaks or oxidised bases. It also has applications in assessing the antioxidant status of cells. In its various forms, the comet assay is now an invaluable tool in human biomonitoring and genotoxicity testing. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn. Copyright © 2013 Elsevier B.V. All rights reserved.

Detoxification of Aflatoxin-Contaminated Maize by Neutral Electrolyzed Oxidizing Water

PubMed Central

Jardon-Xicotencatl, Samantha; Díaz-Torres, Roberto; Marroquín-Cardona, Alicia; Villarreal-Barajas, Tania; Méndez-Albores, Abraham

2015-01-01

Aflatoxins, a group of extremely toxic mycotoxins produced by Aspergillus flavus, A. parasiticus and A. nomius, can occur as natural contaminants of certain agricultural commodities, particularly maize. These toxins have been shown to be hepatotoxic, carcinogenic, mutagenic and cause severe human and animal diseases. The effectiveness of neutral electrolyzed oxidizing water (NEW) on aflatoxin detoxification was investigated in HepG2 cells using several validation methodologies such as the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, the induction of lipid peroxidation, the oxidative damage by means of glutathione modulation, the Ames test and the alkaline Comet assay. Our results showed that, after the aflatoxin-contaminated maize containing 360 ng/g was soaked in NEW (60 mg/L available chlorine, pH 7.01) during 15 min at room temperature, the aflatoxin content did not decrease as confirmed by the immunoaffinity column and ultra performance liquid chromatography methods. Aflatoxin fluorescence strength of detoxified samples was similar to untreated samples. However, aflatoxin-associated cytotoxicity and genotoxicity effects were markedly reduced upon treatment. According to these results, NEW can be effectively used to detoxify aflatoxin-contaminated maize. PMID:26512692

Rosetta/ROSINA observations of the volatiles in the coma of comet 67P/Churyumov-Gerasimenko during the nominal mission

NASA Astrophysics Data System (ADS)

Rubin, M.; Altwegg, K.; Balsiger, H. R.; Berthelier, J. J.; Calmonte, U.; De Keyser, J.; Fiethe, B.; Fuselier, S. A.; Gasc, S.; Gombosi, T. I.; Hässig, M.; Jäckel, A.; Le Roy, L.; Mall, U. A.; Rème, H.; Sémon, T.; Tzou, C. Y.; Wurz, P.

2015-12-01

The European Space Agency's Rosetta spacecraft is in close proximity of comet 67P/Churyumov-Gerasimenko for well over a year now. During this time Rosetta followed the comet from almost 3.5 AU through perihelion at 1.25 AU and away from the Sun again. Part of the scientific payload scrutinizing the comet is the ROSINA experiment, the Rosetta Orbiter Spectrometer for Ion and Neutral Analysis. The suite of instruments consists of the Double Focusing Mass Spectrometer DFMS, the Reflectron Time-Of-Flight mass spectrometer RTOF, and the COmet Pressure Sensor COPS. From the combined measurements by ROSINA, the composition and dynamics of the volatiles in the coma of the comet are derived. On 13 August 2015, comet 67P/Churyumov-Gerasimenko reached perihelion, the point along its orbits that is closest to the Sun. Furthermore equinox occurred in May 2015 leading to a change in season - the previous summer hemisphere is now in winter and vice versa. One of the goals of ROSINA is to track the activity of the comet during its apparition and to investigate potential changes in the chemical composition as the spacecraft orbits around the nucleus. In this presentation we will summarize some key findings obtained during the first year and a half of the nominal mission and present first results comparing the pre- and post perihelion neutral gas coma. The goal of these observations is to gather information about the formation and the composition of the comet and ultimately our early Solar System.

Genotoxicity of doxorubicin in F344 rats by combining the comet assay, flow-cytometric peripheral blood micronucleus test, and pathway-focused gene expression profiling.

PubMed

Manjanatha, Mugimane G; Bishop, Michelle E; Pearce, Mason G; Kulkarni, Rohan; Lyn-Cook, Lascelles E; Ding, Wei

2014-01-01

Doxorubicin (DOX) is an antineoplastic drug effective against many human malignancies. DOX's clinical efficacy is greatly limited because of severe cardiotoxicity. To evaluate if DOX is genotoxic in the heart, ~7-week-old, male F344 rats were administered intravenously 1, 2, and 3 mg/kg bw DOX at 0, 24, 48, and 69 hr and the Comet assays in heart, liver, kidney, and testis and micronucleus (MN) assay in the peripheral blood (PB) erythrocytes using flow cytometry were conducted. Rats were euthanized at 72 hr and PB was removed for the MN assay and single cells were isolated from multiple tissues for the Comet assays. None of the doses of DOX induced a significant DNA damage in any of the tissues examined by the alkaline Comet assay. Contrastingly, the glycosylase enzymes-modified Comet assay showed a significant dose dependent increase in the oxidative DNA damage in the cardiac tissue (P ≤ 0.05). In the liver, only the top dose induced significant increase in the oxidative DNA damage (P ≤ 0.05). The histopathology showed no severe cardiotoxicity but non-neoplastic lesions were present in both untreated and treated samples. A severe toxicity likely occurred in the bone marrow because no viable reticulocytes could be screened for the MN assay. Gene expression profiling of the heart tissues showed a significant alteration in the expression of 11 DNA damage and repair genes. These results suggest that DOX is genotoxic in the heart and the DNA damage may be induced primarily via the production of reactive oxygen species. Copyright © 2013 Wiley Periodicals, Inc.

Comet Assay in Cancer Chemoprevention.

PubMed

Santoro, Raffaela; Ferraiuolo, Maria; Morgano, Gian Paolo; Muti, Paola; Strano, Sabrina

2016-01-01

The comet assay can be useful in monitoring DNA damage in single cells caused by exposure to genotoxic agents, such as those causing air, water, and soil pollution (e.g., pesticides, dioxins, electromagnetic fields) and chemo- and radiotherapy in cancer patients, or in the assessment of genoprotective effects of chemopreventive molecules. Therefore, it has particular importance in the fields of pharmacology and toxicology, and in both environmental and human biomonitoring. It allows the detection of single strand breaks as well as double-strand breaks and can be used in both normal and cancer cells. Here we describe the alkali method for comet assay, which allows to detect both single- and double-strand DNA breaks.

Four-fluid MHD Simulations of the Plasma and Neutral Gas Environment of Comet Churyumov-Gerasimenko Near Perihelio

NASA Astrophysics Data System (ADS)

Huang, Z.; Toth, G.; Gombosi, T. I.; Jia, X.; Rubin, M.; Hansen, K. C.; Fougere, N.; Bieler, A. M.; Shou, Y.; Altwegg, K.; Combi, M. R.; Tenishev, V.

2015-12-01

The neutral and plasma environment is critical in understanding the interaction of comet Churyumov-Gerasimenko (CG), the target of the Rosetta mission, and the solar wind. To serve this need and support the Rosetta mission, we develop a 3-D four fluid model, which is based on BATS-R-US within the SWMF (Space Weather Modeling Framework) that solves the governing multi-fluid MHD equations and the Euler equations for the neutral gas fluid. These equations describe the behavior and interactions of the cometary heavy ions, the solar wind protons, the electrons, and the neutrals. This model incorporates different mass loading processes, including photo and electron impact ionization, charge exchange, dissociative ion-electron recombination, and collisional interactions between different fluids. We simulate the near nucleus plasma and neutral gas environment near perihelion with a realistic shape model of CG and compare our simulation results with Rosetta observations.

Charge and Exchange

NASA Technical Reports Server (NTRS)

2008-01-01

Even though comets are basically giant dirty snowballs, a few years ago they surprised astronomers by emitting X-radiation. These X-rays are not produced by multi-million degree gas (as is often the case) but rather by a process called 'charge exchange'. In this process, ionized atoms (which have lost one or more electrons) which are carried within the solar wind collide with neutral atoms in the comet's coma. The solar wind ion can collide with and capture an electron from the neutral comet atom, and in doing so some of the energy of the collision is observed in the form of X-rays. This produces a glow of X-rays on the sunward side of the comet's atmosphere. Charge exchange can occur in a variety of astrophysical settings, and cometary charge exchange provides astronomers a means to study this process up close. The image above is a pretty picture of comet 73P/Schwassmann-Wachmann 3 passing by the Ring Nebula. This image was obtained by the ultraviolet and optical telescope (UVOT) on the Swift gamma-ray burst hunter. The UVOT observations help astronomers to study the structure and chemistry of the comet, while Swift's X-ray Telescope (XRT) simultaneously monitors the charge exchange process. Comet 73P/Schwassmann-Wachmann 3 is currently in the process of breaking up, and the UVOT observations show important details of how this breakup is occurring.

The development and validation of EpiComet-Chip, a modified high-throughput comet assay for the assessment of DNA methylation status.

PubMed

Townsend, Todd A; Parrish, Marcus C; Engelward, Bevin P; Manjanatha, Mugimane G

2017-08-01

DNA damage and alterations in global DNA methylation status are associated with multiple human diseases and are frequently correlated with clinically relevant information. Therefore, assessing DNA damage and epigenetic modifications, including DNA methylation, is critical for predicting human exposure risk of pharmacological and biological agents. We previously developed a higher-throughput platform for the single cell gel electrophoresis (comet) assay, CometChip, to assess DNA damage and genotoxic potential. Here, we utilized the methylation-dependent endonuclease, McrBC, to develop a modified alkaline comet assay, "EpiComet," which allows single platform evaluation of genotoxicity and global DNA methylation [5-methylcytosine (5-mC)] status of single-cell populations under user-defined conditions. Further, we leveraged the CometChip platform to create an EpiComet-Chip system capable of performing quantification across simultaneous exposure protocols to enable unprecedented speed and simplicity. This system detected global methylation alterations in response to exposures which included chemotherapeutic and environmental agents. Using EpiComet-Chip on 63 matched samples, we correctly identified single-sample hypermethylation (≥1.5-fold) at 87% (20/23), hypomethylation (≥1.25-fold) at 100% (9/9), with a 4% (2/54) false-negative rate (FNR), and 10% (4/40) false-positive rate (FPR). Using a more stringent threshold to define hypermethylation (≥1.75-fold) allowed us to correctly identify 94% of hypermethylation (17/18), but increased our FPR to 16% (7/45). The successful application of this novel technology will aid hazard identification and risk characterization of FDA-regulated products, while providing utility for investigating epigenetic modes of action of agents in target organs, as the assay is amenable to cultured cells or nucleated cells from any tissue. Environ. Mol. Mutagen. 58:508-521, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Ions of Eight Metals from Comet Dust Detected in Mars Atmosphere

NASA Image and Video Library

2014-11-07

These eight graphs present data from the Neutral Gas and Ion Mass Spectrometer on NASA MAVEN orbiter identifying ions of different metals added to the Martian atmosphere shortly after comet C/2013 A1 Siding Spring sped close to Mars.

Influence of experimental conditions on data variability in the liver comet assay.

PubMed

Guérard, M; Marchand, C; Plappert-Helbig, U

2014-03-01

The in vivo comet assay has increasingly been used for regulatory genotoxicity testing in recent years. While it has been demonstrated that the experimental execution of the assay, for example, electrophoresis or scoring, can have a strong impact on the results; little is known on how initial steps, that is, from tissue sampling during necropsy up to slide preparation, can influence the comet assay results. Therefore, we investigated which of the multitude of steps in processing the liver for the comet assay are most critical. All together eight parameters were assessed by using liver samples of untreated animals. In addition, two of those parameters (temperature and storage time of liver before embedding into agarose) were further investigated in animals given a single oral dose of ethyl methanesulfonate at dose levels of 50, 100, and 200 mg/kg, 3 hr prior to necropsy. The results showed that sample cooling emerged as the predominant influence factor, whereas variations in other elements of the procedure (e.g., size of the liver piece sampled, time needed to process the liver tissue post-mortem, agarose temperature, or time of lysis) seem to be of little relevance. Storing of liver samples of up to 6 hr under cooled conditions did not cause an increase in tail intensity. In contrast, storing the tissue at room temperature, resulted in a considerable time-dependent increase in comet parameters. Copyright © 2013 Wiley Periodicals, Inc.

Use of a standardized JaCVAM in vivo rat comet assay protocol to assess the genotoxicity of three coded test compounds; ampicillin trihydrate, 1,2-dimethylhydrazine dihydrochloride, and N-nitrosodimethylamine.

PubMed

McNamee, J P; Bellier, P V

2015-07-01

As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of the in vivo rat alkaline comet assay (comet assay), our laboratory examined ampicillin trihydrate (AMP), 1,2-dimethylhydrazine dihydrochloride (DMH), and N-nitrosodimethylamine (NDA) using a standard comet assay validation protocol (v14.2) developed by the JaCVAM validation management team (VMT). Coded samples were received by our laboratory along with basic MSDS information. Solubility analysis and range-finding experiments of the coded test compounds were conducted for dose selection. Animal dosing schedules, the comet assay processing and analysis, and statistical analysis were conducted in accordance with the standard protocol. Based upon our blinded evaluation, AMP was not found to exhibit evidence of genotoxicity in either the rat liver or stomach. However, both NDA and DMH were observed to cause a significant increase in % tail DNA in the rat liver at all dose levels tested. While acute hepatoxicity was observed for these compounds in the high dose group, in the investigators opinion there were a sufficient number of consistently damaged/measurable cells at the medium and low dose groups to judge these compounds as genotoxic. There was no evidence of genotoxicity from either NDA or DMH in the rat stomach. In conclusion, our laboratory observed increased DNA damage from two blinded test compounds in rat liver (later identified as genotoxic carcinogens), while no evidence of genotoxicity was observed for the third blinded test compound (later identified as a non-genotoxic, non-carcinogen). This data supports the use of a standardized protocol of the in vivo comet assay as a cost-effective alternative genotoxicity assay for regulatory testing purposes. Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.

EVALUATION OF DNA INTEGRITY USING TUNEL AND COMET ASSAY IN HUMAN SEMEN: IMMEDIATE- VERSUS DELAYED-FREEZING

EPA Science Inventory

EVALUATION OF DNA INTEGRITY USING TUNEL AND COMET ASSAY IN HUMAN SEMEN: IMMEDIATE- VERSUS DELAYED-FREEZING
K. Young,* L. Xun,* S. Rothmann,? S. Perreault, ? W. Robbins*
*University of California, Los Angeles, Los Angeles, California; ?Fertility Solutions Inc., Cleveland, ...

Application of DNA comet assay for detection of radiation treatment of grams and pulses.

PubMed

Khan, Hasan M; Khan, Ashfaq A; Khan, Sanaullah

2011-12-01

Several types of whole pulses (green lentils, red lentils, yellow lentils, chickpeas, green peas, cowpeas and yellow peas) and grams (black grams, red grams and white grams) have been investigated for the identification of radiation treatment using microgel electrophoresis of single cells (DNA comet assay). Pulses and grams were exposed to the radiation doses of 0.5, 1.0 and 5 kGy covering the legalized commercial dose range for protection from insect/pest infestations. All irradiated samples showed comet like stretching of fragmented DNA toward anode, which is expected for irradiated samples. Unirradiated samples showed many intact cells/nuclei in form of round stains or with short faint tails, which is typical for unirradiated food samples. The study shows that DNA comet assay can be used as a rapid, inexpensive and highly effective screening test for the detection of radiation treatment of foods, like pulses and grams.

Exposure to pesticide mixtures and DNA damage among rice field workers.

PubMed

Varona-Uribe, Marcela Eugenia; Torres-Rey, Carlos H; Díaz-Criollo, Sonia; Palma-Parra, Ruth Marien; Narváez, Diana María; Carmona, Sandra Patricia; Briceño, Leonardo; Idrovo, Alvaro J

2016-01-01

This study describes the use of pesticides mixtures and their potential association with comet assay results in 223 rice field workers in Colombia. Thirty-one pesticides were quantified in blood, serum, and urine (15 organochlorines, 10 organophosphorus, 5 carbamates, and ethylenethiourea), and the comet assay was performed. Twenty-four (77.42%) pesticides were present in the workers. The use of the maximum-likelihood factor analysis identified 8 different mixtures. Afterwards, robust regressions were used to explore associations between the factors identified and the comet assay. Two groups of mixtures--α-benzene hexachloride (α-BHC), hexachlorobenzene (HCB), and β-BHC (β: 1.21, 95% confidence interval [CI]: 0.33-2.10) and pirimiphos-methyl, malathion, bromophos-methyl, and bromophos-ethyl (β: 11.97, 95% CI: 2.34-21.60)--were associated with a higher percentage of DNA damage and comet tail length, respectively. The findings suggest that exposure to pesticides varies greatly among rice field workers.

Lack of genotoxicity of potassium iodate in the alkaline comet assay and in the cytokinesis-block micronucleus test. Comparison to potassium bromate.

PubMed

Poul, J M; Huet, S; Godard, T; Sanders, P

2004-02-01

Iodine could be added to the diet of human population in the form of iodide or iodate but iodate had not been adequately tested for genotoxicity and carcinogenicity. In the present study, genotoxic effects of potassium iodate were evaluated in vitro using the alkaline comet assay and the cytokinesis-block micronucleus assay on CHO cells and compared to halogenate salt analogues potassium bromate and chlorate and also to their respective reduced forms (potassium iodide, bromide and chloride). The results showed that the comet assay failed to detect the presence of DNA damage after a treatment of cells by potassium iodate for concentrations up to 10 mM. This absence of primary DNA damage was confirmed in the cytokinesis-block micronucleus assay. In the same way, results showed that potassium chlorate as well as potassium iodide, bromide and chloride did not induced DNA damage in the alkaline comet assay for doses up to 10 mM. By contrast, potassium bromate exposure led to an increase in both DNA damage and frequency of micronucleated cells. The repair of bromate-induced DNA damage was incomplete 24 h after the end of treatment. These results seem to indicate that potassium bromate would induce DNA damage by several mechanisms besides oxidative stress.

The comet assay: Reflections on its development, evolution and applications.

PubMed

Singh, Narendra P

2016-01-01

The study of DNA damage and its repair is critical to our understanding of human aging and cancer. This review reflects on the development of a simple technique, now known as the comet assay, to study the accumulation of DNA damage and its repair. It describes my journey into aging research and the need for a method that sensitively quantifies DNA damage on a cell-by-cell basis and on a day-by-day basis. My inspirations, obstacles and successes on the path to developing this assay and improving its reliability and sensitivity are discussed. Recent modifications, applications, and the process of standardizing the technique are also described. What was once untried and unknown has become a technique used around the world for understanding and monitoring DNA damage. The comet assay's use has grown exponentially in the new millennium, as emphasis on studying biological phenomena at the single-cell level has increased. I and others have applied the technique across cell types (including germ cells) and species (including bacteria). As it enters new realms and gains clinical relevance, the comet assay may very well illuminate human aging and its prevention. Copyright © 2016. Published by Elsevier B.V.

Assessing genotoxicity of diuron on Drosophila melanogaster by the wing-spot test and the wing imaginal disk comet assay.

PubMed

Peraza-Vega, Ricardo I; Castañeda-Sortibrán, América N; Valverde, Mahara; Rojas, Emilio; Rodríguez-Arnaiz, Rosario

2017-05-01

The aim of this study was to evaluate the genotoxicity of the herbicide diuron in the wing-spot test and a novel wing imaginal disk comet assay in Drosophila melanogaster. The wing-spot test was performed with standard (ST) and high-bioactivation (HB) crosses after providing chronic 48 h treatment to third instar larvae. A positive dose-response effect was observed in both crosses, but statistically reduced spot frequencies were registered for the HB cross compared with the ST. This latter finding suggests that metabolism differences play an important role in the genotoxic effect of diuron. To verify diuron's ability to produce DNA damage, a wing imaginal disk comet assay was performed after providing 24 h diuron treatment to ST and HB third instar larvae. DNA damage induced by the herbicide had a significantly positive dose-response effect even at very low concentrations in both strains. However, as noted for the wing-spot test, a significant difference between strains was not observed that could be related to the duration of exposure between both assays. A positive correlation between the comet assay and the wing-spot test was found with regard to diuron genotoxicity.

Comet Assay: A Method to Evaluate Genotoxicity of Nano-Drug Delivery System

PubMed Central

Vandghanooni, Somayeh; Eskandani, Morteza

2011-01-01

Introduction Drug delivery systems could induce cellular toxicity as side effect of nanomaterials. The mechanism of toxicity usually involves DNA damage. The comet assay or single cell gel electrophoresis (SCGE) is a sensitive method for detecting strand damages in the DNA of a cell with applications in genotoxicity testing and molecular epidemiology as well as fundamental research in DNA damage and repair. Methods In the current study, we reviewed recent drug delivery researches related to SCGE. Results We found that one preference for choosing the assay is that comet images may result from apoptosis-mediated nuclear fragmentation. This method has been widely used over the last decade in several different areas. Overall cells, such as cultured cells are embedded in agarose on a microscope slide, lysed with detergent, and treated with high salt. Nucleoids are supercoiled DNA form. When the slide is faced to alkaline electrophoresis any breakages present in the DNA cause the supercoiling to relax locally and loops of DNA extend toward the anode as a ‘‘comet tail’’. Conclusion This article provides a relatively comprehensive review upon potentiality of the comet assay for assessment of DNA damage and accordingly it can be used as an informative platform in genotoxicity studies of drug delivery systems. PMID:23678412

Measurement of DNA damage in rat urinary bladder transitional cells: improved selective harvest of transitional cells and detailed Comet assay protocols.

PubMed

Wang, Amy; Robertson, John L; Holladay, Steven D; Tennant, Alan H; Lengi, Andrea J; Ahmed, S Ansar; Huckle, William R; Kligerman, Andrew D

2007-12-01

Urinary bladder transitional epithelium is the main site of bladder cancer, and the use of transitional cells to study carcinogenesis/genotoxicity is recommended over the use of whole bladders. Because the transitional epithelium is only a small fraction of the whole bladder, the alkaline single cell gel electrophoresis assay (Comet assay), which requires only a small number of cells per sample, is especially suitable for measuring DNA damage in transitional cells. However, existed procedures of cell collection did not yield transitional cells with a high purity, and pooling of samples was needed for Comet assay. The goal of this study was to develop an optimized protocol to evaluate DNA damage in the urinary bladder transitional epithelium. This was achieved by an enzymatic stripping method (trypsin-EDTA incubation plus gentle scraping) to selectively harvest transitional cells from rat bladders, and the use of the alkaline Comet assay to detect DNA strand breaks, alkaline labile sites, and DNA-protein crosslinks. Step by step procedures are reported here. Cells collected from a single rat bladder were sufficient for multiple Comet assays. With this new protocol, increases in DNA damage were detected in transitional cells after in vitro exposure to the positive control agents, hydrogen peroxide or formaldehyde. Repair of the induced DNA damage occurred within 4h. This indicated the capacity for DNA repair was maintained in the harvested cells. The new protocol provides a simple and inexpensive method to detect various types of DNA damage and to measure DNA damage repair in urinary bladder transitional cells.

Induction and repair of DNA cross-links induced by sulfur mustard in the A-549 cell line followed by a comet assay.

PubMed

Jost, Petr; Svobodova, Hana; Stetina, Rudolf

2015-07-25

Sulfur mustard is a highly toxic chemical warfare agent with devastating impact on intoxicated tissues. DNA cross-links are probably the most toxic DNA lesions induced in the cell by sulfur mustard. The comet assay is a very sensitive method for measuring DNA damage. In the present study using the A-549 lung cell line, the comet assay protocol was optimized for indirect detection of DNA cross-links induced by sulfur mustard. The method is based on the additional treatment of the assayed cells containing cross-links with the chemical mutagen, styrene oxide. Alkali-labile adducts of styrene oxide cause DNA breaks leading to the formation of comets. A significant dose-dependent reduction of DNA migration of the comet's tail was found after exposing cells to sulfur mustard, indicative of the amount of sulfur mustard induced cross-links. The remarkable decrease of % tail DNA could be observed as early as 5min following exposure to sulfur mustard and the maximal effect was found after 30min, when DNA migration was reduced to the minimum. Sulfur mustard preincubated in culture medium without cells lost its ability to induce cross-links and had a half-life of about 15min. Pre-incubation longer than 30min does not lead to a significant increase in cross-links when applied to cells. However, the amount of cross-links is decreased during further incubation due to repair. The current modification of the comet assay provides a useful tool for detecting DNA cross-links induced by sulfur mustard and could be used for detection of other DNA cross-linking agents such as chemotherapeutic drugs. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Evaluation of DNA-damaging potential of bisphenol A and its selected analogs in human peripheral blood mononuclear cells (in vitro study).

PubMed

Mokra, Katarzyna; Kuźmińska-Surowaniec, Agnieszka; Woźniak, Katarzyna; Michałowicz, Jaromir

2017-02-01

In the present study, we have investigated DNA-damaging potential of BPA and its analogs, i.e. bisphenol S (BPS), bisphenol F (BPF) and bisphenol AF (BPAF) in human peripheral blood mononuclear cells (PBMCs) using the alkaline and neutral versions of the comet assay, which allowed to evaluate DNA single strand-breaks (SSBs) and double strand-breaks (DSBs). The use of the alkaline version of comet assay made also possible to analyze the kinetics of DNA repair in PBMCs after exposure of the cells to BPA or its analogs. We have observed an increase in DNA damage in PBMCs treated with BPA or its analogs in the concentrations ranging from 0.01 to 10 μg/ml after 1 and 4 h incubation. It was noted that bisphenols studied caused DNA damage mainly via SSBs, while DNA fragmentation via double DSBs was low. The strongest changes in DNA damage were provoked by BPA and particularly BPAF, which were capable of inducing SSBs even at 0.01 μg/ml, while BPS caused the lowest changes (only at 10 μg/ml). We have also observed that PBMCs significantly repaired bisphenols-induced DNA damage but they were unable (excluding cells treated with BPS) to repair totally DNA breaks. Copyright © 2016 Elsevier Ltd. All rights reserved.

Search for the 22 GHz water maser emission in selected comets

NASA Astrophysics Data System (ADS)

Cosmovici, C. B.; Pluchino, S.; Montebugnoli, S.; Pogrebenko, S.

2014-06-01

Following the first evidence of planetary water maser emission induced by the collision of comet Shoemaker/Levy with Jupiter and the puzzling detection of the 22 GHz water emission line in Comet Hyakutake we started in the period 2002-2008 systematic observations of selected comets at 22 GHz (1.35 cm) with the aim of clarifying the unusual behavior of the maser line in the cometary “scenario”. Using a fast multichannel spectrometer coupled to the 32 m dish of the Medicina (Bologna, Italy) Radio Telescope we investigated 6 bright or sungrazing comets down to a heliocentric distance of 0.11 AU: 96P/Machholz, 153P/ Ikeya-Zhang, C/2002 V1 (NEAT), C/2002 X5 (Kudo-Fujikawa), C/2002 T7 (Linear), and 73P/Schwassmann-Wachmann 3. All of them, similarly to Comet Hyakutake, demonstrate spectral features that, if real and due to the 1.35 cm water vapor transition, are strongly (up to tens of km/s) shifted relative to the radial velocity of the nucleus and, at least sometimes, seem to be present as two separate peaks. If our interpretation of these spectral peaks is correct, there must be some mechanism of acceleration of neutral water molecules up to the velocities of ions. We discuss here the results achieved and the possible explanation of the chemo-physical constraints. First possible detection of the water maser emission line at 22 GHz in sun-grazing comets Observed puzzling acceleration of neutral water molecules at ion velocities and split of the line in two components. Evidence of plasma-grain interaction in sun-grazing comets. Possible new detections in six peculiar comets.

Double Stranded Sperm DNA Breaks, Measured by Comet Assay, Are Associated with Unexplained Recurrent Miscarriage in Couples without a Female Factor

PubMed Central

Ribas-Maynou, Jordi; García-Peiró, Agustín; Fernandez-Encinas, Alba; Amengual, Maria José; Prada, Elena; Cortés, Pilar; Navarro, Joaquima; Benet, Jordi

2012-01-01

It is known that sperm samples from recurrent pregnancy loss (RPL) couples have an increase in their sperm DNA fragmentation (SDF), but no studies have been performed in order to identify differences between single stranded SDF (ssSDF) and double stranded SDF (dsSDF) in these patients. This could be relevant because the type of DNA damage could have different effects. Semen samples were classified attending their clinical status: 25 fertile donors and 20 RPL patients with at least two unexplained first trimester miscarriages. SDF was analysed using alkaline and neutral Comet assay, SCD test and pulsed-field gel electrophoresis (PFGE), and ROC analysis including data from 105 more infertile patients (n = 150) was performed to establish predictive threshold values. SDF for alkaline and neutral Comet, and the SCD test was analysed in these categories of individuals. Data revealed the presence of two subgroups within fertile donors. The values obtained were 21.10±9.13, 23.35±10.45 and 12.31±4.31, respectively, for fertile donors with low values for both ssSDF and dsSDF; 27.86±12.64, 80.69±12.67 and 12.43±5.22, for fertile donors with low ssSDF and high dsSDF; and 33.61±15.50, 84.64±11.28 and 19.28±6.05, for unexplained RPL patients, also showing a low ssSDF and high dsSDF profile. This latter profile was seen in 85% of unexplained RPL and 33% of fertile donors, suggesting that it may be associated to a male risk factor for undergoing RPL. ROC analysis regarding recurrent miscarriage set the cut-off value at 77.50% of dsDNA SDF. PFGE for low ssSDF and high dsSDF profile samples and positive controls treated with DNase, to induce dsDNA breaks, showed a more intense band of about 48 kb, which fits the toroid model of DNA compaction in sperm, pointing out that some nuclease activity may be affecting their sperm DNA in RPL patients. This work identifies a very specific SDF profile related to the paternal risk of having RPL. PMID:23028579

Optimal dose selection of N-methyl-N-nitrosourea for the rat comet assay to evaluate DNA damage in organs with different susceptibility to cytotoxicity.

PubMed

Kitamoto, Sachiko; Matsuyama, Ryoko; Uematsu, Yasuaki; Ogata, Keiko; Ota, Mika; Yamada, Toru; Miyata, Kaori; Funabashi, Hitoshi; Saito, Koichi

2015-07-01

The in vivo rodent alkaline comet assay (comet assay) is a promising technique to evaluate DNA damage in vivo. However, there is no agreement on a method to evaluate DNA damage in organs where cytotoxicity is observed. As a part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of the comet assay, we examined DNA damage in the liver, stomach, and bone marrow of rats given three oral doses of N-methyl-N-nitrosourea (MNU) up to the maximum tolerated dose based on systemic toxicity. MNU significantly increased the % tail DNA in all the organs. Histopathological analysis showed no cytotoxic effect on the liver, indicating clearly that MNU has a genotoxic potential in the liver. In the stomach, however, the cytotoxic effects were very severe at systemically non-toxic doses. Low-dose MNU significantly increased the % tail DNA even at a non-cytotoxic dose, indicating that MNU has a genotoxic potential also in the stomach. Part of the DNA damage at cytotoxic doses was considered to be a secondary effect of severe cell damage. In the bone marrow, both the % tail DNA and incidence of micronucleated polychromatic erythrocytes significantly increased at non-hematotoxic doses, which were different from the non-cytotoxic doses for liver and stomach. These findings indicate that an optimal dose for detecting DNA damage may vary among organs and that careful attention is required to select an optimum dose for the comet assay based on systemic toxicity such as mortality and clinical observations. The present study shows that when serious cytotoxicity is suggested by increased % hedgehogs in the comet assay, histopathological examination should be included for the evaluation of a positive response. Copyright © 2015 Elsevier B.V. All rights reserved.

Extended atmospheres of outer planet satellites and comets

NASA Technical Reports Server (NTRS)

Smyth, W. H.; Combi, M. R.

1985-01-01

Model analysis of the extended atmospheres of outer planet satellites and comets are discussed. Understanding the neutral hydrogen distribution in the Saturn system concentrated on assessing the spatial dependence of the lifetime of hydrogen atoms and on obtaining appropriately sorted Lyman ALPHA data from the Voyager 1 UVS instrument. Progress in the area of the extended cometary atmospheres included analysis of Pioneer Venus Layman alpha observations of Comet P/Encke with the fully refined hydrogen cloud model, development of the basic carbon and oxygen models, and planning for the Pioneer Venus UVS observations of Comets P/Giacobini-Zinner and P/Halley.

A Dusty Coma Model of Comet Hyakutake

NASA Astrophysics Data System (ADS)

Boice, D. C.; Benkhoff, J.

1996-09-01

We present a multifluid, hydrodynamic model for the gas, dust, and plasma flow in a cometary coma appropriate for Comet Hyakutake. The model accounts for three sources of gas release: sublimation from surface ices, transport of gas from subsurface regions through the surface, and release of gas from dust in the coma. The simulations are based on a spherically symmetric neutral coma model with detailed photo and gas-phase chemistry and dust entrainment by the gas. The model includes a separate energy balance for the electrons, separate flow of the neutral gas, fast neutral atomic and molecular hydrogen, and dust entrainment with fragmentation. The simulations allow a study of how certain features of a cometary coma, e.g., spatial distributions of gas-phase species and dust of various sizes, change with heliocentric distance. Special attention is given to observations of hydrocarbon and sulphur species. In comparison with observations, the model can be used to characterize the environment surrounding Hyakutake and aid in assimilating a variety of diverse observations of this bright comet. A complete description of the model and more extensive results with comparisons to observations where possible will be presented.

Four-fluid MHD Simulations of the Plasma and Neutral Gas Environment of Comet Churyumov-Gerasimenko Near Perihelion

NASA Astrophysics Data System (ADS)

Huang, Z.; Toth, G.; Gombosi, T.; Jia, X.; Rubin, M.; Fougere, N.; Tenishev, V.; Combi, M.; Bieler, A.; Hansen, K.; Shou, Y.; Altwegg, K.

2015-10-01

We develop a 3-D four fluid model to study the plasma environment of comet Churyumov- Gerasimenko (CG), which is the target of the Rosetta mission. Our model is based on BATS-R-US within the SWMF (Space Weather Modeling Framework) that solves the governing multifluid MHD equations and and the Euler equations for the neutral gas fluid. These equations describe the behavior and interactions of the cometary heavy ions, the solar wind protons, the electrons, and the neutrals. This model incorporates mass loading processes, including photo and electron impact ionization, furthermore taken into account are charge exchange, dissociative ion-electron recombination, as well as collisional interactions between different fluids. We simulate the near nucleus plasma and neutral gas environment with a realistic shape model of CG near perihelion and compare our simulation results with Rosetta observations.

The effect of electron collisions on rotational populations of cometary water

NASA Technical Reports Server (NTRS)

Xie, Xingfa; Mumma, Michael J.

1992-01-01

The e-H2O collisional rate for exciting rotational transitions in cometary water is evaluated for conditions found in Comet Halley during the Giotto spacecraft encounter. In the case of the 0(00)-1(11) rotational transition, the e-H2O collisional rate exceeds that for excitation by neutral-neutral collisions at distances exceeding 3000 km from the cometary nucleus. The estimates are based on theoretical and experimental studies of e-H2O collisions, on ion and electron parameters acquired in situ by instruments on the Giotto and Vega spacecraft, and on results obtained from models of the cometary ionosphere. Thus, the rotational temperature of the water molecule in the intermediate coma may be controlled by collisions with electrons rather than with neutral molecules, and the rotational temperature retrieved from high-resolution IR spectra of water in Comet Halley may reflect electron temperatures rather than neutral gas temperatures in the intermediate coma.

ANALYSIS OF DNA DAMAGE AND REPAIR IN SKIN FIBROBLASTS OF INFANT AND OLDER CHILDREN USING THE IN VITRO ALKALINE COMET ASSAY

EPA Science Inventory

ANALYSIS OF DNA DAMAGE AND REPAIR IN SKIN FIBROBLASTS OF INFANT AND OLDER CHILDREN USING THE IN VITRO ALKALINE COMET ASSAY, Alan H. Tennant1, Geremy W. Knapp1 and Andrew D. Kligerman1, 1Environmental Carcinogenesis Division, National Health and Environmental Effects Research Lab...

MUTAGENICITY IN SALMONELLA AND DNA DAMAGE IN THE CHO/COMET ASSAY INDUCED BY NITROHALOMETHANES, A NOVEL CLASS OF DRINKING WATER DISINFECTION BY-PRODUCTS

EPA Science Inventory

Mutagenicity in Salmonella and DNA Damage in the CHO/Comet Assay Induced by Nitrohalomethanes, a Novel Class of Drinking Water Disinfection By-Products.

Halomethanes are a class of drinking water disinfection by-products (DBPs) whose genotoxicity has been studied extensi...

The Use of Bacterial Repair Endonucleases in the Comet Assay.

PubMed

Collins, Andrew R

2017-01-01

The comet assay is a sensitive electrophoretic method for measuring DNA breaks at the level of single cells, used widely in genotoxicity experiments, in biomonitoring, and in fundamental research. Its sensitivity and range of application are increased by the incorporation of an extra step, after lysis of agarose-embedded cells, in which the DNA is digested with lesion-specific endonucleases (DNA repair enzymes of bacterial or phage origin). Enzymes with specificity for oxidized purines, oxidized pyrimidines, alkylated bases, UV-induced cyclobutane pyrimidine dimers, and misincorporated uracil have been employed. The additional enzyme-sensitive sites, over and above the strand breaks detected in the standard comet assay, give a quantitative estimate of the number of specific lesions present in the cells.

Detection of irradiated quail meat by using DNA comet assay and evaluation of comets by image analysis

NASA Astrophysics Data System (ADS)

Erel, Yakup; Yazici, Nizamettin; Özvatan, Sumer; Ercin, Demet; Cetinkaya, Nurcan

2009-09-01

A simple technique of microgel electrophoresis of single cells (DNA comet assay) was used to detect DNA comets in irradiated quail meat samples. Obtained DNA comets were evaluated by both photomicrographic and image analysis. Quail meat samples were exposed to radiation doses of 0.52, 1.05, 1.45, 2.00, 2.92 and 4.00 kGy in gamma cell (gammacell 60Co, dose rate 1.31 kGy/h) covering the permissible limits for enzymatic decay and stored at 2 °C. The cells isolated from muscle (chest, thorax) in cold PBS were analyzed using the DNA comet assay on 1, 2, 3, 4, 7, 8 and 11 day post irradiation. The cells were lysed between 2, 5 and 9 min in 2.5% SDS and electrophorosis was carried out at a voltage of 2 V/cm for 2 min. After propidium iodide staining, the slides were evaluated through a fluorescent microscope. In all irradiated samples, fragmented DNA stretched towards the anode and damaged cells appeared as a comet. All measurement data were analyzed using BS 200 ProP with software image analysis (BS 200 ProP, BAB Imaging System, Ankara, Turkey). The density of DNA in the tails increased with increasing radiation dose. However, in non-irradiated samples, the large molecules of DNA remained relatively intact and there was only minor or no migration of DNA; the cells were round or had very short tails only. The values of tail DNA%, tail length and tail moment were significantly different and identical between 0.9 and 4.0 kGy dose exposure, and also among storage times on day 1, 4 and 8. In conclusion, the DNA Comet Assay EN 13784 standard method may be used not only for screening method for detection of irradiated quail meat depending on storage time and condition but also for the quantification of applied dose if it is combined with image analysis. Image analysis may provide a powerful tool for the evaluation of head and tail of comet intensity related with applied doses.

ON THE ELECTRON-TO-NEUTRAL NUMBER DENSITY RATIO IN THE COMA OF COMET 67P/CHURYUMOV–GERASIMENKO: GUIDING EXPRESSION AND SOURCES FOR DEVIATIONS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vigren, E.; Eriksson, A. I.; Edberg, N. J. T.

2015-10-10

We compute partial photoionization frequencies of H{sub 2}O, CO{sub 2}, and CO, the major molecules in the coma of comet 67P/Churyumov–Gerasimenko, the target comet of the ongoing ESA Rosetta mission. Values are computed from Thermosphere Ionosphere Mesosphere Energy and Dynamics/Solar EUV Experiment solar EUV spectra for 2014 August 1, 2015 March 1, and for perihelion (2015 August, as based on prediction). From the varying total photoionization frequency of H{sub 2}O, as computed from 2014 August 1 to 2015 May 20, we derive a simple analytical expression for the electron-to-neutral number density ratio as a function of cometocentric and heliocentric distance. Themore » underlying model assumes radial movement of the coma constituents and does not account for chemical loss or the presence of electric fields. We discuss various effects/processes that can cause deviations between values from the analytical expression and actual electron-to-neutral number density ratios. The analytical expression is thus not strictly meant as predicting the actual electron-to-neutral number density ratio, but is useful in comparisons with observations as an indicator of processes at play in the cometary coma.« less

Sperm DNA damage output parameters measured by the alkaline Comet assay and their importance.

PubMed

Simon, L; Aston, K I; Emery, B R; Hotaling, J; Carrell, D T

2017-03-01

The alkaline Comet assay has shown high diagnostic value to determine male reproductive health and prognostic ability to predict ART success. Here, spermatozoon was analysed in 47 fertile donors and 238 patients, including 132 couples undergoing ART [semen was collected: Group I - within 3 months of their treatment (n = 79); and Group II - 3 months prior to their treatment (n = 53)]. We introduce four Comet distribution plots (A, B1, B2 and C) by plotting the level of DNA damage (x-axis) and percentage of comets (y-axis). Fertile donors had low mean DNA damage, olive tail moment and per cent of spermatozoa with damage and increased type A plots. Comet parameters were associated with clinical pregnancies in Group I. About 66% of couples with type A distribution plot were successful after ART, whereas couples with type B1, B2 and C distribution plots achieved 56%, 44% and 33% pregnancies respectively. The efficiency of the Comet assay was due to complete decondensation process, where the compact sperm nuclear DNA (28.2 ± 0.2 μm 3 ) is decondensed to ~63 μm 3 (before lysis) and ~1018 μm 3 (after lysis). A combinational analysis of all the Comet output parameters may provide a comprehensive evaluation of patient's reproductive health as these parameters measure different aspects of DNA damage within the spermatozoa. © 2016 Blackwell Verlag GmbH.

Earthworm Comet Assay for Assessing the Risk of Weathered Petroleum Hydrocarbon Contaminated Soils: Need to Look Further than Target Contaminants.

PubMed

Ramadass, Kavitha; Palanisami, Thavamani; Smith, Euan; Mayilswami, Srinithi; Megharaj, Mallavarapu; Naidu, Ravi

2016-11-01

Earthworm toxicity assays contribute to ecological risk assessment and consequently standard toxicological endpoints, such as mortality and reproduction, are regularly estimated. These endpoints are not enough to better understand the mechanism of toxic pollutants. We employed an additional endpoint in the earthworm Eisenia andrei to estimate the pollutant-induced stress. In this study, comet assay was used as an additional endpoint to evaluate the genotoxicity of weathered hydrocarbon contaminated soils containing 520 to 1450 mg hydrocarbons kg -1 soil. Results showed that significantly higher DNA damage levels (two to sixfold higher) in earthworms exposed to hydrocarbon impacted soils. Interestingly, hydrocarbons levels in the tested soils were well below site-specific screening guideline values. In order to explore the reasons for observed toxicity, the contaminated soils were leached with rainwater and subjected to earthworm tests, including the comet assay, which showed no DNA damage. Soluble hydrocarbon fractions were not found originally in the soils and hence no hydrocarbons leached out during soil leaching. The soil leachate's Electrical Conductivity (EC) decreased from an average of 1665 ± 147 to 204 ± 20 µS cm -1 . Decreased EC is due to the loss of sodium, magnesium, calcium, and sulphate. The leachate experiment demonstrated that elevated salinity might cause the toxicity and not the weathered hydrocarbons. Soil leaching removed the toxicity, which is substantiated by the comet assay and soil leachate analysis data. The implication is that earthworm comet assay can be included in future eco (geno) toxicology studies to assess accurately the risk of contaminated soils.

Chemistry in the Dusty Coma of Comet Hale-Bopp

NASA Astrophysics Data System (ADS)

Boice, D. C.; Cochran, A. L.; Disanti, M. A.; Huebner, W. F.

1998-09-01

Recent progress on a multifluid, hydrodynamic model is presented for the dusty gas flow in the inner coma of comet Hale-Bopp at several heliocentric distances. The simulations are based on a 1-D neutral coma model with detailed photo and gas-phase chemistry and dust entrainment by the gas, a separate energy balance for the electrons, separate flow of the neutral gas, fast neutral atomic and molecular hydrogen, and dust entrainment with fragmentation. The model accounts for three sources of gas release: sublimation from surface ices, transport of gas from subsurface regions through the surface, and release of gas from dust in the coma. This permits a consistent study of the importance and strength of each possible source for a variety of gas-phase species. The simulations allow a study of the changes with heliocentric distance of features within a cometary coma, e.g., spatial distributions of gas-phase species and dust of various sizes and the velocity and temperature profiles. In particular, the model is used to probe spatial distributions of gas-phase species (e.g., CN, CH, C_3, C_2, HCN, HNC, CO) and dust, and the velocity and temperature structure to understand the complex gas-phase chemistry that occurs in the inner coma. Comparisons with observations are made where available to characterize the environment surrounding comet Hale-Bopp and to aid in assimilating a variety of diverse observations of this unique comet.

Biomonitoring of agricultural workers exposed to pesticide mixtures in Guerrero state, Mexico, with comet assay and micronucleus test.

PubMed

Carbajal-López, Yolanda; Gómez-Arroyo, Sandra; Villalobos-Pietrini, Rafael; Calderón-Segura, María Elena; Martínez-Arroyo, Amparo

2016-02-01

The aim of this study was to evaluate the genotoxic effect of pesticides in exfoliated buccal cells of workers occupationally exposed in Guerrero, Mexico, using the comet assay and the micronucleus test. The study compared 111 agricultural workers in three rural communities (Arcelia 62, Ajuchitlan 13, and Tlapehuala 36), with 60 non-exposed individuals. All the participants were males. The presence of DNA damage was investigated in the exfoliated buccal cells of study participants with the comet assay and the micronucleus (MN) test; comet tail length was evaluated in 100 nuclei and 3000 epithelial cells of each individual, respectively; other nuclear anomalies such as nuclear buds, karyolysis, karyorrhexis, and binucleate cells were also evaluated. Study results revealed that the tail migration of DNA and the frequency of MN increased significantly in the exposed group, which also showed nuclear anomalies associated with cytotoxic or genotoxic effect. No positive correlation was noted between exposure time and tail length and micronuclei frequencies. No significant effect on genetic damage was observed as a result of age, smoking, and alcohol consumption. The MN and comet assay in exfoliated buccal cells are useful and minimally invasive methods for monitoring genetic damage in individuals exposed to pesticides. This study provided valuable data for establishing the possible risk to human health associated with pesticide exposure.

Direct human DNA protection by Coriolus versicolor (Yunzhi) extract.

PubMed

Szeto, Yim Tong; Lau, Po Chun; Kalle, Wouter; Pak, Sok Cheon

2013-07-01

Scientific evidence has shown Coriolus versicolor (L. ex Fr.) Quel (also known as Yunzhi) has the role of immunomodulator in therapeutic effect. The aim of this in vitro study was to investigate the antioxidative effect of Yunzhi and to explore the mechanisms behind its DNA protection. Commercial Yunzhi extract was dissolved in water and diluted in five concentrations (10(1)-10(5) μg/L) with appropriate buffers. Lymphocytes harvested from three healthy subjects were incubated with Yunzhi extract for 30 min. Cells were then subjected to 5 min oxidant challenge by 45 μM hydrogen peroxide. The standard alkaline comet (SAC) assay and lysed cell comet (LCC) assay were performed in parallel. DNA damage of each treatment was scored under a fluorescence microscope and compared with the cells without Yunzhi pretreatment. U-shaped dose-response was seen in both versions of the comet assay. Yunzhi at 10(4) μg/L demonstrated a genoprotective effect against oxidative damage in the SAC assay (25% decrease in comet score). In the LCC assay, a trend of protection in lymphocytes was observed but it did not reach statistical significance. A direct antioxidant effect of Yunzhi against oxidant challenge on the DNA of lymphocytes was evidenced. The active component in Yunzhi was likely to be membrane permeable.

DNA damage and external lesions in brown bullheads (Ameiurus nebulosus) from contaminated habitats

USGS Publications Warehouse

Yang, X.; Meier, J.; Chang, L.; Rowan, M.; Baumann, P.C.

2006-01-01

The Comet assay was used to compare levels of DNA damage in brown bullheads (Ameiurus nebulosus) collected from three known contaminated locations, the Cuyahoga River (OH, USA), Ashtabula River (OH, USA; both tributaries to Lake Erie, USA), and Ashumet Pond (Cape Cod, MA, USA), with brown bullheads collected from three paired reference sites, Old Woman Creek (OH, USA), Conneaut River (OH, USA; both tributaries to Lake Erie), and Great Herring Pond (mainland MA, USA), respectively. Blood was sampled from each fish, and the Comet assay was conducted on erythrocytes. The assay results demonstrate that fish from the three contaminated sites each suffered higher DNA damage compared with fish from their respective reference sites. The results also show that the genetic damage was associated with the occurrence of external lesions and deformities in fish. The Comet assay is sufficiently sensitive to detect exposure of natural fish populations to environmental levels of genotoxic contaminants. ?? 2006 SETAC.

The comet assay in human biomonitoring.

PubMed

Anderson, Diana; Dhawan, Alok; Laubenthal, Julian

2013-01-01

Human biomonitoring studies aim to identify potential exposures to environmental, occupational, or lifestyle toxicants in human populations and are commonly used by public health decision makers to predict disease risk. The Comet assay measures changes in genomic stability and is one of the most reliable biomarkers to indicate early biological effects, and therefore accepted by various governmental regulatory agencies. The appeal of the Comet assay lies in its relative simplicity, rapidity, sensitivity, and economic efficiency. Furthermore, the assay is known for its broad versatility, as it can be applied to virtually any human cell and easily adapted in order to detect particular biomarkers of interest, such as DNA repair capacity or single- and double-strand breaks. In a standard experiment, isolated single cells are first embedded in agarose, and then lysed in high-salt solutions in order to remove all cellular contents except the DNA attached to a nuclear scaffold. Subsequent electrophoresis results in accumulation of undamaged DNA sequences at the proximity of the nuclear scaffold, while damaged sequences migrate towards the anode. When visualized with fluorochromes, these migrated DNA fragments resemble a comet tail and can be quantified for their intensity and shape according to internationally drafted guidelines.

A Comprehensive Review on Clinical Applications of Comet Assay

PubMed Central

Gunasekarana, Vidya; Chand, Parkash

2015-01-01

Increased levels of DNA damage and ineffective repair mechanisms are the underlying bio-molecular events in the pathogenesis of most of the life-threatening diseases like cancer and degenerative diseases. The sources of DNA damage can be either exogenous or endogenous in origin. Imbalance between the oxidants and antioxidants resulting in increased reactive oxygen species mostly accounts for the endogenously derived attacks on DNA. Among the various methods employed in the estimation of DNA damage, alkaline comet assay is proven to be a relatively simple and versatile tool in the assessment of DNA damage and also in determining the efficacy of DNA repair mechanism. The aim of this article is to review the application of comet assay in the field of medicine towards human biomonitoring, understanding the pathogenesis of cancer and progression of chronic and degenerative diseases, prediction of tumour radio & chemosensitivity and in male infertility. A standardized protocol and analysis system of various variants of comet assay in different types of cells, across the labs will be of useful and reliable clinical tool in the field of Medicine for the estimation of levels of DNA damage and repair mechanisms. PMID:25954633

An improved method for the isolation of rat alveolar type II lung cells: Use in the Comet assay to determine DNA damage induced by cigarette smoke.

PubMed

Dalrymple, Annette; Ordoñez, Patricia; Thorne, David; Dillon, Debbie; Meredith, Clive

2015-06-01

Smoking is a cause of serious diseases, including lung cancer, emphysema, chronic bronchitis and heart disease. DNA damage is thought to be one of the mechanisms by which cigarette smoke (CS) initiates disease in the lung. Indeed, CS induced DNA damage can be measured in vitro and in vivo. The potential of the Comet assay to measure DNA damage in isolated rat lung alveolar type II epithelial cells (AEC II) was explored as a means to include a genotoxicity end-point in rodent sub-chronic inhalation studies. In this study, published AEC II isolation methods were improved to yield viable cells suitable for use in the Comet assay. The improved method reduced the level of basal DNA damage and DNA repair in isolated AEC II. CS induced DNA damage could also be quantified in isolated cells following a single or 5 days CS exposure. In conclusion, the Comet assay has the potential to determine CS or other aerosol induced DNA damage in AEC II isolated from rodents used in sub-chronic inhalation studies. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Assessment of DNA damage in car spray painters exposed to organic solvents by the high-throughput comet assay.

PubMed

Londoño-Velasco, Elizabeth; Martínez-Perafán, Fabián; Carvajal-Varona, Silvio; García-Vallejo, Felipe; Hoyos-Giraldo, Luz Stella

2016-05-01

Occupational exposure as a painter is associated with DNA damage and development of cancer. Comet assay has been widely adopted as a sensitive and quantitative tool for DNA damage assessment at the individual cell level in populations exposed to genotoxics. The aim of this study was to assess the application of the high-throughput comet assay, to determine the DNA damage in car spray painters. The study population included 52 car spray painters and 52 unexposed subjects. A significant increase in the %TDNA median (p <  0.001) was observed in the exposed group in comparison to the unexposed group. Neither age (%TDNA: p =  0.913) nor time of exposure (%TDNA: p = 0.398) were significantly correlated with DNA damage. The car spray painters who consumed alcohol did not show a significant increase in DNA damage compared to nonalcohol consumers (p  > 0.05). The results showed an increase in DNA breaks in car spray painters exposed to organic solvents and paints; furthermore, they demonstrated the application of high-throughput comet assay in an occupational exposure study to genotoxic agents.

Evaluation of p-phenylenediamine, o-phenylphenol sodium salt, and 2,4-diaminotoluene in the rat comet assay as part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiated international validation study of in vivo rat alkaline comet assay.

PubMed

De Boeck, Marlies; van der Leede, Bas-jan; De Vlieger, Kathleen; Geys, Helena; Vynckier, An; Van Gompel, Jacky

2015-07-01

As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiated international validation study of in vivo rat alkaline comet assay (comet assay), p-phenylenediamine dihydrochloride (PPD), o-phenylphenol sodium salt (OPP), and 2,4-diaminotoluene (2,4-DAT), were analyzed in this laboratory as coded test chemicals. Male Sprague-Dawley rats (7-9 weeks of age) were given three oral doses of the test compounds, 24 and 21 h apart and liver and stomach were sampled 3h after the final dose administration. Under the conditions of the test, no increases in DNA damage were observed in liver and stomach with PPD and OPP up to 100 and 1000 mg/kg/day, respectively. 2,4-DAT, a known genotoxic carcinogen, induced a weak but reproducible, dose-related and statistically significant increase in DNA damage in liver cells while no increases were observed in stomach cells. Copyright © 2015 Elsevier B.V. All rights reserved.

Bovine Papillomavirus Clastogenic Effect Analyzed in Comet Assay

PubMed Central

Araldi, R. P.; Melo, T. C.; Diniz, N.; Mazzuchelli-de-Souza, J.; Carvalho, R. F.; Beçak, W.; Stocco, R. C.

2013-01-01

Bovine papillomavirus (BPV) is an oncogenic virus related to serious livestock diseases. Oncoproteins encoded by BPV are involved in several steps of cellular transformation and have been reported as presenting clastogenic effects in peripheral lymphocytes and primary culture cells. The aim of this study was to evaluate the clastogenic potential of BPV types 1, 2, and 4 by comet assay. Peripheral blood was collected from 37 bovines, 32 infected with different levels of papillomatosis (12 animals have no affection) and five calves, virus free (negative control). The viral identification showed presence of more than one virus type in 59.375% of the infected animals. Comet assay was performed according to alkaline technique. The Kruskal-Wallis test showed statistical difference between the negative control group and infected animals (P = 0.0015). The Dunn post hoc test showed difference comparing the infected animals with calves. Mann-Whitney U test verified no difference between animals infected with only one viral type and animals presenting more than one viral type. The comet assay is considered an efficient tool for assessment of damage in the host chromatin due to viral action, specifically highlighting viral activity in blood cells. PMID:23956996

The use of the comet assay in the study of human nutrition and cancer.

PubMed

Wasson, Gillian R; McKelvey-Martin, Valerie J; Downes, C Stephen

2008-05-01

The influence of diet on carcinogenesis is a hugely complex area; not only is the consumption of major dietary factors such as meat, fat and fruits and vegetables associated with increased or decreased risk of a range of cancers but also an increasing number of specific nutrients such as vitamins, minerals and phytochemicals are being proposed as the next 'superfoods' to combat the development of cancer. As well as epidemiological studies to determine the association of these dietary factors with cancer risk, it is also essential to investigate the underlying mechanisms through which these factors may causally influence carcinogenesis. The comet assay provides a relatively simple, cheap and rapid method to examine DNA damage and repair and is, therefore, an ideal biomarker for the study of the effects of nutrition on cancer. This review focuses on the use of the comet assay in studies involving human subjects or human cell lines, which investigate the effects of various nutrients on biomarkers relevant to carcinogenesis, and discusses the potential of the comet assay and its various modifications for use as cancer-related biomarkers suitable for use in nutritional studies.

Moving into advanced nanomaterials. Toxicity of rutile TiO{sub 2} nanoparticles immobilized in nanokaolin nanocomposites on HepG2 cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bessa, Maria João, E-mail: mjbessa8@gmail.com

Immobilization of nanoparticles on inorganic supports has been recently developed, resulting in the creation of nanocomposites. Concerning titanium dioxide nanoparticles (TiO{sub 2} NPs), these have already been developed in conjugation with clays, but so far there are no available toxicological studies on these nanocomposites. The present work intended to evaluate the hepatic toxicity of nanocomposites (C-TiO{sub 2}), constituted by rutile TiO{sub 2} NPs immobilized in nanokaolin (NK) clay, and its individual components. These nanomaterials were analysed by means of FE-SEM and DLS analysis for physicochemical characterization. HepG2 cells were exposed to rutile TiO{sub 2} NPs, NK clay and C-TiO{sub 2}more » nanocomposite, in the presence and absence of serum for different exposure periods. Possible interferences with the methodological procedures were determined for MTT, neutral red uptake, alamar blue (AB), LDH, and comet assays, for all studied nanomaterials. Results showed that MTT, AB and alkaline comet assay were suitable for toxicity analysis of the present materials after slight modifications to the protocol. Significant decreases in cell viability were observed after exposure to all studied nanomaterials. Furthermore, an increase in HepG2 DNA damage was observed after shorter periods of exposure in the absence of serum proteins and longer periods of exposure in their presence. Although the immobilization of nanoparticles in micron-sized supports could, in theory, decrease the toxicity of single nanoparticles, the selection of a suitable support is essential. The present results suggest that NK clay is not the appropriate substrate to decrease TiO{sub 2} NPs toxicity. Therefore, for future studies, it is critical to select a more appropriate substrate for the immobilization of TiO{sub 2} NPs. - Highlights: • Only the MTT and AB assays were found to be suitable for cytotoxicity assessment. • Alkaline comet assay was also appropriate for genotoxicity evaluation. • All nanomaterials decreased the HepG2 cell viability and caused DNA damage. • Nanokaolin is not a suitable clay substrate for the immobilization of TiO{sub 2} NPs. • Further toxicity studies must be performed in other clays to support nanoparticles.« less

Evaluation of a multi-endpoint assay in rats, combining the bone-marrow micronucleus test, the Comet assay and the flow-cytometric peripheral blood micronucleus test.

PubMed

Bowen, Damian E; Whitwell, James H; Lillford, Lucinda; Henderson, Debbie; Kidd, Darren; Mc Garry, Sarah; Pearce, Gareth; Beevers, Carol; Kirkland, David J

2011-05-18

With the publication of revised draft ICH guidelines (Draft ICH S2), there is scope and potential to establish a combined multi-end point in vivo assay to alleviate the need for multiple in vivo assays, thereby reducing time, cost and use of animals. Presented here are the results of an evaluation trial in which the bone-marrow and peripheral blood (via MicroFlow(®) flow cytometry) micronucleus tests (looking at potential chromosome breakage and whole chromosome loss) in developing erythrocytes or young reticulocytes were combined with the Comet assay (measuring DNA strand-breakage), in stomach, liver and blood lymphocytes. This allowed a variety of potential target tissues (site of contact, site of metabolism and peripheral distribution) to be assessed for DNA damage. This combination approach was performed with minimal changes to the standard and regulatory recommended sampling times for the stand-alone assays. A series of eight in vivo genotoxins (2-acetylaminofluorene, benzo[a]pyrene, carbendazim, cyclophosphamide, dimethylnitrosamine, ethyl methanesulfonate, ethyl nitrosourea and mitomycin C), which are known to act via different modes of action (direct- and indirect-acting clastogens, alkylating agents, gene mutagens, cross-linking and aneugenic compounds) were tested. Male rats were dosed at 0, 24 and 45 h, and bone marrow and peripheral blood (micronucleus endpoint), liver, whole blood and stomach (Comet endpoint) were sampled at three hours after the last dose. Comet and micronucleus responses were as expected based on available data for conventional (acute) stand-alone assays. All compounds were detected as genotoxic in at least one of the endpoints. The importance of evaluating both endpoints was highlighted by the uniquely positive responses for certain chemicals (benzo[a]pyrene and 2-acetylaminofluorene) with the Comet endpoint and certain other chemicals (carbendazim and mitomycin C) with the micronucleus endpoint. The data generated from these investigations demonstrate the suitability of the multi-endpoint design. 2011 Elsevier B.V. All rights reserved.

The heterogeneous coma of comet 67P/Churyumov-Gerasimenko as seen by ROSINA: H2O, CO2, and CO from September 2014 to February 2016

NASA Astrophysics Data System (ADS)

Hoang, M.; Altwegg, K.; Balsiger, H.; Beth, A.; Bieler, A.; Calmonte, U.; Combi, M. R.; De Keyser, J.; Fiethe, B.; Fougere, N.; Fuselier, S. A.; Galli, A.; Garnier, P.; Gasc, S.; Gombosi, T.; Hansen, K. C.; Jäckel, A.; Korth, A.; Lasue, J.; Le Roy, L.; Mall, U.; Rème, H.; Rubin, M.; Sémon, T.; Toublanc, D.; Tzou, C.-Y.; Waite, J. H.; Wurz, P.

2017-04-01

Context. The ESA Rosetta mission has been investigating the environment of comet 67P/Churyumov-Gerasimenko (67P) since August 2014. Among the experiments on board the spacecraft, the ROSINA experiment (Rosetta Orbiter Spectrometer for Ion and Neutral Analysis) includes two mass spectrometers to analyse the composition of neutrals and ions and a COmet Pressure Sensor (COPS) to monitor the density and velocity of neutrals in the coma. Aims: We study heterogeneities in the coma during three periods starting in October 2014 (summer in the northern hemisphere) and ending in February 2016 (end of winter in the northern hemisphere). We provide a detailed description of the main volatiles dynamics (H2O, CO2, CO) and their abundance ratios. Methods: We analysed and compared the data of the Reflectron-type Time-Of-Flight (RTOF) mass spectrometer with data from both the Double Focusing Mass Spectrometer (DFMS) and COPS during the comet escort phase. This comparison has demonstrated that the observations performed with each ROSINA sensor are indeed consistent. Furthermore, we used a Direct Simulation Monte Carlo (DSMC) model to compare modelled densitites with in situ detections. Results: Our analysis shows how the active regions of the main volatiles evolve with the seasons with a variability mostly driven by the illumination conditions; this is the case except for an unexpected dichotomy suggesting the presence of a dust layer containing water deposited in the northern hemisphere during previous perihelions hiding the presence of CO2. The influence of various parameters is investigated in detail: distance to the comet, heliocentric distance, longitude and latitude of sub-satellite point, local time, and phase angle.

Evolution of the coma composition at 67P/Churyumov-Gerasimenko as seen by ROSINA/Rosetta from November 2014 to April 2015

NASA Astrophysics Data System (ADS)

Gasc, Sébastien; Altwegg, Kathrin; Balsiger, Hans; Calmonte, Ursina; Galli, André; Jäckel, Annette; Le Roy, Léna; Rubin, Martin; Tzou, Chia-Yu; Wurz, Peter; Berthelier, Jean-Jacques; Fiethe, Björn; Fuselier, Stephen; Gombosi, Tamas; De Keyser, Johan; Mall, Urs; Rème, Henri

2015-04-01

The European Space Agency's Rosetta spacecraft, with the Rosetta Orbiter Spectrometer for Ion and Neutral Analysis (ROSINA) onboard [1], has been following and observing comet 67P/Churyumov-Gerasimenko (67P/C-G) since August 2014. ROSINA has provided new information on the molecular, elemental, and isotopic composition of 67P/C-G's coma [2,3]. ROSINA consists of a pressure sensor (COPS) and two mass spectrometers, the Double Focusing Mass Spectrometer (DFMS) and the Reflectron Time Of Flight mass spectrometer (RTOF). DFMS has a high mass resolution (ca. 3'000 at 1%) and a high sensitivity, whereas RTOF has a wide mass range (from 1 amu/e to >300 amu/e) and a high temporal resolution. Both mass spectrometers are designed to measure cometary neutral gas as well as cometary ions. In this work, we present the first results and discuss the evolution of the composition of the coma measured by ROSINA from November 2014 until the end of March 2015. During this period, Rosetta delivered the lander, then stayed in bound orbits at distances of 20-30 km away from the comet center, and finally performed comet flybys from 10 km up to 250 km away from 67P/C-G. [1] Balsiger, H. et al.: ROSINA-Rosetta Orbiter Spectrometer for Ion and Neutral Analysis, Space Science Reviews, Vol. 128, 745-801, 2007 [2] Altwegg, K. et al.: Comet 67P/Churyumov-Gerasimenko, a true Kuiper belt comet as judged from its D/H in water, Science Express, 2014 [3] Hässig, M. et al.: Time variability and heterogeneity in the coma of 67P/Churyumov-Gerasimenko, Science, in press, 2015

Genotoxicity of waterpipe smoke in buccal cells and peripheral blood leukocytes as determined by comet assay.

PubMed

Al-Amrah, Hadba Jar-Allah; Aboznada, Osama Abdullah; Alam, Mohammad Zubair; ElAssouli, M-Zaki Mustafa; Mujallid, Mohammad Ibrahim; ElAssouli, Sufian Mohamad

2014-12-01

Waterpipe smoke causes DNA damage in peripheral blood leukocytes and in buccal cells of smokers. To determine the exposure effect of waterpipe smoke on buccal cells and peripheral blood leukocytes in regard to DNA damage using comet assay. The waterpipe smoke condensates were analyzed by gas chromatography-mass spectrometry (GC-MS). The study was performed on 20 waterpipe smokers. To perform comet assay on bucaal cells of smokers, 10 µl of cell suspension was mixed with 85 µl of pre-warmed 1% low melting agarose, applied to comet slide and electrophoresed. To analyze the effect of smoke condensate in vitro, 1 ml of peripheral blood was mixed with 10 µl of smoke condensate and subjected for comet assay. The GC-MS analysis revealed the presence of 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4on, nicotine, hydroxymethyl furancarboxaldehyde and 3-ethoxy-4-hydroxybenzaldehyde in the smoke condensates. Waterpipe smoking caused DNA damage in vivo in buccal cells of smokers. The tail moment and tail length in buccal cells of smokers were 186 ± 26 and 456 ± 71, respectively, which are higher than control. The jurak and moassel smoke condensates were found to cause DNA damage in peripheral blood leukocytes. The moassel smoke condensate was more damaging. There is wide misconception that waterpipe smoking is not as harmful as cigarette smoking. This study demonstrated that waterpipe smoke induced DNA damage in exposed cells. Waterpipe smokes cause DNA damage in buccal cells. The smoke condensate of both jurak and moassel caused comet formation suggesting DNA damage in peripheral blood leukocytes.

Evaluation of γ-radiation-induced DNA damage in two species of bivalves and their relative sensitivity using comet assay.

PubMed

Praveen Kumar, M K; Shyama, S K; Sonaye, B S; Naik, U Roshini; Kadam, S B; Bipin, P D; D'costa, A; Chaubey, R C

2014-05-01

Ionizing radiation is known to induce genetic damage in diverse groups of organisms. Under accidental situations, large quantities of radioactive elements get released into the environment and radiation emitted from these radionuclides may adversely affect both the man and the non-human biota. The present study is aimed (a) to know the genotoxic effect of gamma radiation on aquatic fauna employing two species of selected bivalves, (b) to evaluate the possible use of 'Comet assay' for detecting genetic damage in haemocytes of bivalves as a biomarker for environmental biomonitoring and also (c) to compare the relative sensitivity of two species of bivalves viz. Paphia malabarica and Meretrix casta to gamma radiation. The comet assays was optimized and validated using different concentrations (18, 32 and 56 mg/L) of ethyl methanesulfonate (EMS), a direct-acting reference genotoxic agent, to which the bivalves were exposed for various times (24, 48 and 72 h). Bivalves were irradiated (single acute exposure) with 5 different doses (viz. 2, 4, 6, 8 and 10 Gy) of gamma radiation and their genotoxic effects on the haemocytes were studied using the comet assay. Haemolymph was collected from the adductor muscle at 24, 48 and 72 h of both EMS-exposed and irradiated bivalves and comet assay was carried out using standard protocol. A significant increase in DNA damage was observed as indicated by an increase in % tail DNA damage at different concentrations of EMS and all the doses of gamma radiation as compared to controls in both bivalve species. This showed a dose-dependent increase of genetic damage induced in bivalves by EMS as well as gamma radiation. Further, the highest DNA damage was observed at 24h. The damage gradually decreased with time, i.e. was smaller at 48 and 72 h than at 24h post irradiation in both species of bivalves. This may indicate repair of the damaged DNA and/or loss of heavily damaged cells as the post irradiation time advanced. The present study reveals that gamma radiation induces single strand breaks in DNA as measured by alkaline comet assay in bivalves and comet assay serves as a sensitive and rapid method to detect genotoxicity of gamma radiation. This study further indicates that both M. casta and P. malabarica exhibit almost identical sensitivity to gamma radiation as measured by DNA damage. Copyright © 2014 Elsevier B.V. All rights reserved.

Environmental exposure to human carcinogens in teenagers and the association with DNA damage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Franken, Carmen, E-mail: carmen.franken@vito.be

Background: We investigated whether human environmental exposure to chemicals that are labeled as (potential) carcinogens leads to increased (oxidative) damage to DNA in adolescents. Material and methods: Six hundred 14–15-year-old youngsters were recruited all over Flanders (Belgium) and in two areas with important industrial activities. DNA damage was assessed by alkaline and formamidopyrimidine DNA glycosylase (Fpg) modified comet assays in peripheral blood cells and analysis of urinary 8-hydroxydeoxyguanosine (8-OHdG) levels. Personal exposure to potentially carcinogenic compounds was measured in urine, namely: chromium, cadmium, nickel, 1-hydroxypyrene as a proxy for exposure to other carcinogenic polycyclic aromatic hydrocarbons (PAHs), t,t-muconic acid asmore » a metabolite of benzene, 2,5-dichlorophenol (2,5-DCP), organophosphate pesticide metabolites, and di(2-ethylhexyl) phthalate (DEHP) metabolites. In blood, arsenic, polychlorinated biphenyl (PCB) congeners 118 and 156, hexachlorobenzene (HCB), dichlorodiphenyltrichloroethane (DDT) and perfluorooctanoic acid (PFOA) were analyzed. Levels of methylmercury (MeHg) were measured in hair. Multiple linear regression models were used to establish exposure-response relationships. Results: Biomarkers of exposure to PAHs and urinary chromium were associated with higher levels of both 8-OHdG in urine and DNA damage detected by the alkaline comet assay. Concentrations of 8-OHdG in urine increased in relation with increasing concentrations of urinary t,t-muconic acid, cadmium, nickel, 2,5-DCP, and DEHP metabolites. Increased concentrations of PFOA in blood were associated with higher levels of DNA damage measured by the alkaline comet assay, whereas DDT was associated in the same direction with the Fpg-modified comet assay. Inverse associations were observed between blood arsenic, hair MeHg, PCB 156 and HCB, and urinary 8-OHdG. The latter exposure biomarkers were also associated with higher fish intake. Urinary nickel and t,t-muconic acid were inversely associated with the alkaline comet assay. Conclusion: This cross-sectional study found associations between current environmental exposure to (potential) human carcinogens in 14–15-year-old Flemish adolescents and short-term (oxidative) damage to DNA. Prospective follow-up will be required to investigate whether long-term effects may occur due to complex environmental exposures. - Highlights: • Exposure to (potential) carcinogens is associated with (oxidative) damage to DNA. • Most associations of exposures are with urinary 8-OHdG. • 1-Hydroxypyrene and chromium are associated with the comet assay and 8-OHdG. • PFOA is associated with higher levels of DNA damage in the alkaline comet assay.« less

Differential response of DU145 and PC3 prostate cancer cells to ionizing radiation: role of reactive oxygen species, GSH and Nrf2 in radiosensitivity.

PubMed

Jayakumar, Sundarraj; Kunwar, Amit; Sandur, Santosh K; Pandey, Badri N; Chaubey, Ramesh C

2014-01-01

Radioresistance is the major impediment in radiotherapy of many cancers including prostate cancer, necessitating the need to understand the factors contributing to radioresistance in tumor cells. In the present study, the role of cellular redox and redox sensitive transcription factor, Nrf2 in the radiosensitivity of prostate cancer cell lines PC3 and DU145, has been investigated. Differential radiosensitivity of PC3 and DU145 cells was assessed using clonogenic assay, flow cytometry, and comet assay. Their redox status was measured using DCFDA and DHR probes. Expression of Nrf2 and its dependent genes was measured by EMSA and real time PCR. Knockdown studies were done using shRNA transfection. PC3 and DU145 cells differed significantly in their radiosensitivity as observed by clonogenic survival, apoptosis and neutral comet assays. Both basal and inducible levels of ROS were higher in PC3 cells than that of DU145 cells. DU145 cells showed higher level of basal GSH content and GSH/GSSG ratio than that of PC3 cells. Further, significant increase in both basal and induced levels of Nrf2 and its dependent genes was observed in DU145 cells. Knock-down experiments and pharmacological intervention studies revealed the involvement of Nrf2 in differential radio-resistance of these cells. Cellular redox status and Nrf2 levels play a causal role in radio-resistance of prostate cancer cells. The pivotal role Nrf2 has been shown in the radioresistance of tumor cells and this study will further help in exploiting this factor in radiosensitization of other tumor cell types. © 2013.

Genotoxicity of cadmium chloride in the marine gastropod Nerita chamaeleon using comet assay and alkaline unwinding assay.

PubMed

Sarkar, Anupam; Bhagat, Jacky; Ingole, Baban S; Rao, Durga P; Markad, Vijaykumar L

2015-02-01

This paper presents an evaluation of the genotoxic effects of cadmium chloride (CdCl2 ) on marine gastropod, Nerita chamaeleon following the technique of comet assay and the DNA alkaline unwinding assay (DAUA). In this study, the extent of DNA damage in gill cells of N. chamaeleon was measured after in vivo exposure to four different concentrations (10, 25, 50, and 75 µg/L) of CdCl2 . In vitro exposure of hydrogen peroxide (H2 O2 ; 1, 10, 25, and 50 µM) of the gill cells showed a significant increase in the percentage tail DNA, Olive tail moment, and tail length (TL). Significant changes in percentage tail DNA by CdCl2 exposure were observed in all exposed groups of snails with respect to those in control. Exposure to 75 µg/L of CdCl2 produced significant decrease in DNA integrity as measured by DAUA at all duration with respect to control. In vivo exposure to different concentrations of CdCl2 (10, 25, 50, and 75 µg/L) to N. chamaeleon showed considerable increase in DNA damage as observed by both alkaline comet assay and the DAUA. The extent of DNA damage in marine gastropods determined by the application of alkaline comet assay and DAUA clearly indicated the genotoxic responses of marine gastropod, N. chamaeleon to a wide range of cadmium concentration in the marine environment. © 2013 Wiley Periodicals, Inc.

An Overview of the Comet Nucleus TOUR Discovery Mission and a Description of Neutral Gas and Ion Measurements Planned

NASA Technical Reports Server (NTRS)

Mahaffy, Paul; Veverka, Joe; Niemann, Hasso; Harpold, Dan; Chiu, Mary; Reynolds, Edward; Owen, Toby; Kasprzak, Wayne; Patrick, Ed; Raaen, Eric

2001-01-01

The CONTOUR (Comet Nucleus TOUR) Mission led by its Principal Investigator Professor Joseph Veverka of Cornell is presently under development at the Johns Hopkins Applied Physics Laboratory for launch in July of 2002 with a flyby of Comet Encke scheduled for November 3, 2003 at a solar distance of 1.07 au. A robust Whipple dust shield is designed to allow a close nucleus approach distance (less than 150 km). The 2nd nominal CONTOUR target is Comet Schwassmann-Wachmann 3, although the spacecraft can alternately be directed to a new comet if such an interesting target is discovered. CONTOUR contains 4 instruments: an imaging spectrometer (CRISP) developed at APL that will obtain both high resolution nucleus images through 8 filters and IR spectra (800 to 2550 nm) of the nucleus, a narrow field of view forward imager (CFI) to locate the target days before the encounter, a dust composition time of flight mass spectrometer (CIDA) provided by Dr. J. Kissel and von Hoemer & Sulger, GmbH, and a mass spectrometer (NGIMS) provided by Goddard Space Flight Center to measure neutral gas and ambient ions. Laboratory calibration of the NGIMS has now been completed. NGIMS also includes an in-flight calibration system that we plan to exercise before and after each comet encounter. We will provide an overview of the CONTOUR Mission and discuss more specifically the NGIMS measurement goals for this mission.

In vivo Comet assay--statistical analysis and power calculations of mice testicular cells.

PubMed

Hansen, Merete Kjær; Sharma, Anoop Kumar; Dybdahl, Marianne; Boberg, Julie; Kulahci, Murat

2014-11-01

The in vivo Comet assay is a sensitive method for evaluating DNA damage. A recurrent concern is how to analyze the data appropriately and efficiently. A popular approach is to summarize the raw data into a summary statistic prior to the statistical analysis. However, consensus on which summary statistic to use has yet to be reached. Another important consideration concerns the assessment of proper sample sizes in the design of Comet assay studies. This study aims to identify a statistic suitably summarizing the % tail DNA of mice testicular samples in Comet assay studies. A second aim is to provide curves for this statistic outlining the number of animals and gels to use. The current study was based on 11 compounds administered via oral gavage in three doses to male mice: CAS no. 110-26-9, CAS no. 512-56-1, CAS no. 111873-33-7, CAS no. 79-94-7, CAS no. 115-96-8, CAS no. 598-55-0, CAS no. 636-97-5, CAS no. 85-28-9, CAS no. 13674-87-8, CAS no. 43100-38-5 and CAS no. 60965-26-6. Testicular cells were examined using the alkaline version of the Comet assay and the DNA damage was quantified as % tail DNA using a fully automatic scoring system. From the raw data 23 summary statistics were examined. A linear mixed-effects model was fitted to the summarized data and the estimated variance components were used to generate power curves as a function of sample size. The statistic that most appropriately summarized the within-sample distributions was the median of the log-transformed data, as it most consistently conformed to the assumptions of the statistical model. Power curves for 1.5-, 2-, and 2.5-fold changes of the highest dose group compared to the control group when 50 and 100 cells were scored per gel are provided to aid in the design of future Comet assay studies on testicular cells. Copyright © 2014 Elsevier B.V. All rights reserved.

Evaluation of genotoxicity of the acute gamma radiation on earthworm Eisenia fetida using single cell gel electrophoresis technique (Comet assay).

PubMed

Sowmithra, K; Shetty, N J; Jha, S K; Chaubey, R C

2015-12-01

Earthworms (Eisenia fetida) most suitable biological indicators of radioactive pollution. Radiation-induced lesions in DNA can be considered to be molecular markers for early effects of ionizing radiation. Gamma radiation produces a wide spectrum of DNA. Some of these lesions, i.e., DNA strand breaks and alkali labile sites can be detected by the single-cell gel electrophoresis (SCGE) or comet assay by measuring the migration of DNA from immobilized nuclear DNA. E. fetida were exposed to different doses of gamma radiation, i.e., 1, 5, 10, 20, 30, 40 and 50Gy, and comet assay was performed for all the doses along with control at 1, 3 and 5h post irradiation to evaluate the genotoxicity of gamma radiation in this organism. The DNA damage was measured as percentage of comet tail DNA. A significant increase in DNA damage was observed in samples exposed to 5Gy and above, and the increase in DNA damage was dose dependent i.e., DNA damage was increased with increased doses of radiation. The highest DNA damage was noticed at 1h post irradiation and gradually decreased with time, i.e., at 3 and 5h post irradiation. The present study reveals that gamma radiation induces DNA damage in E. fetida and the comet assay is a sensitive and rapid method for its detection to detect genotoxicity of gamma radiation. Copyright © 2015 Elsevier B.V. All rights reserved.

Evolution of Cometary Activity at 67P/Churyumov-Gerasimenko as seen by ROSINA/Rosetta

NASA Astrophysics Data System (ADS)

Jäckel, A.; Altwegg, K.; Balsiger, H.; Calmonte, U.; Gasc, S.; Le Roy, L.; Rubin, M.; Tzou, C. Y.; Wurz, P.; Bieler, A.; Berthelier, J.-J.; Fiethe, B.; Hässig, M.; deKeyser, J.; Mall, U.; Rème, H.

2015-10-01

Since nine months the European Space Agency's spacecraft Rosetta, with the Rosetta Orbiter Spectrometer for Ion and Neutral Analysis (ROSINA) onboard, is in the comet escort phase. ROSINA is a suite of three instruments, consisting of the COmetary Pressure Sensor (COPS), the Double Focusing Mass Spectrometer (DFMS), and the Reflectron-type Time-Of-Flight (RTOF) mass spectrometer [1]. The two mass spectrometers measure in situ the neutral and ionized volatile material in the coma of comet 67P/Churyumov- Gerasimenko (67P/C-G). With COPS we are able to derive the total gas density, bulk velocities and temperatures of the coma.

The formation of magnetic cavities in comets

NASA Technical Reports Server (NTRS)

Klopman, Z.; Eviatar, A.; Goldstein, R.

1992-01-01

In this paper a unidimensional model for the formation of magnetic cavities in comets is presented. This model includes ion-neutral friction, dissociative recombination, photoionization, and thermal energetic ion pressure coupled with a nonconstant velocity profile which was chosen to simulate the flow pattern. The model explains the thermal ion population profile. Conditions under which a cavity may not form are discussed. In the paper the roles of the various processes are studied, and it is shown that focusing on ion-neutral friction as the major process in the creation of the cavity is not in general correct. In the last part of the paper, the limitations of the model are delineated.

Cometary atmospheres: Modeling the spatial distribution of observed neutral radicals

NASA Technical Reports Server (NTRS)

Combi, M. R.

1985-01-01

Progress on modeling the spatial distributions of cometary radicals is described. The Monte Carlo particle-trajectory model was generalized to include the full time dependencies of initial comet expansion velocities, nucleus vaporization rates, photochemical lifetimes and photon emission rates which enter the problem through the comet's changing heliocentric distance and velocity. The effect of multiple collisions in the transition zone from collisional coupling to true free flow were also included. Currently available observations of the spatial distributions of the neutral radicals, as well as the latest available photochemical data were re-evaluated. Preliminary exploratory model results testing the effects of various processes on observable spatial distributions are also discussed.

Ion acoustic waves at comet 67P/Churyumov-Gerasimenko. Observations and computations

NASA Astrophysics Data System (ADS)

Gunell, H.; Nilsson, H.; Hamrin, M.; Eriksson, A.; Odelstad, E.; Maggiolo, R.; Henri, P.; Vallieres, X.; Altwegg, K.; Tzou, C.-Y.; Rubin, M.; Glassmeier, K.-H.; Stenberg Wieser, G.; Simon Wedlund, C.; De Keyser, J.; Dhooghe, F.; Cessateur, G.; Gibbons, A.

2017-04-01

Context. On 20 January 2015 the Rosetta spacecraft was at a heliocentric distance of 2.5 AU, accompanying comet 67P/Churyumov-Gerasimenko on its journey toward the Sun. The Ion Composition Analyser (RPC-ICA), other instruments of the Rosetta Plasma Consortium, and the ROSINA instrument made observations relevant to the generation of plasma waves in the cometary environment. Aims: Observations of plasma waves by the Rosetta Plasma Consortium Langmuir probe (RPC-LAP) can be explained by dispersion relations calculated based on measurements of ions by the Rosetta Plasma Consortium Ion Composition Analyser (RPC-ICA), and this gives insight into the relationship between plasma phenomena and the neutral coma, which is observed by the Comet Pressure Sensor of the Rosetta Orbiter Spectrometer for Ion and Neutral Analysis instrument (ROSINA-COPS). Methods: We use the simple pole expansion technique to compute dispersion relations for waves on ion timescales based on the observed ion distribution functions. These dispersion relations are then compared to the waves that are observed. Data from the instruments RPC-LAP, RPC-ICA and the mutual impedance probe (RPC-MIP) are compared to find the best estimate of the plasma density. Results: We find that ion acoustic waves are present in the plasma at comet 67P/Churyumov-Gerasimenko, where the major ion species is H2O+. The bulk of the ion distribution is cold, kBTI = 0.01 eV when the ion acoustic waves are observed. At times when the neutral density is high, ions are heated through acceleration by the solar wind electric field and scattered in collisions with the neutrals. This process heats the ions to about 1 eV, which leads to significant damping of the ion acoustic waves. Conclusions: In conclusion, we show that ion acoustic waves appear in the H2O+ plasmas at comet 67P/Churyumov-Gerasimenko and how the interaction between the neutral and ion populations affects the wave properties. Computer code for the dispersion analysis is only available at the CDS via anonymous ftp to http://cdsarc.u-strasbg.fr (http://130.79.128.5) or via http://cdsarc.u-strasbg.fr/viz-bin/qcat?J/A+A/600/A3

Leucocytes DNA damage in mice exposed to JS-118 by the comet assay.

PubMed

Zhang, Tao; Hu, Jiye; Zhang, Yuchao; Zhao, Qianfei; Ning, Jun

2011-09-01

JS-118 is an extensively used insecticide in China. The present study investigated the genotoxic effect of JS-118 on whole blood at 24, 48, 72 and 96 h by using alkaline comet assay. Male Kunming mice were given 6.25, 12.5, 25, 50 and 100 mg/kg BW of JS-118 intraperitoneally. A statistically significant increase in all comet parameters indicating DNA damage was observed at 24 h post-treatment (p < 0.05). A clear concentration-dependent increase of DNA damage was revealed as evident by the OTM (arbitrary units), tail length (µm) and tail DNA (%). From 48 h post-treatment, a gradual decrease in mean comet parameters was noted. By 96 h of post-treatment, the mean comet tail length reached control levels indicating repair of damaged DNA. This study on mice showed different DNA damage depending on the concentration of JS-118 and the period of treatment. The present study provided further information of the potential risk of the genetic damage caused by JS-118.

Reliability of plant root comet assay in comparison with human leukocyte comet assay for assessment environmental genotoxic agents.

PubMed

Reis, Gabriela Barreto Dos; Andrade-Vieira, Larissa Fonseca; Moraes, Isabella de Campos; César, Pedro Henrique Souza; Marcussi, Silvana; Davide, Lisete Chamma

2017-08-01

Comet assay is an efficient test to detect genotoxic compounds based on observation of DNA damage. The aim of this work was to compare the results obtained from the comet assay in two different type of cells extracted from the root tips from Lactuca sativa L. and human blood. For this, Spent Pot Liner (SPL), and its components (aluminum and fluoride) were applied as toxic agents. SPL is a solid waste generated in industry from the aluminum mining and processing with known toxicity. Three concentrations of all tested solutions were applied and the damages observed were compared to negative and positive controls. It was observed an increase in the frequency of DNA damage for human leukocytes and plant cells, in all treatments. On human leukocytes, SPL induced the highest percentage of damage, with an average of 87.68%. For root tips cells of L. sativa the highest percentage of damage was detected for aluminum (93.89%). Considering the arbitrary units (AU), the average of nuclei with high levels of DNA fragmentation was significant for both cells type evaluated. The tested cells demonstrated equal effectiveness for detection of the genotoxicity induced by the SPL and its chemical components, aluminum and fluoride. Further, using a unique method, the comet assay, we proved that cells from root tips of Lactuca sativa represent a reliable model to detect DNA damage induced by genotoxic pollutants is in agreement of those observed in human leukocytes as model. So far, plant cells may be suggested as important system to assess the toxicological risk of environmental agents. Copyright © 2017 Elsevier Inc. All rights reserved.

Effects of seven chemicals on DNA damage in the rat urinary bladder: a comet assay study.

PubMed

Wada, Kunio; Yoshida, Toshinori; Takahashi, Naofumi; Matsumoto, Kyomu

2014-07-15

The in vivo comet assay has been used for the evaluation of DNA damage and repair in various tissues of rodents. However, it can give false-positive results due to non-specific DNA damage associated with cell death. In this study, we examined whether the in vivo comet assay can distinguish between genotoxic and non-genotoxic DNA damage in urinary bladder cells, by using the following seven chemicals related to urinary bladder carcinogenesis in rodents: N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), glycidol, 2,2-bis(bromomethyl)-1,3-propanediol (BMP), 2-nitroanisole (2-NA), benzyl isothiocyanate (BITC), uracil, and melamine. BBN, glycidol, BMP, and 2-NA are known to be Ames test-positive and they are expected to produce DNA damage in the absence of cytotoxicity. BITC, uracil, and melamine are Ames test-negative with metabolic activation but have the potential to induce non-specific DNA damage due to cytotoxicity. The test chemicals were administered orally to male Sprague-Dawley rats (five per group) for each of two consecutive days. Urinary bladders were sampled 3h after the second administration and urothelial cells were analyzed by the comet assay and subjected to histopathological examination to evaluate cytotoxicity. In the urinary bladders of rats treated with BBN, glycidol, and BMP, DNA damage was detected. In contrast, 2-NA induced neither DNA damage nor cytotoxicity. The non-genotoxic chemicals (BITC, uracil, and melamine) did not induce DNA damage in the urinary bladders under conditions where some histopathological changes were observed. The results indicate that the comet assay could distinguish between genotoxic and non-genotoxic chemicals and that no false-positive responses were obtained. Copyright © 2014 Elsevier B.V. All rights reserved.

Development of cultures of the marine sponge Hymeniacidon perleve for genotoxicity assessment using the alkaline comet assay.

PubMed

Akpiri, Rachael U; Konya, Roseline S; Hodges, Nikolas J

2017-12-01

Sponges are a potential alternative model species to bivalves in pollution biomonitoring and environmental risk assessment in the aquatic ecosystem. In the present study, a novel in vivo exposure sponge culture model was developed from field-collected and cryopreserved sponge (Hymeniacidon perleve) cells to investigate the genotoxic effects of environmentally relevant metals in the laboratory. Sponge cell aggregates were cultured and exposed to noncytotoxic concentrations (0-0.4 mg/L) of cadmium chloride, nickel chloride, and sodium dichromate as quantified by the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and DNA-strand breaks assessed by the comet assay. Reactive oxygen species (ROS) formation was quantified by oxidation of 2',7'-dichlorofluorescin diacetate in sponge cell aggregates exposed to the same concentrations of Cd, Cr, and Ni. There was a statistically significant (p < 0.05) concentration-dependent increase in the level of DNA-strand breaks and ROS formation in all of the metals investigated. To the best of our knowledge, we have utilized for the first time the alkaline comet assay to detect DNA-strand breaks in marine sponge cells and demonstrated that exposure to noncytotoxic concentrations of Cd, Cr, and Ni for 12 h results in a concentration-dependent increase in DNA damage and levels of ROS production. In conclusion, we have developed a novel in vivo model based on culture of cryopreserved sponge cells that is compatible with the alkaline comet assay. Genotoxicity in marine sponges measured by the comet assay technique may be a useful tool for biomonitoring research and risk assessment in aquatic ecosystems. Environ Toxicol Chem 2017;36:3314-3323. © 2017 SETAC. © 2017 SETAC.

DNA damage in hemodialysis patients with chronic kidney disease; a test of the role of diabetes mellitus; a comet assay investigation.

PubMed

Mamur, Sevcan; Unal, Fatma; Altok, Kadriye; Deger, Serpil Muge; Yuzbasioglu, Deniz

2016-04-01

The incidence of chronic kidney disease (CKD) is increasing rapidly. Diabetes mellitus (DM) is the most important cause of CKD. We studied the possible role of DM in CKD patients with respect to DNA damage, as assessed by the comet assay in 60 CKD patients (with or without DM) undergoing hemodialysis and in 26 controls. Effects of other factors, such as age, sex, hypertension, duration of hemodialysis, body mass index (BMI), and levels of hemoglobin (HB), intact parathormone (iPTH), and ferritin (FER), were also examined. Primary DNA damage measured by the comet assay was significantly higher in CKD patients than in controls. Among CKD patients, the following correlations were observed. (1) There was no difference in comet tail length or tail intensity between diabetic and non-diabetic individuals. (2) Age, sex, hemoglobin, hypertension, duration of hemodialysis, and ferritin levels affected neither tail length nor intensity. (3) BMI values above 25kg/m(2) and iPTH levels above 300pg/ml were associated with significantly greater comet tail length. Our results indicate that primary DNA damage is increased in CKD patients undergoing hemodialysis, compared to controls; however, DM had no additional effect. Copyright © 2016. Published by Elsevier B.V.

Inhibitory effect of grapefruit juice on the genotoxicity induced by hydrogen peroxide in human lymphocytes.

PubMed

Razo-Aguilera, G; Baez-Reyes, R; Alvarez-González, I; Paniagua-Pérez, R; Madrigal-Bujaidar, E

2011-11-01

By means of the comet assay we demonstrated a strong effect by hydrogen peroxide (HP) and no damage by grapefruit juice (GJ) in human lymphocytes. Cells exposed to HP and treated with three concentrations of GJ (10-90 min) showed an increase of DNA damage by HP over the control level, and a decrease of such damage by GJ. With the comet assay plus formamidopyrimidine-DNA-glycosylase we found the strongest increase of DNA damage by HP over the control level, and the strongest reduction of such damage by GJ. By applying the comet/FISH method we determined 98% of the p53 gene signals in the comet head of control cells along the experiment (10-90 min), in contrast with about 90% signals in the comet tail of cells exposed to HP. Cells treated with both agents showed a significant, concentration/time dependent return of p53 signals to the head, suggesting enhancement of the gene repair. Finally, with the annexin V assay we found an increase in apoptosis and necrosis by HP, and no effect by GJ; when GJ was added to HP treated cells no modification was observed in regard to apoptosis, although a decrease of necrosis was observed. Copyright © 2011 Elsevier Ltd. All rights reserved.

Time variability and heterogeneity in the coma of 67P/Churyumov-Gerasimenko

NASA Astrophysics Data System (ADS)

Hässig, M.; Altwegg, K.; Balsiger, H.; Bar-Nun, A.; Berthelier, J. J.; Bieler, A.; Bochsler, P.; Briois, C.; Calmonte, U.; Combi, M.; De Keyser, J.; Eberhardt, P.; Fiethe, B.; Fuselier, S. A.; Galand, M.; Gasc, S.; Gombosi, T. I.; Hansen, K. C.; Jäckel, A.; Keller, H. U.; Kopp, E.; Korth, A.; Kührt, E.; Le Roy, L.; Mall, U.; Marty, B.; Mousis, O.; Neefs, E.; Owen, T.; Rème, H.; Rubin, M.; Sémon, T.; Tornow, C.; Tzou, C.-Y.; Waite, J. H.; Wurz, P.

2015-01-01

Comets contain the best-preserved material from the beginning of our planetary system. Their nuclei and comae composition reveal clues about physical and chemical conditions during the early solar system when comets formed. ROSINA (Rosetta Orbiter Spectrometer for Ion and Neutral Analysis) onboard the Rosetta spacecraft has measured the coma composition of comet 67P/Churyumov-Gerasimenko with well-sampled time resolution per rotation. Measurements were made over many comet rotation periods and a wide range of latitudes. These measurements show large fluctuations in composition in a heterogeneous coma that has diurnal and possibly seasonal variations in the major outgassing species: water, carbon monoxide, and carbon dioxide. These results indicate a complex coma-nucleus relationship where seasonal variations may be driven by temperature differences just below the comet surface.

Evaluation of environmental genotoxicity by comet assay in Columba livia.

PubMed

González-Acevedo, Anahi; García-Salas, Juan A; Gosálvez, Jaime; Fernández, José Luis; Dávila-Rodríguez, Martha I; Cerda-Flores, Ricardo M; Méndez-López, Luis F; Cortés-Gutiérrez, Elva I

2016-01-01

The concentrations of recognized or suspected genotoxic and carcinogenic agents found in the air of large cities and, in particular, developing countries, have raised concerns about the potential for chronic health effects in the populations exposed to them. The biomonitoring of environmental genotoxicity requires the selection of representative organisms as "sentinels," as well as the development of suitable and sensitive assays, such as those aimed at assessing DNA damage. The aim of this study was to evaluate DNA damage levels in erythrocytes from Columba livia living in the metropolitan area of Monterrey, Mexico, compared with control animals via comet assay, and to confirm the results via Micronuclei test (MN) and DNA breakage detection-fluorescence in situ hybridization (DBD-FISH). Our results showed a significant increase in DNA migration in animals from the area assayed compared with that observed in control animals sampled in non-contaminated areas. These results were confirmed by MN test and DBD-FISH. In conclusion, these observations confirm that the examination of erythrocytes from Columba livia via alkaline comet assay provides a sensitive and reliable end point for the detection of environmental genotoxicants.

Assessment of occupational genotoxic risk among Brazilian hairdressers.

PubMed

Galiotte, Maíra Precivalle; Kohler, Priscila; Mussi, Gisele; Gattás, Gilka J F

2008-10-01

To evaluate the genotoxic risk to hairdressers exposed daily to chemical substances such as hair dyes, waving and straightening preparations and manicurists' products by the Comet assay test (single-cell gel electrophoresis). The Comet assay was performed on blood samples from 69 female hairdressers (36.4 +/- 10.7 years old) currently employed in 21 different beauty institutes in São Paulo, Brazil, and on 55 female control blood donors (32.6 +/- 10.0 years old) from the São Paulo University Clinical Hospital blood bank. All the control subjects had occupations other than hairdresser. Comet assays were performed by evaluating 100 blood lymphocytes per individual and graded by visual score according to comet tail length. The hairdressers showed a higher frequency of DNA damage revealed by Comet Score (159.8 +/- 71) when compared to the control group (125.4 +/- 64.1), and the difference was statistically significant by the Student's t-test (P = 0.005). Multiple regression analysis showed that in addition to the hairdressers' profession, tobacco use contributed to the higher frequency of cells with comets (P < 0.05). The observed DNA damage could be associated with the hairdressers' occupational environment, where different chemicals are chronically manipulated and inhaled. Considering that this profession in many countries, including Brazil, is not officially regulated, more attention should focus on these professionals not only by legislative bodies but also by multidisciplinary teams able to develop and implement risk prevention and control strategies for chemical, physical and biological agents to which hairdressers are exposed.

Introducing a true internal standard for the Comet assay to minimize intra- and inter-experiment variability in measures of DNA damage and repair

PubMed Central

Zainol, Murizal; Stoute, Julia; Almeida, Gabriela M.; Rapp, Alexander; Bowman, Karen J.; Jones, George D. D.

2009-01-01

The Comet assay (CA) is a sensitive/simple measure of genotoxicity. However, many features of CA contribute variability. To minimize these, we have introduced internal standard materials consisting of ‘reference’ cells which have their DNA substituted with BrdU. Using a fluorescent anti-BrdU antibody, plus an additional barrier filter, comets derived from these cells could be readily distinguished from the ‘test’-cell comets, present in the same gel. In experiments to evaluate the reference cell comets as external and internal standards, the reference and test cells were present in separate gels on the same slide or mixed together in the same gel, respectively, before their co-exposure to X-irradiation. Using the reference cell comets as internal standards led to substantial reductions in the coefficient of variation (CoV) for intra- and inter-experimental measures of comet formation and DNA damage repair; only minor reductions in CoV were noted when the reference and test cell comets were in separate gels. These studies indicate that differences between individual gels appreciably contribute to CA variation. Further studies using the reference cells as internal standards allowed greater significance to be obtained between groups of replicate samples. Ultimately, we anticipate that development will deliver robust quality assurance materials for CA. PMID:19828597

Electron plasma environment at comet Grigg-Skjellerup: General observations and comparison with the environment at comet Halley

NASA Technical Reports Server (NTRS)

Reme, H.; Mazelle, C.; Sauvaud, J. A.; D'Uston, C.; Froment, F.; Lin, R. P.; Anderson, K. A.; Carlson, C. W.; Larson, D. E.; Korth, A.

1993-01-01

The three-dimensional electron spectrometer of the Reme plasma analyzer-complete positive ion, electron and ram negative ion measurements near comet Halley (RPA-COPERNIC) experiment aboard the Giotto spacecraft, although damaged during the comet Halley encounter in March 1986, has provided very new results during the encounter on July 10, 1992, with the weakly active comet Grigg-Skjellerup (G-S). The main characteristic features of the highly structured interaction region extending from approximately 26,500 km inbound to approximately 37,200 km outbound are presented. These results are compared to the results obtained by the same instrument during the Giotto comet Halley fly-by. Despite the large difference in the size of the interaction regions (approximately 60,000 km for G-S, approximately 2000,000 km for Halley) due to 2 orders of magnitude difference in cometary neutral gas production rate, there are striking similarities in the solar wind interactions with the two comets.

Drosophila comet assay: insights, uses, and future perspectives

PubMed Central

Gaivão, Isabel; Sierra, L. María

2014-01-01

The comet assay, a very useful tool in genotoxicity and DNA repair testing, is being applied to Drosophila melanogaster since around 15 years ago, by several research groups. This organism is a valuable model for all kind of processes related to human health, including DNA damage response. The assay has been performed mainly in vivo using different larvae cell types (from brain, midgut, hemolymph, and imaginal disk), but also in vitro with the S2 cell line. Since its first application, it has been used to analyze the genotoxicity and action mechanisms of different chemicals, demonstrating good sensitivity and proving its usefulness. Moreover, it is the only assay that can be used to analyze DNA repair in somatic cells in vivo, comparing the effects of chemicals in different repair strains, and to quantitate repair activities in vitro. Additionally, the comet assay in Drosophila, in vivo and in vitro, has been applied to study the influence of protein overexpression on genome integrity and degradation. Although the assay is well established, it could benefit from some research to determine optimal experimental design to standardize it, and then to allow comparisons among laboratories independently of the chosen cell type. PMID:25221574

Genotoxicity testing of two lead-compounds in somatic cells of Drosophila melanogaster.

PubMed

Carmona, Erico R; Creus, Amadeu; Marcos, Ricard

2011-09-18

The in vivo genotoxic activity of two inorganic lead compounds was studied in Drosophila melanogaster by measurement of two different genetic endpoints. We used the wing-spot test and the comet assay. The comet assay was conducted with larval haemocytes. The results from the wing-spot test showed that neither lead chloride, PbCl(2), nor lead nitrate, Pb(NO(3))(2), were able to induce significant increases in the frequency of mutant spots. In addition, the combined treatments with gamma-radiation and PbCl(2) or Pb(NO(3))(2) did not show significant variations in the frequency of the three categories of mutant spots recorded, compared with the frequency induced by gamma-radiation alone. This seems to indicate that the lead compounds tested do not interact with the repair of the genetic damage induced by ionizing radiation. When the lead compounds were evaluated in the in vivo comet assay with haemocytes, Pb(NO(3))(2) was effective in inducing significant increases of DNA damage with a direct dose-response pattern. These results confirm the usefulness of the comet assay with haemocytes as an in vivo model and support the assumption that there is a genotoxic risk associated with lead exposure. Copyright © 2011 Elsevier B.V. All rights reserved.

A CO2-rich coma model applied to the neutral coma of Comet West

NASA Technical Reports Server (NTRS)

Mitchell, G. F.; Swift, M. B.; Huntress, W. T.

1982-01-01

Models of the cometary coma in which the dominant volatile is CO2 have been constructed for a range of heliocentric distances. Model coma abundances of C2, C3, and CN are compared with the abundances observed in Comet West and are found to be in good agreement. Furthermore, the variation with heliocentric distance of C2, C3, and CN model abundances agree well with the observed variation in Comet West. The present work lends detailed support to a previous suggestion that a substance more volatile than water, such as CO2, controls the evaporation of the nucleus of Comet West. The implications for cometary formation are briefly discussed.

JaCVAM-organized international validation study of the in vivo rodent alkaline comet assay for detection of genotoxic carcinogens: II. Summary of definitive validation study results.

PubMed

Uno, Yoshifumi; Kojima, Hajime; Omori, Takashi; Corvi, Raffaella; Honma, Masamistu; Schechtman, Leonard M; Tice, Raymond R; Beevers, Carol; De Boeck, Marlies; Burlinson, Brian; Hobbs, Cheryl A; Kitamoto, Sachiko; Kraynak, Andrew R; McNamee, James; Nakagawa, Yuzuki; Pant, Kamala; Plappert-Helbig, Ulla; Priestley, Catherine; Takasawa, Hironao; Wada, Kunio; Wirnitzer, Uta; Asano, Norihide; Escobar, Patricia A; Lovell, David; Morita, Takeshi; Nakajima, Madoka; Ohno, Yasuo; Hayashi, Makoto

2015-07-01

The in vivo rodent alkaline comet assay (comet assay) is used internationally to investigate the in vivo genotoxic potential of test chemicals. This assay, however, has not previously been formally validated. The Japanese Center for the Validation of Alternative Methods (JaCVAM), with the cooperation of the U.S. NTP Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM)/the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), the European Centre for the Validation of Alternative Methods (ECVAM), and the Japanese Environmental Mutagen Society/Mammalian Mutagenesis Study Group (JEMS/MMS), organized an international validation study to evaluate the reliability and relevance of the assay for identifying genotoxic carcinogens, using liver and stomach as target organs. The ultimate goal of this exercise was to establish an Organisation for Economic Co-operation and Development (OECD) test guideline. The study protocol was optimized in the pre-validation studies, and then the definitive (4th phase) validation study was conducted in two steps. In the 1st step, assay reproducibility was confirmed among laboratories using four coded reference chemicals and the positive control ethyl methanesulfonate. In the 2nd step, the predictive capability was investigated using 40 coded chemicals with known genotoxic and carcinogenic activity (i.e., genotoxic carcinogens, genotoxic non-carcinogens, non-genotoxic carcinogens, and non-genotoxic non-carcinogens). Based on the results obtained, the in vivo comet assay is concluded to be highly capable of identifying genotoxic chemicals and therefore can serve as a reliable predictor of rodent carcinogenicity. Copyright © 2015 Elsevier B.V. All rights reserved.

Detection of Irradiation Treatment of Foods Using DNA `Comet Assay'

NASA Astrophysics Data System (ADS)

Khan, Hasan M.; Delincée, Henry

1998-06-01

Microgel electrophoresis of single cells (DNA comet assay) has been investigated to detect irradiation treatment of some food samples. These samples of fresh and frozen rainbow trout, red lentil, gram and sliced almonds were irradiated to 1 or 2 kGy using 10 MeV electron beam from a linear accelerator. Rainbow trout samples yielded good results with samples irradiated to 1 or 2 kGy showing fragmentation of DNA and, therefore, longer comets with no intact cells. Unirradiated samples showed shorter comets with a significant number of intact cells. For rainbow trout stored in a freezer for 11 days the irradiated samples can still be discerned by electrophoresis from unirradiated samples, however, the unirradiated trouts also showed some longer comets besides some intact cells. Radiation treatment of red lentils can also be detected by this method, i.e. no intact cells in 1 or 2 kGy irradiated samples and shorter comets and some intact cells in unirradiated samples. However, the results for gram and sliced almond samples were not satisfactory since some intact DNA cells were observed in irradiated samples as well. Probably, incomplete lysis has led to these deviating results.

Application of cytogenetic endpoints and comet assay on human lymphocytes treated with atorvastatin in vitro.

PubMed

Gajski, Goran; Garaj-Vrhovac, Vera

2008-01-01

This study investigated the genotoxic potential of atorvastatin on human lymphocytes using comet assay, structural chromosome aberrations (CA) and sister-chromatid exchange (SCE) analysis. Lymphocyte cultures were treated with a single drug at a concentration of 30.21 ng/mL. For comet assay, cells exposed to atorvastatin for 24 h, 48 h and 72 h were embedded in agarose slides, lysed with alkaline lysis solution and exposed to an electric field. DNA migrated within the agarose and formed comets whose length depends on the amount of DNA damage. For analysis of structural CA, cells were grown on medium for 48 h and for SCE analysis for 72 h. Structural CA did not induce significant damage to the genome, although a higher CA frequency was observed in cells treated with atorvastatin for 3 h, 20 h and 48 h than in control samples. Results of the SCE analysis did show statistically significant differences in the mean SCE number between atorvastatin-exposed and control human lymphocytes and between different exposure times. Comet assay also showed increased DNA damage caused in atorvastatin-exposed human lymphocytes than in corresponding control cells for exposure times of 24 h, 48 h and 72 h for the tail length and for 72 h for the tail moment. Results obtained in this study point to the significance of biological indicators providing information on the primary genome damage after long-term exposure, which can help to establish drug therapeutic concentrations that do not put patients with high blood cholesterol to a greater treatment-related risk.

2 years with comet 67P/Churyumov-Gerasimenko: H2O, CO2, CO as seen by ROSINA RTOF

NASA Astrophysics Data System (ADS)

Hoang, M.; Garnier, P.; Lasue, J.; Reme, H.; Altwegg, K.; Balsiger, H. R.; Bieler, A. M.; Calmonte, U.; Capria, M. T.; Combi, M. R.; De Keyser, J. M.; Fiethe, B.; Fougere, N.; Fuselier, S. A.; Galli, A.; Gasc, S.; Gombosi, T. I.; Hansen, K. C.; Jäckel, A.; Korth, A.; Mall, U.; Migliorini, A.; Rubin, M.; Sémon, T.; Tzou, C. Y.; Waite, J. H., Jr.; Wurz, P.

2017-12-01

The Rosetta space mission investigated comet 67P/Churyumov-Gerasimenko (67P) over two years from August 2014 to September 2016. Onboard the spacecraft, the ROSINA experiment included two mass spectrometers to derive the composition of neutrals and ions, and a COmet Pressure Sensor (COPS) to monitor the density and velocity of the neutrals in the coma. We will here analyse and discuss data from the Reflectron-type Time-Of-Flight instrument during the comet escort phase. The RTOF mass spectrometer possessed a wide mass range and a high temporal resolution (Balsiger et al., 2007). The analysis of 67P/C-G's coma major molecules over the mission showed strong variability of the comet coma's main volatiles concentrations (H2O, CO2, CO) and their relative abundances. The 2 years long Rosetta mission allowed us to observe the seasonal evolution in the atmosphere of 67P, in particular the change occurring during the equinoxes and at perihelion. In this work, we analyze the asymmetry in the outgassing rate before and after the perihelion (13/08/2015), the evolution of abundance ratios through the whole mission, and in particular the behavior of the very volatile CO molecules. Density maps projected on the surface of 67P demonstrate the evolution of the three main coma species after the outbound equinox. We will present first results of our comet nucleus thermal modelling used to simulate the internal structure and temperature evolution of 67P at characteristic surface areas. These results will be compared with the coma composition measurements obtained by ROSINA.

Three dimensional Particle-in-Cell (PIC) simulations of the 67P environment

NASA Astrophysics Data System (ADS)

Divin, Andrey; Deca, Jan; Henri, Pierre; Horanyi, Mihaly; Markidis, Stefano; Lapenta, Giovanni; Olshevsky, Vyacheslav; Eriksson, Anders

2017-04-01

ESA's Rosetta orbiter spacecraft escorted comet 67P/Churyumov-Gerasimenko for two years, carrying 21 scientific instruments. Five of those were dedicated to plasma measurements. The mission revealed for the first time, and in unprecedented detail, the fascinating evolution of a comet and its interaction with our Sun as it races along its 6.45yr elliptical orbit around the Sun. Using a self-consistent 3-D fully kinetic electromagnetic particle-in-cell approach, we focus on the global cometary environment and, in particular, on the collisionless electron-kinetic interaction. We include cometary ions and electrons produced by the ionization of the outgassing cometary atmosphere in addition to the solar wind ion and electron plasma flow. We approximate mass-loading of the cold cometary ion and electron populations using a 1/r relation with distance to the comet with a total neutral production rate of Q = 1026 s-1. Our simulation results disentangle for the first time the kinetic ion and electron dynamics of the solar wind interaction with a weakly outgassing comet. The simulated global structure of the solar wind-comet interaction confirms the results reported in hybrid simulations of the induced cometary magnetosphere. Moreover, we show that cometary and solar wind electrons neutralize the solar wind protons and cometary ions, respectively, in the region of influence around the comet, representing to first order a four-fluid behavior. The electron energy distribution close to the comet is shown to be a mix of cometary and solar wind electrons that appear as, respectively, a thermal and a suprathermal components. Analyzing ion and electron energy distribution functions, and comparing with plasma measurements from ESA's Rosetta mission to comet 67P/Churyumov-Gerasimenko, we conclude that a detailed kinetic treatment of the electron dynamics is critical to fully capture the complex physics of mass-loading plasmas.

Evaluation of 4,4'-diaminodiphenyl ether in the rat comet assay: Part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of in vivo rat alkaline comet assay.

PubMed

Priestley, Catherine C; Walker, Joanne S; O'Donovan, Michael R; Doherty, Ann T

2015-07-01

As a part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of the in vivo rat alkaline comet assay, 4,4'-diaminodiphenyl ether (DPE), a known rodent genotoxic carcinogen, was tested in this laboratory. Sprague Dawley rats (7-9 weeks of age) were given three oral doses of DPE, 24 and 21 h apart and liver or stomach sampled 3h after the final dose. Under the conditions of the test, no increases in DNA damage in liver and stomach were observed with DPE (up to 200 mg/kg/day). A dose-dependent decrease in DNA migration, compared to vehicle controls, was noted for DPE in rat stomach. Further analysis is required to elucidate fully whether this decrease is a consequence of the mode of action or due to the toxicity of DPE. What is perhaps surprising is the inability of the comet assay to detect a known rat genotoxic carcinogen in liver. Further investigation is needed to clarify whether this apparent lack of response results from limited tissue exposure or metabolic differences between species. This finding highlights a need for careful consideration of study design when evaluating assay performance as a measure of in vivo genotoxicity. Copyright © 2015 Elsevier B.V. All rights reserved.

DNA Damage among Wood Workers Assessed with the Comet Assay

PubMed Central

Bruschweiler, Evin Danisman; Wild, Pascal; Huynh, Cong Khanh; Savova-Bianchi, Dessislava; Danuser, Brigitta; Hopf, Nancy B.

2016-01-01

Exposure to wood dust, a human carcinogen, is common in wood-related industries, and millions of workers are occupationally exposed to wood dust worldwide. The comet assay is a rapid, simple, and sensitive method for determining DNA damage. The objective of this study was to investigate the DNA damage associated with occupational exposure to wood dust using the comet assay (peripheral blood samples) among nonsmoking wood workers (n = 31, furniture and construction workers) and controls (n = 19). DNA damage was greater in the group exposed to composite wood products compared to the group exposed to natural woods and controls (P < 0.001). No difference in DNA damage was observed between workers exposed to natural woods and controls (P = 0.13). Duration of exposure and current dust concentrations had no effect on DNA damage. In future studies, workers’ exposures should include cumulative dust concentrations and exposures originating from the binders used in composite wood products. PMID:27398027

Identification of gamma-irradiated papaya, melon and watermelon

NASA Astrophysics Data System (ADS)

Marín-Huachaca, Nélida S.; Mancini-Filho, Jorge; Delincée, Henry; Villavicencio, Anna Lúcia C. H.

2004-09-01

Ionizing radiation can be used to control spoilage microorganisms and to increase the shelf life of fresh fruits and vegetables in replacement for the treatment with chemical fumigants. In order to enforce labelling regulations, methods for detecting the irradiation treatment directly in the produce are required. Recently, a number of detection methods for irradiated food have been adopted by the Codex Comission. A rapid screening method for qualitative detection of irradiation is the DNA Comet Assay. The applicability of the DNA Comet Assay for distinguishing irradiated papaya, melon, and watermelon was evaluated. The samples were treated in a 60Co facility at dose levels of 0.0, 0.5, 0.75, and 1.0kGy. The irradiated samples showed typical DNA fragmentation whereas cells from non-irradiated ones appeared intact. In addition to the DNA Comet Assay also the half-embryo test was applied in melon and watermelon to detect the irradiation treatment.

Genotoxicity of TiO2 nanoparticles assessed by mini-gel comet assay and micronucleus scoring with flow cytometry.

PubMed

Di Bucchianico, Sebastiano; Cappellini, Francesca; Le Bihanic, Florane; Zhang, Yuning; Dreij, Kristian; Karlsson, Hanna L

2017-01-01

The widespread production and use of nanoparticles calls for faster and more reliable methods to assess their safety. The main aim of this study was to investigate the genotoxicity of three reference TiO 2 nanomaterials (NM) within the frame of the FP7-NANoREG project, with a particular focus on testing the applicability of mini-gel comet assay and micronucleus (MN) scoring by flow cytometry. BEAS-2B cells cultured under serum-free conditions were exposed to NM100 (anatase, 50-150nm), NM101 (anatase, 5-8nm) and NM103 (rutile, 20-28nm) for 3, 24 or 48h mainly at concentrations 1-30 μg/ml. In the mini-gel comet assay (eight gels per slide), we included analysis of (i) DNA strand breaks, (ii) oxidised bases (Fpg-sensitive sites) and (iii) light-induced DNA damage due to photocatalytic activity. Furthermore, MN assays were used and we compared the results of more high-throughput MN scoring with flow cytometry to that of cytokinesis-block MN cytome assay scored manually using a microscope. Various methods were used to assess cytotoxic effects and the results showed in general no or low effects at the doses tested. A weak genotoxic effect of the tested TiO 2 materials was observed with an induction of oxidised bases for all three materials of which NM100 was the most potent. When the comet slides were briefly exposed to lab light, a clear induction of DNA strand breaks was observed for the anatase materials, but not for the rutile. This highlights the risk of false positives when testing photocatalytically active materials if light is not properly avoided. A slight increase in MN formation for NM103 was observed in the different MN assays at the lower doses tested (1 and 5 μg/ml). We conclude that mini-gel comet assay and MN scoring using flow cytometry successfully can be used to efficiently study cytotoxic and genotoxic properties of nanoparticles. © The Author 2016. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society.

Genotoxicity of TiO2 nanoparticles assessed by mini-gel comet assay and micronucleus scoring with flow cytometry

PubMed Central

Di Bucchianico, Sebastiano; Cappellini, Francesca; Le Bihanic, Florane; Zhang, Yuning; Dreij, Kristian; Karlsson, Hanna L.

2017-01-01

The widespread production and use of nanoparticles calls for faster and more reliable methods to assess their safety. The main aim of this study was to investigate the genotoxicity of three reference TiO2 nanomaterials (NM) within the frame of the FP7-NANoREG project, with a particular focus on testing the applicability of mini-gel comet assay and micronucleus (MN) scoring by flow cytometry. BEAS-2B cells cultured under serum-free conditions were exposed to NM100 (anatase, 50–150nm), NM101 (anatase, 5–8nm) and NM103 (rutile, 20–28nm) for 3, 24 or 48h mainly at concentrations 1–30 μg/ml. In the mini-gel comet assay (eight gels per slide), we included analysis of (i) DNA strand breaks, (ii) oxidised bases (Fpg-sensitive sites) and (iii) light-induced DNA damage due to photocatalytic activity. Furthermore, MN assays were used and we compared the results of more high-throughput MN scoring with flow cytometry to that of cytokinesis-block MN cytome assay scored manually using a microscope. Various methods were used to assess cytotoxic effects and the results showed in general no or low effects at the doses tested. A weak genotoxic effect of the tested TiO2 materials was observed with an induction of oxidised bases for all three materials of which NM100 was the most potent. When the comet slides were briefly exposed to lab light, a clear induction of DNA strand breaks was observed for the anatase materials, but not for the rutile. This highlights the risk of false positives when testing photocatalytically active materials if light is not properly avoided. A slight increase in MN formation for NM103 was observed in the different MN assays at the lower doses tested (1 and 5 μg/ml). We conclude that mini-gel comet assay and MN scoring using flow cytometry successfully can be used to efficiently study cytotoxic and genotoxic properties of nanoparticles. PMID:27382040

Assessment of DNA Damage and Telomerase Activity in Exfoliated Urinary Cells as Sensitive and Noninvasive Biomarkers for Early Diagnosis of Bladder Cancer in Ex-Workers of a Rubber Tyres Industry

PubMed Central

Pira, Enrico; Romano, Canzio; Fresegna, Anna Maria; Ciervo, Aureliano; Buresti, Giuliana; Zoli, Wainer; Calistri, Daniele

2014-01-01

The aim of the present study was to identify sensitive and noninvasive biomarkers of early carcinogenic effect at target organ to use in biomonitoring studies of workers at risk for previous occupational exposure to potential carcinogens. Standard urine cytology (Papanicolaou staining test), comet assay, and quantitative telomerase repeat amplification protocol (TRAP) assay were performed in 159 ex-rubber workers employed in tyres production and 97 unexposed subjects. In TRAP positive cases, a second level analysis using FISH (Urovysion) was done. Cystoscopy results were available for 11 individuals whose 6 FISH/TRAP/comet positive showed in 3 cases a dysplastic condition confirmed by biopsy, 1 comet positive resulted in infiltrating UBC to the biopsy and with hyperplasia and slight dysplasia to the urinary cytology, 1 comet positive resulted in papillary superficial UBC to the biopsy, 1 FISH/TRAP positive showed a normal condition, and 2 TRAP positive showed in one case a phlogosis condition. The results evidenced good concordance of TRAP, comet, and FISH assays as early biomarkers of procarcinogenic effect confirmed by the dysplastic condition and UBC found by cystoscopy-biopsy analysis. The analysis of these markers in urine cells could be potentially more accurate than conventional cytology in monitoring workers exposed to mixture of bladder potential carcinogens. PMID:24877087

Assessment of DNA damage and telomerase activity in exfoliated urinary cells as sensitive and noninvasive biomarkers for early diagnosis of bladder cancer in ex-workers of a rubber tyres industry.

PubMed

Cavallo, Delia; Casadio, Valentina; Bravaccini, Sara; Iavicoli, Sergio; Pira, Enrico; Romano, Canzio; Fresegna, Anna Maria; Maiello, Raffaele; Ciervo, Aureliano; Buresti, Giuliana; Zoli, Wainer; Calistri, Daniele

2014-01-01

The aim of the present study was to identify sensitive and noninvasive biomarkers of early carcinogenic effect at target organ to use in biomonitoring studies of workers at risk for previous occupational exposure to potential carcinogens. Standard urine cytology (Papanicolaou staining test), comet assay, and quantitative telomerase repeat amplification protocol (TRAP) assay were performed in 159 ex-rubber workers employed in tyres production and 97 unexposed subjects. In TRAP positive cases, a second level analysis using FISH (Urovysion) was done. Cystoscopy results were available for 11 individuals whose 6 FISH/TRAP/comet positive showed in 3 cases a dysplastic condition confirmed by biopsy, 1 comet positive resulted in infiltrating UBC to the biopsy and with hyperplasia and slight dysplasia to the urinary cytology, 1 comet positive resulted in papillary superficial UBC to the biopsy, 1 FISH/TRAP positive showed a normal condition, and 2 TRAP positive showed in one case a phlogosis condition. The results evidenced good concordance of TRAP, comet, and FISH assays as early biomarkers of procarcinogenic effect confirmed by the dysplastic condition and UBC found by cystoscopy-biopsy analysis. The analysis of these markers in urine cells could be potentially more accurate than conventional cytology in monitoring workers exposed to mixture of bladder potential carcinogens.

Detection of molecular microwave transitions in the 3 mm wavelength range in comet Kohoutek (1973f)

NASA Technical Reports Server (NTRS)

Buhl, D.; Huebner, W. F.; Snyder, L. E.

1976-01-01

Observations of comet Kohoutek made with a 3-mm line receiver mounted on the 11-m NRAO radio dish at Kitt Peak are presented. The detection of line transitions of hydrogen cyanide and methyl cyanide is reported and discussed along with the variability of neutral gas jets. Microwave transitions in molecules of cometary origin are also examined.

Ion cyclotron waves near comet C/2013 A1 (Siding Spring) and Mars

NASA Astrophysics Data System (ADS)

Crary, F. J.; Dols, V. J.; Connerney, J. E. P.; Espley, J. R.

2014-12-01

On October 19, 2014, comet C/2013 A1 (Siding Spring) passed approximately 135,000 km from Mars. Previously,we predicted the amplitude of ion cyclotron waves which might be observed during the Siding Spring encounter. Ioncyclotron waves have been observed both in the vicinity of comets and of Mars. These waves are generated by theionization of neutrals in the flowing solar wind, which produces an unstable ring-beam velocity distribution. We estimated that, for a production rate of 2x1028 s-1, ion cyclotron wave with amplitudes over 0.1 nT would be present within ‡5 hours (1.2 million km) of closest approach. We will compare the actual observations made by the MAVEN spacecraft with these predictions. The spacecraft was close to or downstream of the martian bow shock, which complicates the interpretation of the data. Taking thisinto account, we will describe the observations and their implications for wave activity and cometary neutral production. We also present updated hybrid simulations of ion cyclotron wave generation. The simulations use our best estimate of solar wind conditions at the time of the encounter and a variable injection of 18 AMU pickup ions, at a rates consistent a model of the cometary neutrals.

A Search for Rarely Seen Ultraviolet Coma Emissions and New Species Upper Limits at Comet 67P/Churyumov-Gerasimenko Using the Rosetta-Alice Ultraviolet Spectrograph

NASA Astrophysics Data System (ADS)

Noonan, J.; Stern, S. A.; Parker, J. W.; Keeney, B. A.; Weaver, H. A., Jr.; Feldman, P.; Steffl, A.; Feaga, L. M.; Bertaux, J. L.

2017-12-01

The Alice far/extreme-UV spectrograph aboard Rosetta is one of three US instruments provided by NASA; it is the first UV spectrograph to reach any comet. Numerous scientific results have been obtained regarding 67P/Churyumov-Gerasimenko by this instrument. Here we summarize two new sets of results from a search for rarely appearing atomic and molecular spectral emission features and a grand sum spectrum allowing us to place new atomic and molecular neutral and ionized species upper limits in the comet's coma.

Organic material: Asteroids, meteorites, and planetary satellites

NASA Technical Reports Server (NTRS)

Cruikshank, Dale P.; Kerridge, John F.

1992-01-01

Telescopic observations in in situ spacecraft investigations over the last two decades have shown that many planetary satellites, asteroids, and comets have surfaces containing very dark material that is either neutral (black) or red in color. Although comets are not the focus of this paper, the possible relationship of comets to asteroids, meteorites, and interplanetary dust is briefly discussed in the context of their dark-matter component. The following topics are discussed with respect to their organic content: carbonaceous chondrites; asteroids; low-albedo planetary satellites; and Pluto, Charon, and Triton. Laboratory studies and a summary are also presented.

Detection of radiation treatment of beans using DNA comet assay

NASA Astrophysics Data System (ADS)

Khan, Ashfaq A.; Khan, Hasan M.; Delincée, Henry

2002-03-01

A simple technique of microgel electrophoresis of single cells (DNA Comet Assay) enabled a quick detection of radiation treatment of several kinds of leguminous beans (azuki, black, black eye, mung, pinto, red kidney and white beans). Each variety was exposed to radiation doses of 0.5, 1 and 5kGy covering the permissible limits for insect disinfestation. The cells or nuclei from beans were extracted in cold PBS, embedded in agarose on microscope slides, lysed between 15 and 60min in 2.5% SDS and electrophoresis was carried out at a voltage of 2V/cm for 2-2.5min. After silver staining, the slides were evaluated through an ordinary transmission microscope. In irradiated samples, fragmented DNA stretched towards the anode and the damaged cells appeared as a comet. The density of DNA in the tails increased with increasing radiation dose. However, in non-irradiated samples, the large molecules of DNA remained relatively intact and there was only minor or no migration of DNA; the cells were round or had very short tails only. Hence, the DNA comet assay provides an inexpensive, rapid and relatively simple screening method for the detection of irradiated beans.

BENZO[A]PYRENE AND ITS K-REGION DIOL INDUCE DNA DAMAGE IN C3H10T1/2C18 CELLS AS MEASURED BY THE ALKALINE SINGLE CELL GEL (COMET) ASSAY

EPA Science Inventory

160. Benzo[a]pyrene and its K-region diol induce DNA damage in C3HlOTl/2Cl8 cells as measured by the alkaline single cell gel (Comet) assay

In a continuing series of studies on the genotoxicity ofK-region dihydrodiols of polycyclic aromatic hydrocarbons, we have repo...

Genotoxicity evaluation of HMG CoA reductase inhibitor rosuvastatin.

PubMed

Berber, Ahmet Ali; Celik, Mustafa; Aksoy, Hüseyin

2014-07-01

The genotoxic potential of rosuvastatin as one of the statin drugs was assessed by chromosomal aberrations (CAs), micronucleus (MN) and DNA damage by comet assay in the human peripheral blood lymphocytes. Rosuvastatin was used at concentrations of 0.0625, 0.125, 0.25, 0.5 and 1 µg/mL for these in vitro assays. In all assays, a negative and positive control were also included. CA frequencies were significantly increased in all concentrations at 24 hours and significantly increased in all concentrations except 0.0625 µg/mL at 48 hours, compared to the negative control. Rosuvastatin has a decreased mitotic index (MI) at 0.5- and 1-µg/mL concentrations at 24 hours and at 0.25, 0.5 and 1 µg/mL at 48 hours. A significant increase was observed for induction of MN in all treatments, compared to the negative control. Cytokinesis-block proliferation indices were not affected by treatments with rosuvastatin. In the comet assay, significant increases in comet tail length and tail moment were observed at 0.0625-, 0.5- and 1-µg/mL concentrations. Comet intensity was significantly increased in all concentrations except 0.0625 µg/mL. According to these results, rosuvastatin is cytotoxic and clastogenic/aneugenic in human peripheral lymphocytes. Further studies should be conducted in other test systems to evaluate the full genotoxic potential of rosuvastatin.

Cometary science. Time variability and heterogeneity in the coma of 67P/Churyumov-Gerasimenko.

PubMed

Hässig, M; Altwegg, K; Balsiger, H; Bar-Nun, A; Berthelier, J J; Bieler, A; Bochsler, P; Briois, C; Calmonte, U; Combi, M; De Keyser, J; Eberhardt, P; Fiethe, B; Fuselier, S A; Galand, M; Gasc, S; Gombosi, T I; Hansen, K C; Jäckel, A; Keller, H U; Kopp, E; Korth, A; Kührt, E; Le Roy, L; Mall, U; Marty, B; Mousis, O; Neefs, E; Owen, T; Rème, H; Rubin, M; Sémon, T; Tornow, C; Tzou, C-Y; Waite, J H; Wurz, P

2015-01-23

Comets contain the best-preserved material from the beginning of our planetary system. Their nuclei and comae composition reveal clues about physical and chemical conditions during the early solar system when comets formed. ROSINA (Rosetta Orbiter Spectrometer for Ion and Neutral Analysis) onboard the Rosetta spacecraft has measured the coma composition of comet 67P/Churyumov-Gerasimenko with well-sampled time resolution per rotation. Measurements were made over many comet rotation periods and a wide range of latitudes. These measurements show large fluctuations in composition in a heterogeneous coma that has diurnal and possibly seasonal variations in the major outgassing species: water, carbon monoxide, and carbon dioxide. These results indicate a complex coma-nucleus relationship where seasonal variations may be driven by temperature differences just below the comet surface. Copyright © 2015, American Association for the Advancement of Science.

The neutral coma of comets: A review

NASA Technical Reports Server (NTRS)

Delsemme, A. H.

1976-01-01

The hypothesis that water snow controls the vaporization of the nucleus of some comets seems verified from the general order of magnitude of the size of their nucleus and of their nuclear albedo; the largest observed production rates are H and OH which both seem to originate from the photodissociation of H2O, as also confirmed by the scale length of the invisible parent molecule producing OH. However, comet Encke is not uniformly covered by water snow, as it produces only one tenth of the expected vaporization. Early results on comet Kohoutek suggest that the conclusions could be slightly different for some of the new comets in Oort's sense. If the far ultraviolet observations confirm the early assessments of the production rates of C, O and H, then at least another major constituent competing with water has not yet been detected. Such a major constituent is suggested by the ratios C/O = 0.24 and H/O = 2.5.

E-cigarette vapour is not inert and exposure can lead to cell damage.

PubMed

Holliday, Richard; Kist, Ralf; Bauld, Linda

2016-03-01

In vitro experiments were performed on normal epithelial cells as well as head and neck squamous cell carcinoma (HNSCC) cell lines. The widely available cell line HaCat, a spontaneously transformed immortal keratinocyte and the HNSCC cell lines HN30 and UMSCC10B were used. Cells were exposed to nicotine-containing and nicotine-free vapour extract from two popular e-cigarette brands for periods ranging from 48 hours to eight weeks. Cytotoxicity was assessed using Annexin V flow cytometric analysis, trypan blue exclusion and clonogenic assays. Genotoxicity in the form of DNA strand breaks was quantified using the neutral comet assay and γ-H2AX immunostaining. E-cigarette-exposed cells showed significantly reduced cell viability and clonogenic survival, along with increased rates of apoptosis and necrosis, regardless of e-cigarette vapour nicotine content. They also exhibited significantly increased comet tail length and accumulation of γ-H2AX foci, demonstrating increased DNA strand breaks. In conclusion, our study strongly suggests that electronic cigarettes are not as safe as their marketing makes them appear to the public. Our in vitro experiments employing two brands of e-cigs show that at biologically relevant doses, vapourised e-cig liquids induce increased DNA strand breaks and cell death, and decreased clono- genic survival in both normal epithelial and HNSCC cell lines independently of nicotine content. Further research is needed to definitively determine the long-term effects of e-cig usage, as well as whether the DNA damage shown in our study as a result of e-cig exposure will lead to mutations that ultimately result in cancer.

Monitoring regulation of DNA repair activities of cultured cells in-gel using the comet assay

PubMed Central

Nickson, Catherine M.; Parsons, Jason L.

2014-01-01

Base excision repair (BER) is the predominant cellular mechanism by which human cells repair DNA base damage, sites of base loss, and DNA single strand breaks of various complexity, that are generated in their thousands in every human cell per day as a consequence of cellular metabolism and exogenous agents, including ionizing radiation. Over the last three decades the comet assay has been employed in scientific research to examine the cellular response to these types of DNA damage in cultured cells, therefore revealing the efficiency and capacity of BER. We have recently pioneered new research demonstrating an important role for post-translational modifications (particularly ubiquitylation) in the regulation of cellular levels of BER proteins, and that subtle changes (∼20–50%) in protein levels following siRNA knockdown of E3 ubiquitin ligases or deubiquitylation enzymes can manifest in significant changes in DNA repair capacity monitored using the comet assay. For example, we have shown that the E3 ubiquitin ligase Mule, the tumor suppressor protein ARF, and the deubiquitylation enzyme USP47 modulate DNA repair by controlling cellular levels of DNA polymerase β, and also that polynucleotide kinase phosphatase levels are controlled by ATM-dependant phosphorylation and Cul4A–DDB1–STRAP-dependent ubiquitylation. In these studies we employed a modification of the comet assay whereby cultured cells, following DNA damage treatment, are embedded in agarose and allowed to repair in-gel prior to lysis and electrophoresis. Whilst this method does have its limitations, it avoids the extensive cell culture-based processing associated with the traditional approach using attached cells and also allows for the examination of much more precise DNA repair kinetics. In this review we will describe, using this modified comet assay, our accumulating evidence that ubiquitylation-dependant regulation of BER proteins has important consequences for overall cellular DNA repair capacity. PMID:25076968

The impact of lymphocyte isolation on induced DNA damage in human blood samples measured by the comet assay.

PubMed

Bausinger, Julia; Speit, Günter

2016-09-01

The comet assay is frequently used in human biomonitoring for the detection of exposure to genotoxic agents. Peripheral blood samples are most frequently used and tested either as whole blood or after isolation of lymphocytes (i.e. peripheral blood mononuclear cells, PBMC). To investigate a potential impact of lymphocyte isolation on induced DNA damage in human blood samples, we exposed blood ex vivo to mutagens with different modes of genotoxic action. The comet assay was performed either directly with whole blood at the end of the exposure period or with lymphocytes isolated directly after exposure. In addition to the recommended standard protocol for lymphocyte isolation, a shortened protocol was established to optimise the isolation procedure. The results indicate that the effects of induced DNA strand breaks and alkali-labile sites induced by ionising radiation and alkylants, respectively, are significantly reduced in isolated lymphocytes. In contrast, oxidative DNA base damage (induced by potassium bromate) and stable bulky adducts (induced by benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide; BPDE) seem to be less affected. Our findings suggest that in vivo-induced DNA damage might also be reduced in isolated lymphocytes in comparison with the whole blood depending of the types of DNA damage induced. Because only small genotoxic effects can generally be expected in human biomonitoring studies with the comet assay after occupational and environmental exposure to genotoxic agents, any loss might be relevant and should be avoided. The possibility of such effects and their potential impact on variability of comet assay results in human biomonitoring should be considered when performing or evaluating such kind of studies. © The Author 2016. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Detection of hypoxic fractions in murine tumors by comet assay: Comparison with other techniques

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hu, Q.; Kavanagh, M.C.; Newcombe, D.

1995-12-01

The alkaline comet assay was used to detect the hypoxic fractions of murine tumors. A total of four tumor types were tested using needle aspiration biopsies taken immediately after a radiation dose of 15 Gy. Initial studies confirmed that the normalized tail moment, a parameter reflecting single-strand DNA breaks induced by the radiation, was linearly related to radiation dose. Further, it was shown that for a mixed population (1:1) of cells irradiated under air-breathing or hypoxic conditions, the histogram of normal tail moment values obtained from analyzing 400 cells in the population had a double peak which, when fitted withmore » two Gaussian distributions, gave a good estimate of the proportion of the two subpopulations. For the four tumor types, the means of the calculated hypoxic fractions from four or five individual tumors were 0.15 {+-} 0.04 for B16F1, 0.08 {+-} 0.04 for KHT-LP1, 0.17 {+-} 0.04 for RIF-1 and 0.04 {+-} 0.01 for SCCVII. Analysis of variance showed that the hypoxic fraction in KHT-LP1 tumors is significantly lower than those of the other three tumors (P = 0.026) but that there is no significant difference in hypoxic fraction between B16F1, RIF-1 and SCCVII tumors (P = 0.574). Results from multiple samples taken from each of five RIF-1 tumors showed that the intertumor heterogeneity of hypoxic fractions was greater than that within the same tumor. The mean hypoxic fraction obtained using the comet assay for the four tumor types was compared with the hypoxic fraction determined by the clonogenic assay, or median pO{sub 2} values, or [{sup 3}H]misonidazole binding in the same tumor types. The values of hypoxic fraction obtained with the comet assay were two to four times lower than those measured by the paired survival method. Preliminary results obtained with a dose of 5 Gy were consistent with those obtained using 15 Gy. These results suggest the further development of the comet assay for clinical studies. 21 refs., 7 figs., 5 tabs.« less

Electron impact ionization in the vicinity of comets

NASA Astrophysics Data System (ADS)

Cravens, T. E.; Kozyra, J. U.; Nagy, A. F.; Gombosi, T. I.; Kurtz, M.

1987-07-01

The solar wind interacts very strongly with the extensive cometary coma, and the various interaction processes are initiated by the ionization of cometary neutrals. The main ionization mechanism far outside the cometary bow shock is photoionization by solar extreme ultraviolet radiation.Electron distributions measured in the vicinity of comets Halley and Giacobini-Zinner by instruments on the VEGA and ICE spacecraft, respectively, are used to calculate electron impact ionization frequencies. Ionization by electrons is of comparable importance to photoionization in the magnetosheaths of Comets Halley and Giacobini-Zinner. The ionization frequency in the inner part of the cometary plasma region of comet Halley is several times greater than the photoionization value. Tables of ionization frequencies as functions of electron temperature are presented for H2O, CO2, CO, O, N2, and H.

Modeling the cometary environment using a fluid approach

NASA Astrophysics Data System (ADS)

Shou, Yinsi

Comets are believed to have preserved the building material of the early solar system and to hold clues to the origin of life on Earth. Abundant remote observations of comets by telescopes and the in-situ measurements by a handful of space missions reveal that the cometary environments are complicated by various physical and chemical processes among the neutral gases and dust grains released from comets, cometary ions, and the solar wind in the interplanetary space. Therefore, physics-based numerical models are in demand to interpret the observational data and to deepen our understanding of the cometary environment. In this thesis, three models using a fluid approach, which include important physical and chemical processes underlying the cometary environment, have been developed to study the plasma, neutral gas, and the dust grains, respectively. Although models based on the fluid approach have limitations in capturing all of the correct physics for certain applications, especially for very low gas density environment, they are computationally much more efficient than alternatives. In the simulations of comet 67P/Churyumov-Gerasimenko at various heliocentric distances with a wide range of production rates, our multi-fluid cometary neutral gas model and multi-fluid cometary dust model have achieved comparable results to the Direct Simulation Monte Carlo (DSMC) model, which is based on a kinetic approach that is valid in all collisional regimes. Therefore, our model is a powerful alternative to the particle-based model, especially for some computationally intensive simulations. Capable of accounting for the varying heating efficiency under various physical conditions in a self-consistent way, the multi-fluid cometary neutral gas model is a good tool to study the dynamics of the cometary coma with different production rates and heliocentric distances. The modeled H2O expansion speeds reproduce the general trend and the speed's nonlinear dependencies of production rate and heliocentric distance, which are found in remote observations. In the multi-fluid dust model, we use a newly developed numerical mesh to resolve the real shaped nucleus in the center and to facilitate prescription of the outer boundary conditions that accommodate the rotating frame. The model studies the effects of the rotating nucleus and the cometary activity in time-dependent simulations for the first time. The result also suggests that the rotation of the nucleus explains why there is no clear dust speed dependence on size in some of the dust observations. We developed a new multi-species comet MHD model to simulate the plasma environment of comet C/2006 P1 (McNaught) over a wide range of heliocentric distances from 0.17 AU to 1.75 AU, with the constraints provided by remote and in situ observations. Typical subsolar standoff distances of bow shock and contact surface are modeled and presented to characterize the solar wind interaction of the comet at various heliocentric distances. In addition, the model is also the first one to be used to study the composition and dynamics in the distant cometary tail. The results agree well with the measured water group ion abundances from the Ulysses/SWICS 1.7 AU down-tail from the comet and the velocity and temperature measured by Ulysses/SWOOPS.

Inventory of Volatiles in the Coma of Comet 67P/Churyumov-Gerasimenko from Rosetta ROSINA - An Overview of First Results

NASA Astrophysics Data System (ADS)

Altwegg, K.; Rubin, M.; Balsiger, H. R.; Jäckel, A.; Le Roy, L.; Wurz, P.; Gasc, S.; Calmonte, U.; Tzou, C. Y.; Mall, U. A.; Fiethe, B.; De Keyser, J. M.; Berthelier, J. J.; Reme, H.; Gombosi, T. I.; Fuselier, S.

2014-12-01

The European Space Agency's Rosetta spacecraft is now close in a bound orbit around comet 67P/Churyumov-Gerasimenko (67P/C-G). On board is the Rosetta Orbiter Spectrometer for Ion and Neutral Analysis (ROSINA) instrument suite. ROSINA consists of two mass spectrometers, the Double Focusing Mass Spectrometer (DFMS) and the Reflectron-type Time-Of-Flight (RTOF), as well as the COmet Pressure Sensor (COPS). ROSINA is designed to detect and monitor the neutral gas and thermal plasma environment in the comet's coma by in situ investigation. The two mass spectrometers have high dynamic ranges and complement each other with high mass resolution (DFMS) and high time resolution and large mass range (RTOF). Especially the unprecedented sensitivity and mass resolution of DFMS together with the large mass range of RTOF will allow determining precisely light species (e.g. isotopologues) as well as detecting heavy organics. The pressure sensor COPS is capable to derive total gas densities, velocities, and temperatures. To date only limited data for the composition of cometary comae at heliocentric distances of more than 2.5 AU are available. The set is dominated by CO and daughter species of water from bright comets originating in the Oort cloud. While some molecules can be detected from far by remote sensing (e.g. CO) other molecules are much more difficult to observe from ground (e.g. CO2). The Rosetta mission presents a unique opportunity to directly probe the parent species in the thin cometary atmosphere of a Kuiper-belt object at more than 2.5 AU from the Sun and relate it to ground-based observations. Distances that far from the Sun are of particular interest as the comet's activity transitions from being super volatiles dominated to being water dominated. We will report on the first measurements of the volatile inventory obtained from ROSINA observations as Rosetta is following comet 67P/C-G in close vicinity.

PREDICTIONS OF ION PRODUCTION RATES AND ION NUMBER DENSITIES WITHIN THE DIAMAGNETIC CAVITY OF COMET 67P/CHURYUMOV-GERASIMENKO AT PERIHELION

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vigren, E.; Galand, M., E-mail: e.vigren@imperial.ac.uk

2013-07-20

We present a one-dimensional ion chemistry model of the diamagnetic cavity of comet 67P/Churyumov-Gerasimenko, the target comet for the ESA Rosetta mission. We solve the continuity equations for ionospheric species and predict number densities of electrons and selected ions considering only gas-phase reactions. We apply the model to the subsolar direction and consider conditions expected to be encountered by Rosetta at perihelion (1.29 AU) in 2015 August. Our default simulation predicts a maximum electron number density of {approx}8 Multiplication-Sign 10{sup 4} cm{sup -3} near the surface of the comet, while the electron number densities for cometocentric distances r > 10more » km are approximately proportional to 1/r {sup 1.23} assuming that the electron temperature is equal to the neutral temperature. We show that even a small mixing ratio ({approx}0.3%-1%) of molecules having higher proton affinity than water is sufficient for the proton transfer from H{sub 3}O{sup +} to occur so readily that other ions than H{sub 3}O{sup +}, such as NH{sub 4} {sup +} or CH{sub 3}OH{sub 2} {sup +}, become dominant in terms of volume mixing ratio in part of, if not throughout, the diamagnetic cavity. Finally, we test how the predicted electron and ion densities are influenced by changes of model input parameters, including the neutral background, the impinging EUV solar spectrum, the solar zenith angle, the cross sections for photo- and electron-impact processes, the electron temperature profile, and the temperature dependence of ion-neutral reactions.« less

Evaluation of methyl methanesulfonate, 2,6-diaminotoluene and 5-fluorouracil: Part of the Japanese center for the validation of alternative methods (JaCVAM) international validation study of the in vivo rat alkaline comet assay.

PubMed

Plappert-Helbig, Ulla; Junker-Walker, Ursula; Martus, Hans-Joerg

2015-07-01

As a part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of the in vivo rat alkaline comet assay (comet assay), we examined methyl methanesulfonate, 2,6-diaminotoluene, and 5-fluorouracil under coded test conditions. Rats were treated orally with the maximum tolerated dose (MTD) and two additional descending doses of the respective compounds. In the MMS treated groups liver and stomach showed significantly elevated DNA damage at each dose level and a significant dose-response relationship. 2,6-diaminotoluene induced significantly elevated DNA damage in the liver at each dose and a statistically significant dose-response relationship whereas no DNA damage was obtained in the stomach. 5-fluorouracil did not induce DNA damage in either liver or stomach. Copyright © 2015 Elsevier B.V. All rights reserved.

Genotoxicity of AMPA, the environmental metabolite of glyphosate, assessed by the Comet assay and cytogenetic tests.

PubMed

Mañas, F; Peralta, L; Raviolo, J; García Ovando, H; Weyers, A; Ugnia, L; Gonzalez Cid, M; Larripa, I; Gorla, N

2009-03-01

Formulations containing glyphosate are the most widely used herbicides in the world. AMPA is the major environmental breakdown product of glyphosate. The purpose of this study is to evaluate the in vitro genotoxicity of AMPA using the Comet assay in Hep-2 cells after 4h of incubation and the chromosome aberration (CA) test in human lymphocytes after 48h of exposition. Potential in vivo genotoxicity was evaluated through the micronucleus test in mice. In the Comet assay, the level of DNA damage in exposed cells at 2.5-7.5mM showed a significant increase compared with the control group. In human lymphocytes we found statistically significant clastogenic effect AMPA at 1.8mM compared with the control group. In vivo, the micronucleus test rendered significant statistical increases at 200-400mg/kg. AMPA was genotoxic in the three performed tests. Very scarce data are available about AMPA potential genotoxicity.

Primary DNA damage assessed with the comet assay and comparison to the absorbed dose of diagnostic X-rays in children.

PubMed

Milkovic, Durdica; Garaj-Vrhovac, Vera; Ranogajec-Komor, Mária; Miljanic, Saveta; Gajski, Goran; Knezevic, Zeljka; Beck, Natko

2009-01-01

The aim of this work is to assess DNA damage in peripheral blood lymphocytes of children prior to and following airway X-ray examinations of the chest using the alkaline comet assay and to compare data with the measured absorbed dose. Twenty children with pulmonary diseases, between the ages of 5 and 14 years, are assessed. Absorbed dose measurements are conducted for posterior-anterior projection on the forehead, thyroid gland, gonads, chest, and back. Doses are measured using thermoluminescent and radiophotoluminescent dosimetry systems. Differences between tail lengths, tail intensity, and tail moments as well as for the long-tailed nuclei before and after exposures are statistically significant and are dependent on the individual. The results demonstrate the usefulness of the comet assay as a measure of X-ray damage to lymphocytes in a clinical setting. Doses measured with both dosimeters show satisfactory agreement (0.01 mSv) and are suitable for dosimetric measurements in X-ray diagnostics.

The spectrum of comet austin from 910 to 1180 a.

PubMed

Green, J C; Cash, W; Cook, T A; Stern, S A

1991-01-25

A spectrum of comet Austin (1988 c(1)) has been obtained from 910 to 1180 A. Three bright emission lines were detected, including a forbidden oxygen line (1128 A), which are attributable to radiative pumping of neutral oxygen by solar Lyman beta. The relative strengths of the observed features should prove to be a useful diagnostic of the physical conditions and radiation fields in cometary comae. In addition, the absence of strong spectral features from highly volatile species such as He, Ar, or N(2) can be used to place constraints on the thermal environment under which the comet was formed and has been processed.

Angular and energy distribution of low energy cometary ions measured in the outer coma of Comet Halley

NASA Technical Reports Server (NTRS)

Berthelier, J. J.; Illiano, J. M.; Hodges, R. R.; Krankowsky, D.; Eberhardt, P.; Laemmerzahl, P.; Hoffman, J. H.; Herrwerth, I.; Woweries, J.; Dolder, U.

1986-01-01

During the early phase of the Giotto encounter with comet Halley, at distances from the nucleus greater than 350,000 km, the neutral mass spectrometer was operated in a mode allowing the measurement of low energy ions. Data reveal two important features of the outer coma: the presence of a sharp discontinuity in the plasma flow at 550,000 km from the nucleus which results in a significant decrease of the plasma flow accompanied by an increase in temperature; and the detection of newly born ions identified as O(+) and CO(+), at distances from the comet greater than 800,000 km.

Detection of genotoxic effects of drinking water disinfection by-products using Vicia faba bioassay.

PubMed

Hu, Yu; Tan, Li; Zhang, Shao-Hui; Zuo, Yu-Ting; Han, Xue; Liu, Na; Lu, Wen-Qing; Liu, Ai-Lin

2017-01-01

Plant-based bioassays have gained wide use among the toxicological and/or ecotoxicological assessment procedures because of their simplicity, sensitivity, low cost, and reliability. The present study describes the use of Vicia faba (V. faba) micronucleus (MN) test and V. faba comet assay in the evaluation of the genotoxic potential of disinfection by-products (DBPs) commonly found in chlorine-disinfected drinking water. Five haloacetic acids and three halogenated acetonitriles were chosen as representatives of DBPs in this study because they are of potentially great public health risk. Results of the MN test indicated that monochloroacetic acid (MCA), monobromoacetic acid (MBA), dichloroacetic acid (DCA), dibromoacetic acid (DBA), trichloroacetic acid (TCA), and trichloroacetonitrile (TCAN) caused a statistically significant increase in MN frequency in V. faba root tip cells. However, no genotoxic response was observed for dichloroacetonitrile (DCAN) and dibromoacetonitrile (DBAN). Results of the comet assay showed that all tested DBPs induced a statistically significant increase in genomic DNA damage to V. faba root tip cells. On considering the capacity to detect genomic damage of a different nature, we suggest that a combination of V. faba MN test and V. faba comet assay is a useful tool for the detection of genotoxic effects of DBPs. It is worthy of assessing the feasibility of using V. faba comet assay combined with V. faba MN test to screen for the genotoxic activity of chlorinated drinking water in future work.

Ultrastructural Interactions and Genotoxicity Assay of Cerium Dioxide Nanoparticles on Mouse Oocytes

PubMed Central

Courbiere, Blandine; Auffan, Mélanie; Rollais, Raphaël; Tassistro, Virginie; Bonnefoy, Aurélie; Botta, Alain; Rose, Jérôme; Orsière, Thierry; Perrin, Jeanne

2013-01-01

Cerium dioxide nanoparticles (CeO2 ENPs) are on the priority list of nanomaterials requiring evaluation. We performed in vitro assays on mature mouse oocytes incubated with CeO2 ENPs to study (1) physicochemical biotransformation of ENPs in culture medium; (2) ultrastructural interactions with follicular cells and oocytes using Transmission Electron Microscopy (TEM); (3) genotoxicity of CeO2 ENPs on follicular cells and oocytes using a comet assay. DNA damage was quantified as Olive Tail Moment. We show that ENPs aggregated, but their crystal structure remained stable in culture medium. TEM showed endocytosis of CeO2 ENP aggregates in follicular cells. In oocytes, CeO2 ENP aggregates were only observed around the zona pellucida (ZP). The comet assay revealed significant DNA damage in follicular cells. In oocytes, the comet assay showed a dose-related increase in DNA damage and a significant increase only at the highest concentrations. DNA damage decreased significantly both in follicular cells and in oocytes when an anti-oxidant agent was added in the culture medium. We hypothesise that at low concentrations of CeO2 ENPs oocytes could be protected against indirect oxidative stress due to a double defence system composed of follicular cells and ZP. PMID:24185910

Genotoxicity assessment of amaranth and allura red using Saccharomyces cerevisiae.

PubMed

Jabeen, Hafiza Sumara; ur Rahman, Sajjad; Mahmood, Shahid; Anwer, Sadaf

2013-01-01

Amaranth (E123) and Allura red (E129), very important food azo dyes used in food, drug, paper, cosmetic and textile industries, were assessed for their genotoxic potential through comet assay in yeast cells. Comet assay was standardized by with different concentration of H(2)O(2). Concentrations of Amaranth and Allura red were maintained in sorbitol buffer starting from 9.76 to 5,000 μg/mL and 1 × 10(4) cells were incubated at two different incubation temperatures 28 and 37°C. Amaranth (E123) and Allura red (E129) were found to exhibit their genotoxic effect directly in Saccharomyces cerevisiae. No significant genotoxic activity was observed for Amaranth and Allura red at 28°C but at 37°C direct relation of Amaranth concentration with comet tail was significant and no positive relation was seen with time exposure factor. At 37°C the minimum concentration of Amaranth and Allura red at which significant DNA damage observed through comet assay was 1,250 μg/mL in 2nd h post exposure time. The results indicated that food colors should be carefully used in baking products as heavy concentration of food colors could affect the fermentation process of baking.

Ion composition at comet 67P near perihelion: Rosetta/ROSINA measurements and modeling

NASA Astrophysics Data System (ADS)

Beth, Arnaud; Altwegg, Kathrin; Balsiger, Hans; Berthelier, Jean-Jacques; Calmonte, Ursina; Combi, Michael R.; De Keyser, Johan; Dhooghe, Frederik; Fiethe, Björn; Fuselier, Stephen; Galand, Marina; Gasc, Sébastien; Gombosi, T. I.; Hansen, Kenneth C.; Hässig, Myrtha; Héritier, Kévin; Kopp, Ernest; Le Roy, Léna; Peroy, Solène; Rubin, Martin; Sémon, Thierry; Tzou, Chia-Yu; Vigren, Erik

2016-10-01

On August 13th, 2015, comet 67P/Churyumov-Gerasimenko reached its perihelion at 1.24 AU, a milestone for its cometary activity observed by the European Space Agency's Rosetta spacecraft which arrived in August 2014. The Rosetta Orbiter Spectrometer for Ion and Neutral Analysis (ROSINA)/Comet Pressure Sensor (COPS) instrument onboard Rosetta measured local outgassing rates over 1028 molecules.s-1 in summer 2015. In the meantime, the ROSINA/Double Focusing Mass Spectrometer (DFMS) instrument measured the ion composition in the coma which was expected to be more diversified than during the early phase of the mission. Indeed, the increase in the cometary activity is expected to trigger new chemical pathways, yielding the formation of new cometary ions, other than the major water ions observed at larger heliocentric distances. Such new ion species can be produced from minor neutral species, such as those with proton affinity higher than that of water. This includes NH4+ whose detection has been recently reported (Beth et al., 2016).In this study, we propose to investigate other ion species during the perihelion period by:- analysing DFMS data to find any signature of substantial ion species,- modeling the ionosphere of 67P by driving the model with the neutral densities measured by DFMS and COPS to support or constrain the absence or the presence of these ion species,- discussing any discrepancy between observations and simulations.

MOLECULAR OXYGEN IN OORT CLOUD COMET 1P/HALLEY

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rubin, M.; Altwegg, K.; Dishoeck, E. F. van

2015-12-10

Recently, the ROSINA mass spectrometer suite on board the European Space Agency's Rosetta spacecraft discovered an abundant amount of molecular oxygen, O{sub 2}, in the coma of Jupiter family comet 67P/Churyumov–Gerasimenko of O{sub 2}/H{sub 2}O = 3.80 ± 0.85%. It could be shown that O{sub 2} is indeed a parent species and that the derived abundances point to a primordial origin. Crucial questions are whether the O{sub 2} abundance is peculiar to comet 67P/Churyumov–Gerasimenko or Jupiter family comets in general, and also whether Oort cloud comets such as comet 1P/Halley contain similar amounts of molecular oxygen. We investigated mass spectra obtained bymore » the Neutral Mass Spectrometer instrument during the flyby by the European Space Agency's Giotto probe of comet 1P/Halley. Our investigation indicates that a production rate of O{sub 2} of 3.7 ± 1.7% with respect to water is indeed compatible with the obtained Halley data and therefore that O{sub 2} might be a rather common and abundant parent species.« less

Line Ratios for Solar Wind Charge Exchange with Comets

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mullen, P. D.; Cumbee, R. S.; Lyons, D.

Charge exchange (CX) has emerged in X-ray emission modeling as a significant process that must be considered in many astrophysical environments—particularly comets. Comets host an interaction between solar wind ions and cometary neutrals to promote solar wind charge exchange (SWCX). X-ray observatories provide astronomers and astrophysicists with data for many X-ray emitting comets that are impossible to accurately model without reliable CX data. Here, we utilize a streamlined set of computer programs that incorporate the multi-channel Landau–Zener theory and a cascade model for X-ray emission to generate cross sections and X-ray line ratios for a variety of bare and non-baremore » ion single electron capture (SEC) collisions. Namely, we consider collisions between the solar wind constituent bare and H-like ions of C, N, O, Ne, Na, Mg, Al, and Si and the cometary neutrals H{sub 2}O, CO, CO{sub 2}, OH, and O. To exemplify the application of this data, we model the X-ray emission of Comet C/2000 WM1 (linear) using the CX package in SPEX and find excellent agreement with observations made with the XMM-Newton RGS detector. Our analyses show that the X-ray intensity is dominated by SWCX with H, while H{sub 2}O plays a secondary role. This is the first time, to our knowledge, that CX cross sections have been implemented into a X-ray spectral fitting package to determine the H to H{sub 2}O ratio in cometary atmospheres. The CX data sets are incorporated into the modeling packages SPEX and Kronos .« less

The enigmatic object 2201 Oljato - Is it an asteroid or an evolved comet?

NASA Technical Reports Server (NTRS)

Mcfadden, Lucy A.; Cochran, Anita L.; Barker, Edwin S.; Cruikshank, Dale P.; Hartmann, William K.

1993-01-01

The orbital properties of near-earth object 2201 have been associated with meteor showers, and its modeled orbital evolution is chaotic - a property which might indicate a history related to comets. Telescopic observations of its visible and near-infrared spectral reflectance, broad-band visible and near-infrared photometry, infrared radiometric measurements, and radar echoes are reported here from two apparitions, 1979 and 1983. This asteroid has a high radiometric albedo, a property not associated with comet nuclei. In certain wavelength regimes it is classified as an S-type asteroid, in others, an E-type, but its overall spectral reflectance is not typical of either taxonomic type, and neither type is thought of as cometlike. Unexpectedly high ultraviolet reflectance at the 1979 apparition was suggested to be the result of residual outgassing as in a comet. The UV photometric data are modeled as fluorescent emission from neutral species found in comets. The resulting calculations indicate a plausible value for OH and CN emission at 0.3085 and 0.38 micron relative to the observed range of active comets.

Genotoxicity of citrate-coated silver nanoparticles to human keratinocytes assessed by the comet assay and cytokinesis blocked micronucleus assay.

PubMed

Bastos, V; Duarte, I F; Santos, C; Oliveira, H

2017-02-01

Silver nanoparticles (AgNPs) are widely used in industrial, cosmetic, and biomedical products, and humans are frequently exposed to these products through the skin. It is widely recognized that the characteristics of AgNPs (e.g., size, coating) may influence their cytotoxic effects, but their correlation with DNA damage and mitotic disorders remains poorly explored. In this study, human keratinocytes (HaCaT cell line) were exposed to well-characterized 30 nm AgNPs coated with citrate, and their effects on viability, DNA fragmentation (assessed by the comet assay), and micronuclei (MNi) induction (assessed by the cytokinesis-block micronucleus cytome assays, CBMN) were investigated. The results showed that 10 and 40 μg/mL AgNPs decreased cell proliferation and viability, and induced a significant genetic damage. This was observed by an increase of DNA amount in comet tail, which linearly correlated with dose and time of exposure. Also, cytostaticity (increase of mononucleated cells) and MNi rates increased in treated cells. In contrast, no significant changes were observed in nucleoplasmatic bridges (NPBs) or nuclear buds (NBUDs), although NBUDs tended to increase in all conditions and periods. The cytostatic effects on HaCaT cells were also shown by the decrease of their nuclear division index. Thus, both comet and CBMN assays supported the observation that citrate-AgNPs induced genotoxic effects on HaCaT cells. Considering that AgNPs are present in a vast number of consumer products and also in multiple nanomedicine skin applications and formulations, more research is needed to determine the properties that confer less toxicity of AgNPs to different cell lines.

Comet Assay on Daphnia magna in eco-genotoxicity testing.

PubMed

Pellegri, Valerio; Gorbi, Gessica; Buschini, Annamaria

2014-10-01

Detection of potentially hazardous compounds in water bodies is a priority in environmental risk assessment. For the evaluation and monitoring of water quality, a series of methodologies may be applied. Among them, the worldwide used toxicity tests with organisms of the genus Daphnia is one of the most powerful. In recent years, some attempts were made to utilize Daphnia magna in genotoxicity testing as many of the new environmental contaminants are described as DNA-damaging agents in aquatic organisms. The aim of this research was to develop a highly standardized protocol of the Comet Assay adapted for D. magna, especially regarding the isolation of cells derived from the same tissue (haemolymph) from newborn organisms exposed in vivo. Several methods for haemolymph extraction and different Comet Assay parameters were compared. Electrophoretic conditions were adapted in order to obtain minimum DNA migration in cells derived from untreated organisms and, at the same time, maximum sensitivity in specimens treated with known genotoxicants (CdCl2 and H2O2). Additional tests were performed to investigate if life-history traits of the cladoceran (such as the age of adult organisms that provide newborns, the clutch size of origin, the number of generations reared in standard conditions) and the water composition as well, might influence the response of the assay. This study confirms the potential application of the Comet Assay in D. magna for assessing genotoxic loads in aqueous solution. The newly developed protocol could integrate the acute toxicity bioassay, thus expanding the possibility of using this model species in freshwater monitoring (waters, sediment and soil elutriates) and is in line with the spirit of the EU Water Framework Directive in reducing the number of bioassays that involve medium-sized species. Copyright © 2014 Elsevier B.V. All rights reserved.

Genotoxicity of Styrene–Acrylonitrile Trimer in Brain, Liver, and Blood Cells of Weanling F344 Rats

PubMed Central

Hobbs, Cheryl A.; Chhabra, Rajendra S.; Recio, Leslie; Streicker, Michael; Witt, Kristine L.

2012-01-01

Styrene–acrylonitrile Trimer (SAN Trimer), a by-product in production of acrylonitrile styrene plastics, was identified at a Superfund site in Dover Township, NJ, where childhood cancer incidence rates were elevated for a period of several years. SAN Trimer was therefore tested by the National Toxicology Program in a 2-year perinatal carcinogenicity study in F344/N rats and a bacterial mutagenicity assay; both studies gave negative results. To further characterize its genotoxicity, SAN Trimer was subsequently evaluated in a combined micronucleus (MN)/Comet assay in juvenile male and female F344 rats. SAN Trimer (37.5, 75, 150, or 300 mg/kg/day) was administered by gavage once daily for 4 days. Micronucleated reticulocyte (MN-RET) frequencies in blood were determined by flow cytometry, and DNA damage in blood, liver, and brain cells was assessed using the Comet assay. Highly significant dose-related increases (P < 0.0001) in MN-RET were measured in both male and female rats administered SAN Trimer. The RET population was reduced in high dose male rats, suggesting chemical-related bone marrow toxicity. Results of the Comet assay showed significant, dose-related increases in DNA damage in brain cells of male (P < 0.0074) and female (P < 0.0001) rats; increased levels of DNA damage were also measured in liver cells and leukocytes of treated rats. Chemical-related cytotoxicity was not indicated in any of the tissues examined for DNA damage. The results of this subacute MN/Comet assay indicate induction of significant genetic damage in multiple tissues of weanling F344 male and female rats after oral exposure to SAN Trimer. PMID:22351108

In vitro assessment of the cytotoxic, DNA damaging, and cytogenetic effects of hydroquinone in human peripheral blood lymphocytes.

PubMed

Jurica, Karlo; Karačonji, Irena Brčić; Benković, Vesna; Kopjar, Nevenka

2017-12-20

This study investigated the mechanisms of hydroquinone toxicity and assessed the relationships between its cytotoxic, genotoxic, and cytogenetic effects tested at 8, 140, and 280 μg mL-1 in human peripheral blood lymphocytes exposed for 24 h. The outcomes of the treatments were evaluated using the apoptosis/necrosis assay, the alkaline comet assay, and the cytokinesis-block micronucleus (CBMN) cytome assay. The tested hydroquinone concentrations produced relatively weak cytotoxicity in resting lymphocytes, which mostly died via apoptosis. Hydroquinone's marked genotoxic effects were detected using the alkaline comet assay. Significantly decreased values of all comet parameters compared to controls indicated specific mechanisms of hydroquinone-DNA interactions. Our results suggest that the two higher hydroquinone concentrations possibly led to cross-linking and adduct formation. Increased levels of DNA breakage measured following exposure to the lowest concentration suggested mechanisms related to oxidative stress and inhibition of topoisomerase II. At 8 μg mL-1, hydroquinone did not significantly affect MN formation. At 140 and 280 μg mL-1, it completely blocked lymphocyte division. The two latter concentrations also led to erythrocyte stabilization and prevented their lysis. At least two facts contribute to this study's relevance: (I) this is the first study that quantifies the degree of reduction in total comet area measured in lymphocyte DNA after hydroquinone treatment, (II) it is also the first one on a lymphocyte model that adopted the "cytome" protocol in an MN assay and found that lymphocytes exposure even to low hydroquinone concentration resulted in a significant increase of nuclear bud frequency. Considering the limitations of the lymphocyte model, which does not possess intrinsic metabolic activation, in order to unequivocally prove the obtained results further studies using other appropriate cell lines are advised.

Evaluation of DNA damage in flight personnel by Comet assay.

PubMed

Cavallo, Delia; Tomao, Paola; Marinaccio, Alessandro; Perniconi, Barbara; Setini, Andrea; Palmi, Silvana; Iavicoli, Sergio

2002-04-26

There have been some suggestions that air-crew are at a higher-than-normal risk of developing cancer, since they are exposed to potential genotoxic factors. These include cosmic radiations, airborne pollutants such as the combustion products of jet propulsion, ozone, and electromagnetic fields. We used the Comet assay to investigate DNA damage in flight personnel with the aim of assessing potential health hazards in this occupational category. We studied 40 civil air-crew members who had been flying long-haul routes for at least 5 years, and compared them with a homogeneous control group of 40 healthy male ground staff. The Comet assay, or single-cell gel electrophoresis (SCGE), detects DNA single- and double-strand breaks (DSBs) and alkali-labile lesions in individual cells, and is a powerful and sensitive technique for detecting genetic damage induced by different genotoxic agents. Taking into consideration occupational risk and possible confounding factors, this assay showed a small increase, that did not reach statistical significance, of DNA damage in long-haul crew members compared to controls, indicating a lack of evident genotoxic effects. An association, although again not statistically significant, was found between reduced DNA damage and use of protective drugs (antioxidants).

Analysis of the Fc Gamma Receptor-Dependent Component of Neutralization Measured by Anthrax Toxin Neutralization Assays▿

PubMed Central

Verma, Anita; Ngundi, Miriam M.; Meade, Bruce D.; De Pascalis, Roberto; Elkins, Karen L.; Burns, Drusilla L.

2009-01-01

Anthrax toxin neutralization assays are used to measure functional antibody levels elicited by anthrax vaccines in both preclinical and clinical studies. In this study, we investigated the magnitude and molecular nature of Fc gamma (Fcγ) receptor-dependent toxin neutralization observed in commonly used forms of the anthrax toxin neutralization assay. Significantly more Fcγ receptor-dependent neutralization was observed in the J774A.1 cell-based assay than in the RAW 264.7 cell-based assay, a finding that could be due to the larger numbers of Fcγ receptors that we found on J774A.1 cells by using flow cytometry. Thus, the extent to which Fcγ receptor-dependent neutralization contributes to the total neutralization measured by the assay depends on the specific cell type utilized in the assay. Using Fcγ receptor blocking monoclonal antibodies, we found that at least three murine Fcγ receptor classes, IIB, III, and IV, can contribute to Fcγ receptor-dependent neutralization. When antibodies elicited by immunization of rabbits with protective-antigen-based anthrax vaccines were analyzed, we found that the magnitude of Fcγ receptor-dependent neutralization observed in the J774A.1 cell-based assay was dependent on the concentration of protective antigen utilized in the assay. Our results suggest that the characteristics of the antibodies analyzed in the assay (e.g., species of origin, isotype, and subclass), as well as the assay design (e.g., cell type and protective antigen concentration), could significantly influence the extent to which Fcγ receptor-dependent neutralization contributes to the total neutralization measured by anthrax toxin neutralization assays. These findings should be considered when interpreting anthrax toxin neutralization assay output. PMID:19656993

Early Activity of Cometary Species from ROSINA/DFMS at 67P/ Churyumov-Gerasimenko

NASA Astrophysics Data System (ADS)

Hässig, Myrtha; Fuselier, Stephen A.; Altwegg, Kathrin; Balsiger, Hans; Berthelier, Jean-Jacques; Bieler, André; Calmonte, Ursina; Dhooghe, Frederik; Fiethe, Björn; Gasc, Sébastien; Gombosi, Tamas I.; Jäckel, Annette; Korth, Axel; Le Roy, Léna; Rème, Henri; Rubin, Martin; Tzou, Chia-Yu; Wurz, Peter

2014-11-01

The European Space Agency’s Rosetta spacecraft arrived after a journey of more than 10 years at comet 67P/Churyumov-Gerasimenko. ROSINA is an instrument package on board Rosetta. It consists of two mass spectrometers and a COmetary Pressure Sensor (COPS). The two mass spectrometers, the Double Focusing Mass Spectrometer (DFMS) and the Reflectron Time of Flight (RTOF) complement each other with high mass resolution (e.g to resolve 13C from CH), high dynamic range (to detect low abundant isotopes and species), high mass range (to detect organics), and high time resolution. ROSINA is designed to measure the neutral gas and plasma composition in the coma of 67P/Churyumov-Gerasimenko in addition to the physical properties of the neutral component of the coma. For the first time, a comet can be observed in situ from its early activity towards and after perihelion. Little is known about what drives initial cometary activity very far from the Sun. Remote sensing observations to date are highly constrained to a limited number of a few bright comets (e.g. Hale-Bopp) and a limited number of species. Rosetta provides the first measurements of the early activity of a comet in situ and detected the first cometary molecules early August. We will focus on early activity of cometary species from the high resolution mass spectrometer ROSINA/DFMS.

Detection of argon in the coma of comet 67P/Churyumov-Gerasimenko.

PubMed

Balsiger, Hans; Altwegg, Kathrin; Bar-Nun, Akiva; Berthelier, Jean-Jacques; Bieler, Andre; Bochsler, Peter; Briois, Christelle; Calmonte, Ursina; Combi, Michael; De Keyser, Johan; Eberhardt, Peter; Fiethe, Björn; Fuselier, Stephen A; Gasc, Sébastien; Gombosi, Tamas I; Hansen, Kenneth C; Hässig, Myrtha; Jäckel, Annette; Kopp, Ernest; Korth, Axel; Le Roy, Lena; Mall, Urs; Marty, Bernard; Mousis, Olivier; Owen, Tobias; Rème, Henri; Rubin, Martin; Sémon, Thierry; Tzou, Chia-Yu; Waite, J Hunter; Wurz, Peter

2015-09-01

Comets have been considered to be representative of icy planetesimals that may have contributed a significant fraction of the volatile inventory of the terrestrial planets. For example, comets must have brought some water to Earth. However, the magnitude of their contribution is still debated. We report the detection of argon and its relation to the water abundance in the Jupiter family comet 67P/Churyumov-Gerasimenko by in situ measurement of the Rosetta Orbiter Spectrometer for Ion and Neutral Analysis (ROSINA) mass spectrometer aboard the Rosetta spacecraft. Despite the very low intensity of the signal, argon is clearly identified by the exact determination of the mass of the isotope (36)Ar and by the (36)Ar/(38)Ar ratio. Because of time variability and spatial heterogeneity of the coma, only a range of the relative abundance of argon to water can be given. Nevertheless, this range confirms that comets of the type 67P/Churyumov-Gerasimenko cannot be the major source of Earth's major volatiles.

Detection of argon in the coma of comet 67P/Churyumov-Gerasimenko

PubMed Central

Balsiger, Hans; Altwegg, Kathrin; Bar-Nun, Akiva; Berthelier, Jean-Jacques; Bieler, Andre; Bochsler, Peter; Briois, Christelle; Calmonte, Ursina; Combi, Michael; De Keyser, Johan; Eberhardt, Peter; Fiethe, Björn; Fuselier, Stephen A.; Gasc, Sébastien; Gombosi, Tamas I.; Hansen, Kenneth C.; Hässig, Myrtha; Jäckel, Annette; Kopp, Ernest; Korth, Axel; Le Roy, Lena; Mall, Urs; Marty, Bernard; Mousis, Olivier; Owen, Tobias; Rème, Henri; Rubin, Martin; Sémon, Thierry; Tzou, Chia-Yu; Waite, J. Hunter; Wurz, Peter

2015-01-01

Comets have been considered to be representative of icy planetesimals that may have contributed a significant fraction of the volatile inventory of the terrestrial planets. For example, comets must have brought some water to Earth. However, the magnitude of their contribution is still debated. We report the detection of argon and its relation to the water abundance in the Jupiter family comet 67P/Churyumov-Gerasimenko by in situ measurement of the Rosetta Orbiter Spectrometer for Ion and Neutral Analysis (ROSINA) mass spectrometer aboard the Rosetta spacecraft. Despite the very low intensity of the signal, argon is clearly identified by the exact determination of the mass of the isotope 36Ar and by the 36Ar/38Ar ratio. Because of time variability and spatial heterogeneity of the coma, only a range of the relative abundance of argon to water can be given. Nevertheless, this range confirms that comets of the type 67P/Churyumov-Gerasimenko cannot be the major source of Earth’s major volatiles. PMID:26601264

Protective Effect of Boric Acid on Oxidative DNA Damage In Chinese Hamster Lung Fibroblast V79 Cell Lines

PubMed Central

Yılmaz, Sezen; Ustundag, Aylin; Cemiloglu Ulker, Ozge; Duydu, Yalcın

2016-01-01

Objective Many studies have been published on the antioxidative effects of boric acid (BA) and sodium borates in in vitro studies. However, the boron (B) concentrations tested in these in vitro studies have not been selected by taking into account the realistic blood B concentrations in humans due to the lack of comprehensive epidemiological studies. The recently published epidemiological studies on B exposure conducted in China and Turkey provided blood B concentrations for both humans in daily life and workers under extreme exposure conditions in occupational setting. The results of these studies have made it possible to test antioxidative effects of BA in in vitro studies within the concentra- tion range relevant to humans. The aim of this study was to investigate the protective ef- fects of BA against oxidative DNA damage in V79 (Chinese hamster lung fibroblast) cells. The concentrations of BA tested for its protective effect was selected by taking the blood B concentrations into account reported in previously published epidemiological studies. Therefore, the concentrations of BA tested in this study represent the exposure levels for humans in both daily life and occupational settings. Materials and Methods In this experimental study, comet assay and neutral red uptake (NRU) assay methods were used to determinacy to toxicity and genotoxicity of BA and hydrogen peroxide (H2O2). Results The results of the NRU assay showed that BA was not cytotoxic within the tested concentrations (3, 10, 30, 100 and 200 µM). These non-cytotoxic concentrations were used for comet assay. BA pre-treatment significantly reduced (P<0.05, one-way ANOVA) the DNA damaging capacity of H2O2 at each tested BA concentrations in V79 cells. Conclusion Consequently, pre-incubation of V79 cells with BA has significantly reduced the H2O2-induced oxidative DNA damage in V79 cells. The protective effect of BA against oxidative DNA damage in V79 cells at 5, 10, 50, 100 and 200 μM (54, 108, 540, 1080, and 2161 ng/ml B equivalents) concentrations was proved in this in vitro study. PMID:26862534

Protective Effect of Boric Acid on Oxidative DNA Damage In Chinese Hamster Lung Fibroblast V79 Cell Lines.

PubMed

Yılmaz, Sezen; Ustundag, Aylin; Cemiloglu Ulker, Ozge; Duydu, Yalcın

2016-01-01

Many studies have been published on the antioxidative effects of boric acid (BA) and sodium borates in in vitro studies. However, the boron (B) concentrations tested in these in vitro studies have not been selected by taking into account the realistic blood B concentrations in humans due to the lack of comprehensive epidemiological studies. The recently published epidemiological studies on B exposure conducted in China and Turkey provided blood B concentrations for both humans in daily life and workers under extreme exposure conditions in occupational setting. The results of these studies have made it possible to test antioxidative effects of BA in in vitro studies within the concentra- tion range relevant to humans. The aim of this study was to investigate the protective ef- fects of BA against oxidative DNA damage in V79 (Chinese hamster lung fibroblast) cells. The concentrations of BA tested for its protective effect was selected by taking the blood B concentrations into account reported in previously published epidemiological studies. Therefore, the concentrations of BA tested in this study represent the exposure levels for humans in both daily life and occupational settings. In this experimental study, comet assay and neutral red uptake (NRU) assay methods were used to determinacy to toxicity and genotoxicity of BA and hydrogen peroxide (H2O2). The results of the NRU assay showed that BA was not cytotoxic within the tested concentrations (3, 10, 30, 100 and 200 µM). These non-cytotoxic concentrations were used for comet assay. BA pre-treatment significantly reduced (P<0.05, one-way ANOVA) the DNA damaging capacity of H2O2 at each tested BA concentrations in V79 cells. Consequently, pre-incubation of V79 cells with BA has significantly reduced the H2O2-induced oxidative DNA damage in V79 cells. The protective effect of BA against oxidative DNA damage in V79 cells at 5, 10, 50, 100 and 200 μM (54, 108, 540, 1080, and 2161 ng/ml B equivalents) concentrations was proved in this in vitro study.

Model-Observation Comparisons of Electron Number Densities in the Coma of 67P/Churyumov-Gerasimenko during January 2015

NASA Astrophysics Data System (ADS)

Vigren, E.; Altwegg, K.; Edberg, N. J. T.; Eriksson, A. I.; Galand, M.; Henri, P.; Johansson, F.; Odelstad, E.; Tzou, C.-Y.; Valliéres, X.

2016-09-01

During 2015 January 9-11, at a heliocentric distance of ˜2.58-2.57 au, the ESA Rosetta spacecraft resided at a cometocentric distance of ˜28 km from the nucleus of comet 67P/Churyumov-Gerasimenko, sweeping the terminator at northern latitudes of 43°N-58°N. Measurements by the Rosetta Orbiter Spectrometer for Ion and Neutral Analysis/Comet Pressure Sensor (ROSINA/COPS) provided neutral number densities. We have computed modeled electron number densities using the neutral number densities as input into a Field Free Chemistry Free model, assuming H2O dominance and ion-electron pair formation by photoionization only. A good agreement (typically within 25%) is found between the modeled electron number densities and those observed from measurements by the Mutual Impedance Probe (RPC/MIP) and the Langmuir Probe (RPC/LAP), both being subsystems of the Rosetta Plasma Consortium. This indicates that ions along the nucleus-spacecraft line were strongly coupled to the neutrals, moving radially outward with about the same speed. Such a statement, we propose, can be further tested by observations of H3O+/H2O+ number density ratios and associated comparisons with model results.

Dicholesteroyl diselenide: cytotoxicity, genotoxicity and mutagenicity in the yeast Saccharomyces cerevisiae and in Chinese hamster lung fibroblasts.

PubMed

de Oliveira, Iuri Marques; Degrandi, Tiago Hoerbe; Jorge, Patrícia Mendes; Saffi, Jenifer; Rosa, Renato Moreira; Guecheva, Temenouga Nikolova; Henriques, João Antonio Pêgas

2014-03-15

The organoselenium compound, dicholesteroyl diselenide (DCDS) is a structural analogue of diphenyl diselenide (DPDS) and may be considered as a promising antioxidant drug in vivo. Nevertheless, little is known about the toxicological properties of DCDS. In the present study we evaluated the cytotoxic, genotoxic and mutagenic properties of DCDS in Chinese hamster lung fibroblasts (V79) and in strains of the yeast Saccharomyces cerevisiae, proficient and deficient in several DNA-repair pathways. The results with V79 cells show that DCDS induced cytotoxicity, GSH depletion and elevation of lipid peroxidation at lower concentrations than did DPDS. DCDS also generated single- and double-strand DNA breaks in V79 cells, both in the presence and in the absence of metabolic activation, as revealed by alkaline and neutral comet assays. Moreover, the induction of oxidative DNA base-damage was demonstrated by means of a modified comet assay with formamidopyrimidine-DNA glycosylase and endonuclease III. Treatment with DCDS also induced micronucleus formation in V79 cells as well as point and frame-shift mutations in a haploid wild-type strain of S. cerevisiae. Yeast mutants defective in base excision-repair proteins were the most sensitive to DCDS. Pre-incubation with N-acetylcysteine reduced DCDS's oxidative, genotoxic and mutagenic effects in yeast and in V79 cells. Our findings indicate that the presence of cholesteroyl substituents in DCDS results in elevation of its cytotoxic and genotoxic potential compared with that of DPDS in yeast and in V79 cells. However, due to dose-dependent contrasting behaviour of organoselenium compounds and differences in their toxicity in in vitro and in vivo systems, further studies are needed in order to establish the non-toxic concentration range for treatment in mammals. Copyright © 2014 Elsevier B.V. All rights reserved.

Environmental exposure to human carcinogens in teenagers and the association with DNA damage.

PubMed

Franken, Carmen; Koppen, Gudrun; Lambrechts, Nathalie; Govarts, Eva; Bruckers, Liesbeth; Den Hond, Elly; Loots, Ilse; Nelen, Vera; Sioen, Isabelle; Nawrot, Tim S; Baeyens, Willy; Van Larebeke, Nicolas; Boonen, Francis; Ooms, Daniëlla; Wevers, Mai; Jacobs, Griet; Covaci, Adrian; Schettgen, Thomas; Schoeters, Greet

2017-01-01

We investigated whether human environmental exposure to chemicals that are labeled as (potential) carcinogens leads to increased (oxidative) damage to DNA in adolescents. Six hundred 14-15-year-old youngsters were recruited all over Flanders (Belgium) and in two areas with important industrial activities. DNA damage was assessed by alkaline and formamidopyrimidine DNA glycosylase (Fpg) modified comet assays in peripheral blood cells and analysis of urinary 8-hydroxydeoxyguanosine (8-OHdG) levels. Personal exposure to potentially carcinogenic compounds was measured in urine, namely: chromium, cadmium, nickel, 1-hydroxypyrene as a proxy for exposure to other carcinogenic polycyclic aromatic hydrocarbons (PAHs), t,t-muconic acid as a metabolite of benzene, 2,5-dichlorophenol (2,5-DCP), organophosphate pesticide metabolites, and di(2-ethylhexyl) phthalate (DEHP) metabolites. In blood, arsenic, polychlorinated biphenyl (PCB) congeners 118 and 156, hexachlorobenzene (HCB), dichlorodiphenyltrichloroethane (DDT) and perfluorooctanoic acid (PFOA) were analyzed. Levels of methylmercury (MeHg) were measured in hair. Multiple linear regression models were used to establish exposure-response relationships. Biomarkers of exposure to PAHs and urinary chromium were associated with higher levels of both 8-OHdG in urine and DNA damage detected by the alkaline comet assay. Concentrations of 8-OHdG in urine increased in relation with increasing concentrations of urinary t,t-muconic acid, cadmium, nickel, 2,5-DCP, and DEHP metabolites. Increased concentrations of PFOA in blood were associated with higher levels of DNA damage measured by the alkaline comet assay, whereas DDT was associated in the same direction with the Fpg-modified comet assay. Inverse associations were observed between blood arsenic, hair MeHg, PCB 156 and HCB, and urinary 8-OHdG. The latter exposure biomarkers were also associated with higher fish intake. Urinary nickel and t,t-muconic acid were inversely associated with the alkaline comet assay. This cross-sectional study found associations between current environmental exposure to (potential) human carcinogens in 14-15-year-old Flemish adolescents and short-term (oxidative) damage to DNA. Prospective follow-up will be required to investigate whether long-term effects may occur due to complex environmental exposures. Copyright © 2016 Elsevier Inc. All rights reserved.

Genotoxicity induced by metal oxide nanoparticles: a weight of evidence study and effect of particle surface and electronic properties.

PubMed

Golbamaki, Azadi; Golbamaki, Nazanin; Sizochenko, Natalia; Rasulev, Bakhtiyor; Leszczynski, Jerzy; Benfenati, Emilio

2018-06-09

The genetic toxicology of nanomaterials is a crucial toxicology issue and one of the least investigated topics. Substantially, the genotoxicity of metal oxide nanomaterials' data is resulting from in vitro comet assay. Current contributions to the genotoxicity data assessed by the comet assay provide a case-by-case evaluation of different types of metal oxides. The existing inconsistency in the literature regarding the genotoxicity testing data requires intelligent assessment strategies, such as weight of evidence evaluation. Two main tasks were performed in the present study. First, the genotoxicity data from comet assay for 16 noncoated metal oxide nanomaterials with different core composition were collected. An evaluation criterion was applied to establish which of these individual lines of evidence were of sufficient quality and what weight could have been given to them in inferring genotoxic results. The collected data were surveyed on (1) minimum necessary characterization points for nanomaterials and (2) principals of correct comet assay testing for nanomaterials. Second, in this study the genotoxicity effect of metal oxide nanomaterials was investigated by quantitative nanostructure-activity relationship approach. A set of quantum-chemical descriptors was developed for all investigated metal oxide nanomaterials. A classification model based on decision tree was developed for the investigated dataset. Thus, three descriptors were identified as the most responsible factors for genotoxicity effect: heat of formation, molecular weight, and surface area of the oxide cluster based on the conductor-like screening model. Conclusively, the proposed genotoxicity assessment strategy is useful to prioritize the study of the nanomaterials for further risk assessment evaluations.

Evaluation of genotoxicity of coal fly ash in Allium cepa root cells by combining comet assay with the Allium test.

PubMed

Chakraborty, Rajarshi; Mukherjee, Ashit Kumar; Mukherjee, Anita

2009-06-01

Fly ash is a by-product of coal-fired electricity generation plants. Its utilization and disposal is of utmost importance. Using onion (Allium cepa) root tip system, the present study was carried out to evaluate the potential toxic and genotoxic effects of fly ash, collected from a thermal power plant in West Bengal, India. Prior to testing, the collected fly ash sample was mixed with sand in different proportions. Allium bulbs were allowed to germinate directly in fly ash and after five days the germinating roots were processed for the Allium test. Additionally, the Allium test was adapted for detecting DNA damage through comet assay. The results from the Allium test indicate that fly ash at 100% concentration inhibits root growth and mitotic indices; induces binucleated cells as a function of the proportion, but is not toxic at very low concentration. In the comet assay, a statistical increase for DNA strand breaks was found only at higher concentrations. The sample was analyzed by flame atomic absorption spectrometer for Zn, Pb, Cu, Ni, Cd and As, whose presence could partly be responsible for the toxicity of fly ash. The study concludes that the classical Allium test can give a more comprehensive data when done in combination with the comet assay, which is faster, simpler and independent of mitosis. Also when fly ash is used for other purposes in combination with soils, it should be judiciously used at very low concentrations in order to protect the ecosystem health from any potential adverse effects.

Novel Strategies for the Treatment of Estrogen Receptor-Negative Breast Cancer

DTIC Science & Technology

2007-10-01

quercetin on benzo[a]pyrene induced DNA damage in HepG2 cells as measured by the comet assay” at the Annual Intermountain Meeting of the American Society...Anti-genotoxic effects of Garlic extract and Quercetin as Measured by the Comet Assay, The Journal of the Intermountain Branch of the American

Genotoxicity evaluation of carvacrol in rats using a combined micronucleus and comet assay.

PubMed

Llana-Ruiz-Cabello, María; Maisanaba, Sara; Puerto, María; Prieto, Ana I; Pichardo, Silvia; Moyano, Rosario; González-Pérez, José A; Cameán, Ana M

2016-12-01

Genotoxic data of substances which could be incorporated into food packaging are required by the European Food Safety Authority. Due to its antioxidant and antibacterial properties carvacrol is one of these compounds. This work aims to study for the first time the in vivo genotoxic effects produced in rats orally exposed to 81, 256 or 810 mg cavacrol/kg body weight (bw) at 0, 24 and 45 h. A combination of the micronucleus assay (OECD 474) in bone marrow and the standard (OECD 489) and enzyme-modified comet assay was used to determine the genotoxicity on cells isolated from stomach and liver of exposed animals. In addition, a histopathological study was performed on the assayed tissues, and also in the lungs due to the volatility of carvacrol. Direct analytical pyrolysis was used to search for carvacrol in viscera and to ensure that the compound reaches stomach and liver cells. Results from MN-comet assay revealed that carvacrol (81-810 mg/kg bw) did not induce in vivo genotoxicity or oxidative DNA damage in any of the tissues investigated. Moreover, no histopathological changes were observed. Altogether, these results suggest lack of genotoxicity of carvacrol and therefore its good profile for its potential application as food preservative. Copyright © 2016 Elsevier Ltd. All rights reserved.

Evaluation of Genotoxic Pressure along the Sava River

PubMed Central

Kračun-Kolarević, Margareta; Kostić, Jovana; Simonović, Predrag; Simić, Vladica; Milošković, Aleksandra; Reischer, Georg; Farnleitner, Andreas; Gačić, Zoran; Milačič, Radmila; Zuliani, Tea; Vidmar, Janja; Pergal, Marija; Piria, Marina; Paunović, Momir; Vuković-Gačić, Branka

2016-01-01

In this study we have performed a comprehensive genotoxicological survey along the 900 rkm of the Sava River. In total, 12 sites were chosen in compliance with the goals of GLOBAQUA project dealing with the effects of multiple stressors on biodiversity and functioning of aquatic ecosystems. The genotoxic potential was assessed using a complex battery of bioassays performed in prokaryotes and aquatic eukaryotes (freshwater fish). Battery comprised evaluation of mutagenicity by SOS/umuC test in Salmonella typhimurium TA1535/pSK1002. The level of DNA damage as a biomarker of exposure (comet assay) and biomarker of effect (micronucleus assay) and the level of oxidative stress as well (Fpg—modified comet assay) was studied in blood cells of bleak and spirlin (Alburnus alburnus/Alburnoides bipunctatus respectively). Result indicated differential sensitivity of applied bioassays in detection of genotoxic pressure. The standard and Fpg—modified comet assay showed higher potential in differentiation of the sites based on genotoxic potential in comparison with micronucleus assay and SOS/umuC test. Our data represent snapshot of the current status of the river which indicates the presence of genotoxic potential along the river which can be traced to the deterioration of quality of the Sava River by communal and industrial wastewaters. The major highlight of the study is that we have provided complex set of data obtained from a single source (homogeneity of analyses for all samples). PMID:27631093

Genotoxicity assessment of membrane concentrates of landfill leachate treated with Fenton reagent and UV-Fenton reagent using human hepatoma cell line.

PubMed

Wang, Guifang; Lu, Gang; Yin, Pinghe; Zhao, Ling; Yu, Qiming Jimmy

2016-04-15

Membrane concentrates of landfill leachates contain organic and inorganic contaminants that could be highly toxic and carcinogenic. In this paper, the genotoxicity of membrane concentrates before and after Fenton and UV-Fenton reagent was assessed. The cytotoxicity and genotoxicity was determined by using the methods of methyltetrazolium (MTT), cytokinesis-block micronucleus (CBMN) and comet assay in human hepatoma cells. MTT assay showed a cytotoxicity of 75% after 24h of exposure to the highest tested concentration of untreated concentrates, and no cytotoxocity for UV-Fenton and Fenton treated concentrates. Both CBMN and comet assays showed increased levels of genotoxicity in cells exposed to untreated concentrates, compared to those occurred in cells exposed to UV-Fenton and Fenton reagent treated concentrates. There was no significant difference between negative control and UV-Fenton treated concentrates for micronucleus and comet assay parameters. UV-Fenton and Fenton treatment, especially the former, were effective methods for degradation of bisphenol A and nonylphenol in concentrates. These findings showed UV-Fenton and Fenton reaction were effective methods for treatment of such complex concentrates, UV-Fenton reagent provided toxicological safety of the treated effluent, and the genotoxicity assays were found to be feasible tools for assessment of toxicity risks of complex concentrates. Copyright © 2016 Elsevier B.V. All rights reserved.

Genotoxicity Assessment of Volatile Organic Compounds in Landfill Gas Emission Using Comet Assay in Higher Terrestrial Plant.

PubMed

Na Roi-Et, Veerapas; Chiemchaisri, Wilai; Chiemchaisri, Chart

2017-02-01

Genotoxicity model is developed to assess the individual subacute toxicity of benzene, toluene, ethylbenzene, and xylene (BTEX) at very low levels as in a landfill gas. Golden Pothos (Epipremnum aureum), a higher plant, was tested under variation of benzene 54-5656 ng/L, toluene 10-4362 ng/L, ethylbenzene 28-4997 ng/L, xylene 53-4845 ng/L, for 96 h. DNA fragmentation in plant leaves were investigated via comet assay. The results show that DNA migration ratio increased with the BTEX concentrations, but at different rates. The 50% effective concentration (EC 50 ) of DNA fragmentation from the dose-response relationships indicated toluene has the highest EC 50 value and followed by benzene, xylene and ethylbenzene. Alternatively, ethylbenzene has the highest toxicity unit and followed by xylene, benzene and toluene as described by toxicity unit (TU). In conclusion, comet assay of Pothos can be used in differentiating DNA fragmentation against very low levels of BTEX in the atmosphere. Pothos is recommended for genotoxicity assessment of a low BTEX contaminated atmosphere.

Investigation of sodium arsenite, thioacetamide, and diethanolamine in the alkaline comet assay: Part of the JaCVAM comet validation exercise.

PubMed

Beevers, Carol; Henderson, Debbie; Lillford, Lucinda

2015-07-01

As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of the in vivo rat alkaline comet assay (comet assay), we examined sodium arsenite, thioacetamide, and diethanolamine. Using the JaCVAM approved study protocol version 14.2, each chemical was tested in male rats up to maximum tolerated dose levels and DNA damage in the liver and stomach was assessed approximately 3h after the final administration by gavage. Histopathology assessments of liver and stomach sections from the same animals were also examined for evidence of cytotoxicity or necrosis. No evidence of DNA damage was observed in the stomach of animals treated with sodium arsenite at 7.5, 15, or 30 mg/kg/day. However, equivocal findings were found in the liver, where increases in DNA migration were observed in two independent experiments, but not in all treated animals and not at the same dose levels. Thioacetamide caused an increase in DNA migration in the stomach of rats treated at 19, 38, and 75 mg/kg/day, but not in the liver, despite evidence of marked hepatotoxicity following histopathology assessments. No evidence of DNA damage was observed in the stomach or liver of animals treated with diethanolamine at 175, 350, or 700 mg/kg/day. Copyright © 2015 Elsevier B.V. All rights reserved.

In vitro antioxidation activity and genoprotective effect of selected Chinese medicinal herbs.

PubMed

Szeto, Yim Tong; Wong, Shirley Ching Yee; Wong, Julia Wai Ming; Kalle, Wouter; Pak, Sok Cheon

2011-01-01

Some traditional Chinese medicinal seeds and fruits are well known for their antioxidant properties. This research aims to investigate whether Fructus Lycii, Fructus Schisandrae Chinensis, Fructus Ligustri Lucidi and Semen Cuscutae protect DNA from oxidant challenge by hydrogen peroxide (H(2)O(2)). The standard comet assay was used to assess the genoprotective effect of these medicinal herbs. Blood was taken from three healthy adults, aged from 36 to 42. Lymphocytes were isolated and treated with different concentrations of aqueous herbal extracts, while controls were treated with phosphate buffered saline. The lymphocytes were stressed with 50 μM H(2)O(2). Treated cells were embedded in agarose and layered on slides. These sandwiched lymphocytes were lysed and afterwards subjected to an electric field in an alkaline environment. Damaged DNA was pulled out from the nucleus towards the positive electrode as a comet tail; its density was related to the degree of DNA damage. Finally, the slides were stained with fluorescence dye and tails were visually scored for 100 cells. The experiment was repeated three times and DNA damage in treated cells was compared to the controls. There was no statistical difference in DNA damage among the herb treated cells and untreated cells in the comet assay. Our data demonstrated that the selected medicinal herbs did not show in vitro DNA protection in the comet assay against oxidant challenge.

Impact of host cell variation on the neutralization of HIV-1 in vitro.

PubMed

Polonis, Victoria R; Schuitemaker, Hanneke; Bunnik, Evelien M; Brown, Bruce K; Scarlatti, Gabriella

2009-09-01

In this review we present current advances in our understanding of HIV-1 neutralization assays that employ primary cell types, as compared with those that utilize cell lines and the newer, more standardized pseudovirus assays. A commentary on the challenges of standardizing in-vitro neutralization assays using primary cells is included. The data from reporter cell line neutralization assays may agree with results observed in primary cells; however, exceptions have recently been reported. Multiple variables exist in primary cell assays using peripheral blood mononuclear cells from HIV-seronegative donors; in-vitro neutralization titers can vary significantly based on the donor cells used for assay targets and for virus propagation. Thus, more research is required to achieve validated primary cell neutralization assays. HIV-vaccine-induced antibody performance in the current neutralization assays may function as a 'gatekeeper' for HIV-1 subunit vaccine advancement. Development of standardized platforms for reproducible measurement of in-vitro neutralization is therefore a high priority. Given the considerable variation in results obtained from some widely applied HIV neutralization platforms, parallel evaluation of new antibodies using different host cells for assay targets, as well as virus propagation, is recommended until immune correlates of protection are identified.

Swift Gamma Ray Observatory Observations Of The Comet 73P/Schwassmann-Wachmann 3

NASA Astrophysics Data System (ADS)

Brown, Gregory V.; Beiersdorfer, P.; Bodewits, D.; Porter, F.; Willingale, R.

2007-05-01

The XRT on the Swift Gamma Ray Observatory has been used to observe fragment C of the comet 73P/Schwassmann-Wachmann 3 on 19 different days over the course of May and June of 2006. During these observations, comet 73P/SW3C was near perihelion and passed within 0.1 AU of the Earth. The XRT spectra show distinct line emission from helium-like and hydrogenic oxygen. This line emission is caused by charge exchange recombination between solar wind ions and cometary neutrals. Our observations also include monitoring of the comet with Swift's UV/Optical Telescope. An overview of our observation, the XRT spectra, and the current status of our data analysis will be presented. Work at LLNL was completed under the auspices of the U.S. D.o.E by the University of California Lawrence Livermore National Laboratory under contract W-7405-Eng-48.

Respiratory Syncytial Virus (RSV): Neutralizing Antibody, a Correlate of Immune Protection.

PubMed

Piedra, Pedro A; Hause, Anne M; Aideyan, Letisha

2016-01-01

Assays that measure RSV-specific neutralizing antibody activity are very useful for evaluating vaccine candidates, performing seroprevalence studies, and detecting infection. Neutralizing antibody activity is normally measured by a plaque reduction neutralization assay or by a microneutralization assay with or without complement. These assays measure the functional capacity of serum (or other fluids) to neutralize virus infectivity in cells as compared to ELISA assays that only measure the binding capacity against an antigen. This chapter discusses important elements in standardization of the RSV-specific microneutralization assay for use in the laboratory.

Interaction of Comets and the Solar Wind

NASA Technical Reports Server (NTRS)

Wagner, William (Technical Monitor); Raymond, John C.

2003-01-01

We had originally planned to analyze UVCS observations of Comet Machholz, but we obtained higher quality observations of Comet Kudo-Fujikawa in January 2003 at its 0.19 AU perihelion. Besides a large and rapidly increasing water outgassing rate, we detected a bright tail in doubly ionized carbon. The amount of carbon was greater than could be accounted for by GO photodissociation, and we attribute the carbon to evaporation of organics from dust. A spectacular disconnection event was apparent in the C III tail, and it coincides within the uncertainties with the position of the heliospheric current sheet. A paper is in press in Science, and it will be the subject of a press release. We are also analyzing two sungrazing comets. Comet C/2001 C2 shows evidence for sub-fragments and for a very long lasting source of neutrals, which we tentatively identify as evaporation of pyroxene dust grains. Comet C/2002 S2 shows a sudden 2 magnitude drop in optical brightness and an equally sudden recovery. UVCS observations during that time show a steadily increasing outgassing rate. We have derived solar wind densities for both comets, but we are still sorting out the ambiguities involving the fragmentation and optical behavior. We are collaborating with Philippe Lamy on the LASCO measurements.

Preliminary Inventory in the Early Coma of Comet 67P/Churyumov-Gerasimenko

NASA Astrophysics Data System (ADS)

Calmonte, Ursina; Altwegg, Kathrin; Le Roy, Léna; Rubin, Martin; Berthelier, Jean-Jacques; De Keyser, Johan; Fiethe, Björn; Fuselier, Steve A; Combi, Mike

2014-11-01

After a 10-year journey, the European Space Agency’s Rosetta mission encountered its target comet Churyumov-Gerasimenko. Rosetta will accompany the comet to perihelion and beyond. On board the Rosetta spacecraft is the ROSINA (Rosetta Orbiter Spectrometer for Ion and Neutral Analysis) experiment. ROSINA consists of a pressure sensor and two complementary mass spectrometers. One is the Double Focusing Mass Spectrometer, which has high dynamic range and a mass resolution m/Δm = 3000 at 1% peak height (m/Δm = 9000 at 50% peak height) at mass 28 amu/q. It is therefore well suited to detect minor species in the lower mass range up to mass 140 amu [1].ROSINA has been taking data since May 2014 and first signals of the comet were detected at the beginning of August. We will present a preliminary inventory of species seen by ROSINA in the early coma of comet Churyumov-Gerasimenko.References[1] Balsiger, H., Altwegg, K., Bochsler, P., Eberhardt, P., Fischer, J., Graf, S., Jäckel, A., Kopp, E., Langer, U., Mildner, M., Müller, J., Riesen, T., Rubin, M., Scherer, S., Wurz, P., Wüthrich, S., Arijs, E., Delanoye, S., de Keyser, J., Neefs, E., Nevejans, D., Rème, H., Aoustin, C., Mazelle, C., Médale, J.-L., Sauvaud, J.A., Berthelier, J.-J., Bertaux, J.-L., Duvet, L., Illiano, J.-M., Fuselier, S.A., Ghielmetti, A.G., Magoncelli, T., Shelley, E.G., Korth, A., Heerlein, K., Lauche, H., Livi, S., Loose, A., Mall, U., Wilken, B., Gliem, F., Fiethe, B., Gombosi, T.I., Block, B., Carignan, G.R., Fisk, L.A., Waite, J.H., Young, D.T. and Wollnik, H.,. Rosina Rosetta Orbiter Spectrometer for Ion and Neutral Analysis, Space Science Reviews 128, pp745-801, 2007.

Modeling the Thermodynamic Properties of the Inner Comae of Comets

NASA Astrophysics Data System (ADS)

Boice, Daniel C.

2017-10-01

Introduction: Modeling is central to understand the important properties of the cometary environment. We have developed a comet model, SUISEI, that self-consistently includes the relevant physicochemical processes within a global modeling framework, from the porous subsurface layers of the nucleus to the interaction with the solar wind. Our goal is to gain valuable insights into the intrinsic properties of cometary nuclei so we can better understand observations and in situ measurements. SUISEI includes a multifluid, reactive gas dynamics simulation of the dusty coma (ComChem) and a suite of other coupled numerical simulations. This model has been successfully applied to a variety of comets in previous studies over the past three decades. We present results from a quantitative study of the thermodynamic properties and chemistry of cometary comae as a function of cometocentric and heliocentric distance to aid in interpretation of observations and in situ measurements of comets.Results and Discussion: ComChem solves the fluid dynamic equations for the mass, momentum, and energy of three neutral fluids (H, H2, and the heavier bulk fluid), ions, and electrons. In the inner coma, the gas expands, cools, accelerates, and undergoes many photolytic and gas-phase chemical reactions tracking hundreds of sibling species. The code handles the transition to free molecular flow and describes the spatial distribution of species in the coma of a comet. Variations of neutral gas temperature and velocity with cometocentric distance and heliocentric distance for a comet approaching the Sun from 2.5 to 0.3 AU are presented. Large increases in the gas temperatures (>400 K) due to photolytic heating in the coma within ~0.5 AU are noted, with dramatic effects on the chemistry, optical depth, and other coma properties. Results are compared to observations when available.Conclusions: SUISEI has proven to be a unique and valuable model to understand the relevant physical processes and properties of small Solar System bodies, including near-Sun comets and asteroids.Acknowledgments: This work was supported by FAPESP under Grant No. 2015/03176-8 and the National Science Foundation Planetary Astronomy Program Grant No. 0908529.

Genotoxic effects of styrene-7,8-oxide in human white blood cells: comet assay in relation to the induction of sister-chromatid exchanges and micronuclei.

PubMed

Laffon, B; Pásaro, E; Méndez, J

2001-04-05

Styrene is used in the production of plastics, resins and rubber. The highest human exposures to styrene take place by inhalation during the production of fiberglass reinforced plastics. Styrene is metabolized mainly in the liver to styrene-7,8-oxide (SO), its principal in vivo mutagenic metabolite. In this study, human peripheral white blood cells were exposed to several SO concentrations (10-200 microM) in order to evaluate its genotoxic properties by means of comet assay, sister-chromatid exchanges (SCE) and cytokinesis-blocked micronucleus (MN) test, in addition to determine its clastogenic or aneugenic properties by combining MN with fluorescence in situ hybridization (FISH) procedures. Our results show that SO induces DNA damage, SCE and MN in human leukocytes in vitro at concentrations above 50 microM, and that there is a strong relationship between DNA damage, as measured by the comet assay, and cytogenetic damage induced by SO at the doses employed. SO shows preferentially a clastogenic activity and produces a cytostatic effect at high doses, reflected by the significant decrease of the calculated proliferation indices. A good dose-effect relationship is obtained in the three tests performed at the concentration range assayed.

Laboratory Measurements of Solar-Wind/Comet X-Ray Emission and Charge Exchange Cross Sections

NASA Technical Reports Server (NTRS)

Chutjian, A.; Cadez, I.; Greenwood, J. B.; Mawhorter, R. J.; Smith, S. J.; Lozano, J.

2002-01-01

The detection of X-rays from comets such as Hyakutake, Hale-Bopp, d Arrest, and Linear as they approach the Sun has been unexpected and exciting. This phenomenon, moreover, should be quite general, occurring wherever a fast solar or stellar wind interacts with neutrals in a comet, a planetary atmosphere, or a circumstellar cloud. The process is, O(+8) + H2O --> O(+7*) + H2O(+), where the excited O(+7*) ions are the source of the X-ray emissions. Detailed modeling has been carried out of X-ray emissions in charge-transfer collisions of heavy solar-wind Highly Charged Ions (HCIs) and interstellar/interplanetary neutral clouds. In the interplanetary medium the solar wind ions, including protons, can charge exchange with interstellar H and He. This can give rise to a soft X-ray background that could be correlated with the long-term enhancements seen in the low-energy X-ray spectrum of ROSAT. Approximately 40% of the soft X-ray background detected by Exosat, ROSAT, Chandra, etc. is due to Charge Exchange (CXE): our whole heliosphere is glowing in the soft X-ray due to CXE.

Herschel/SPIRE observations of water production rates and ortho-to-para ratios in comets★

NASA Astrophysics Data System (ADS)

Wilson, Thomas G.; Rawlings, Jonathan M. C.; Swinyard, Bruce M.

2017-04-01

This paper presents Herschel/SPIRE (Spectral and Photometric Imaging Receiver) spectroscopic observations of several fundamental rotational ortho- and para-water transitions seen in three Jupiter-family comets and one Oort-cloud comet. Radiative transfer models that include excitation by collisions with neutrals and electrons, and by solar infrared radiation, were used to produce synthetic emission line profiles originating in the cometary coma. Ortho-to-para ratios (OPRs) were determined and used to derived water production rates for all comets. Comparisons are made with the water production rates derived using an OPR of 3. The OPR of three of the comets in this study is much lower than the statistical equilibrium value of 3; however they agree with observations of comets 1P/Halley and C/2001 A2 (LINEAR), and the protoplanetary disc TW Hydrae. These results provide evidence suggesting that OPR variation is caused by post-sublimation gas-phase nuclear-spin conversion processes. The water production rates of all comets agree with previous work and, in general, decrease with increasing nucleocentric offset. This could be due to a temperature profile, additional water source or OPR variation in the comae, or model inaccuracies.

Systematic random sampling of the comet assay.

PubMed

McArt, Darragh G; Wasson, Gillian R; McKerr, George; Saetzler, Kurt; Reed, Matt; Howard, C Vyvyan

2009-07-01

The comet assay is a technique used to quantify DNA damage and repair at a cellular level. In the assay, cells are embedded in agarose and the cellular content is stripped away leaving only the DNA trapped in an agarose cavity which can then be electrophoresed. The damaged DNA can enter the agarose and migrate while the undamaged DNA cannot and is retained. DNA damage is measured as the proportion of the migratory 'tail' DNA compared to the total DNA in the cell. The fundamental basis of these arbitrary values is obtained in the comet acquisition phase using fluorescence microscopy with a stoichiometric stain in tandem with image analysis software. Current methods deployed in such an acquisition are expected to be both objectively and randomly obtained. In this paper we examine the 'randomness' of the acquisition phase and suggest an alternative method that offers both objective and unbiased comet selection. In order to achieve this, we have adopted a survey sampling approach widely used in stereology, which offers a method of systematic random sampling (SRS). This is desirable as it offers an impartial and reproducible method of comet analysis that can be used both manually or automated. By making use of an unbiased sampling frame and using microscope verniers, we are able to increase the precision of estimates of DNA damage. Results obtained from a multiple-user pooled variation experiment showed that the SRS technique attained a lower variability than that of the traditional approach. The analysis of a single user with repetition experiment showed greater individual variances while not being detrimental to overall averages. This would suggest that the SRS method offers a better reflection of DNA damage for a given slide and also offers better user reproducibility.

Assessment of DNA damage and repair efficiency in drug naïve schizophrenia using comet assay.

PubMed

Muraleedharan, Aparna; Menon, Vikas; Rajkumar, Ravi Philip; Chand, Parkash

2015-09-01

The etiology of schizophrenia continues to be confounding and elusive. Some knowledge gaps exist in the neurodegenerative theory of schizophrenia. Oxidative DNA damage and repair deficits are relevant to the mechanisms of neurodegeneration but have not been studied in drug naïve schizophrenia. The present study used the comet assay technique to study the extent of DNA damage in circulating peripheral lymphocytes of patients with drug naïve schizophrenia (n = 40) along with an age and gender matched control group (n = 40). We also assessed the DNA repair efficiency in cases following incubation in a nutrient medium. All the assayed comet parameters demonstrated significantly greater baseline DNA damage in cases in comparison to the controls except for head diameter (p < 0.001 for all significant results, p = 0.32 for head diameter). Gender, age and duration of illness (p = 0.21, 0.69 and 0.12 respectively for tail length) did not influence any of the parameters significantly. Significant decrease was noted in the comet tail length and percentage of DNA in comet tail (p < 0.001 for both) in cases following incubation suggesting that the DNA repair machinery was preserved. No difference in DNA repair efficiency was noted between the genders (p = 0.23 for tail length). Our findings confirm the presence of significant baseline DNA damage in schizophrenia even prior to the initiation of anti-psychotic treatment. Additionally, intact genomic repair efficiency was noted in this group as a whole. These results provide some evidence for oxidative DNA damage as molecular link underpinning neurodegeneration in drug naïve schizophrenia. Copyright © 2015 Elsevier Ltd. All rights reserved.

[Study on three kinds of gasoline oxygenates-induced DNA damage in mice fibroblasts].

PubMed

Song, Chonglin; Zhang, Zhifu; Chen, Xue; Zhang, Yanfeng; Wang, Chunhua; Liu, Keming

2002-10-01

To study DNA damage of three kinds of gasoline oxygenates. Single cell gel electrophoresis assay(Comet assay) was used to detect the damage effects of three gasoline oxygenates[methyl tertiary butyl ether(MTBE), ethanol anhydrous(EA) and dimethyl carbonate(DMC)] on DNA in L-929 mice fibroblasts. In certain concentation(37.500-150.000 mg/ml), MTBE could directly cause DNA damage of L-929 mice fibroblasts. There was obvious dose-effect relationship, i.e. when the concentration of MTBE was increased from 9.375 to 150.000 mg/ml, the comet rate also increased from 4% to 85%, and the length of comet tail changed correspondingly. The results of EA and DMC were negative. Under the condition of this experiment(150.000 mg/ml), MTBE could directly cause DNA damage while the effect of EA and DMC on DNA damage was not found.

Automated detection of irradiated food with the comet assay.

PubMed

Verbeek, F; Koppen, G; Schaeken, B; Verschaeve, L

2008-01-01

Food irradiation is the process of exposing food to ionising radiation in order to disinfect, sanitise, sterilise and preserve food or to provide insect disinfestation. Irradiated food should be adequately labelled according to international and national guidelines. In many countries, there are furthermore restrictions to the product-specific maximal dose that can be administered. Therefore, there is a need for methods that allow detection of irradiated food, as well as for methods that provide a reliable dose estimate. In recent years, the comet assay was proposed as a simple, rapid and inexpensive method to fulfil these goals, but further research is required to explore the full potential of this method. In this paper we describe the use of an automated image analysing system to measure DNA comets which allow the discrimination between irradiated and non-irradiated food as well as the set-up of standard dose-response curves, and hence a sufficiently accurate dose estimation.

The comet assay in Folsomia candida: A suitable approach to assess genotoxicity in collembolans.

PubMed

Cardoso, Diogo N; Silva, Ana Rita R; Cruz, Andreia; Lourenço, Joana; Neves, Joana; Malheiro, Catarina; Mendo, Sónia; Soares, Amadeu M V M; Loureiro, Susana

2017-09-01

The present study shows the comet assay technique being successfully applied for the first time to one of the most widely used soil organisms in standardized ecotoxicological tests, Folsomia candida, providing a step forward in assessing the genotoxicity induced by xenobiotics. Because collembolans have a high content of chitin, a new methodology was developed in which the heads of the collembolans were separated from the rest of the body, allowing the hemolymph to leak out. This procedure allows the cells to be released, and after lysis the genetic material is available for the comet assay. Among other key procedures, the use of 30 organisms (20- to 22-d-old adults) per replicate and the correct amount of cells with genetic material (translated as 10 μL of suspension) applied on the agarose gel were determinants for the success of the results obtained. The methodology was validated by exposing F. candida to a representative metallic element (cadmium) and a representative of organophosphates, the insecticide dimethoate, for a shorter time period of 10 d, compared with the 28 d for the International Organization for Standardization 11267 method. Within this method, the relatively low percentage of DNA damage (30%) observed in controls and the significant increase in terms of percentage of DNA damage for almost all the concentrations of dimethoate and Cd (reaching 52% and 56% of damage in the highest concentrations, respectively) confirmed the genotoxic effect of both compounds and validated this technique. The comet assay proved to be a sensitive technique to detect DNA strand breaks in collembolans' cells. Environ Toxicol Chem 2017;36:2514-2520. © 2017 SETAC. © 2017 SETAC.

High-throughput screening platform for engineered nanoparticle-mediated genotoxicity using CometChip technology.

PubMed

Watson, Christa; Ge, Jing; Cohen, Joel; Pyrgiotakis, Georgios; Engelward, Bevin P; Demokritou, Philip

2014-03-25

The likelihood of intentional and unintentional engineered nanoparticle (ENP) exposure has dramatically increased due to the use of nanoenabled products. Indeed, ENPs have been incorporated in many useful products and have enhanced our way of life. However, there are many unanswered questions about the consequences of nanoparticle exposures, in particular, with regard to their potential to damage the genome and thus potentially promote cancer. In this study, we present a high-throughput screening assay based upon the recently developed CometChip technology, which enables evaluation of single-stranded DNA breaks, abasic sites, and alkali-sensitive sites in cells exposed to ENPs. The strategic microfabricated, 96-well design and automated processing improves efficiency, reduces processing time, and suppresses user bias in comparison to the standard comet assay. We evaluated the versatility of this assay by screening five industrially relevant ENP exposures (SiO2, ZnO, Fe2O3, Ag, and CeO2) on both suspension human lymphoblastoid (TK6) and adherent Chinese hamster ovary (H9T3) cell lines. MTT and CyQuant NF assays were employed to assess cellular viability and proliferation after ENP exposure. Exposure to ENPs at a dose range of 5, 10, and 20 μg/mL induced dose-dependent increases in DNA damage and cytotoxicity. Genotoxicity profiles of ZnO>Ag>Fe2O3>CeO2>SiO2 in TK6 cells at 4 h and Ag>Fe2O3>ZnO>CeO2>SiO2 in H9T3 cells at 24 h were observed. The presented CometChip platform enabled efficient and reliable measurement of ENP-mediated DNA damage, therefore demonstrating the efficacy of this powerful tool in nanogenotoxicity studies.

Cr(VI) induces DNA damage, cell cycle arrest and polyploidization: a flow cytometric and comet assay study in Pisum sativum.

PubMed

Rodriguez, Eleazar; Azevedo, Raquel; Fernandes, Pedro; Santos, Conceição

2011-07-18

Chromium(VI) is recognized as the most toxic valency of Cr, but its genotoxicity and cytostaticity in plants is still poorly studied. In order to analyze Cr(VI) cyto- and gentotoxicity, Pisum sativum L. plants were grown in soil and watered with solutions with different concentrations of Cr up to 2000 mg/L. After 28 days of exposure, leaves showed no significant variations in either cell cycle dynamics or ploidy level. As for DNA damage, flow cytometric (FCM) histograms showed significant differences in full peak coefficient of variation (FPCV) values, suggesting clastogenicity. This is paralleled by the Comet assay results, showing an increase in DNA damage for 1000 and 2000 mg/L. In roots, exposure to 2000 mg/L resulted in cell cycle arrest at the G(2)/M checkpoint. It was also verified that under the same conditions 40% of the individuals analyzed suffered polyploidization having both 2C and 4C levels. DNA damage analysis by the Comet assay and FCM revealed dose-dependent increases in DNA damage and FPCV. Through this, we have unequivocally demonstrated for the first time in plants that Cr exposure can result in DNA damage, cell cycle arrest, and polyploidization. Moreover, we critically compare the validity of the Comet assay and FCM in evaluating cytogenetic toxicity tests in plants and demonstrate that the data provided by both techniques complement each other and present high correlation levels. In conclusion, the data presented provides new insight on Cr effects in plants in general and supports the use of the parameters tested in this study as reliable endpoints for this metal toxicity in plants. © 2011 American Chemical Society

Comet assay: a reliable tool for the assessment of DNA damage in different models.

PubMed

Dhawan, Alok; Bajpayee, Mahima; Parmar, Devendra

2009-02-01

New chemicals are being added each year to the existing burden of toxic substances in the environment. This has led to increased pollution of ecosystems as well as deterioration of the air, water, and soil quality. Excessive agricultural and industrial activities adversely affect biodiversity, threatening the survival of species in a particular habitat as well as posing disease risks to humans. Some of the chemicals, e.g., pesticides and heavy metals, may be genotoxic to the sentinel species and/or to non-target species, causing deleterious effects in somatic or germ cells. Test systems which help in hazard prediction and risk assessment are important to assess the genotoxic potential of chemicals before their release into the environment or commercial use as well as DNA damage in flora and fauna affected by contaminated/polluted habitats. The Comet assay has been widely accepted as a simple, sensitive, and rapid tool for assessing DNA damage and repair in individual eukaryotic as well as some prokaryotic cells, and has increasingly found application in diverse fields ranging from genetic toxicology to human epidemiology. This review is an attempt to comprehensively encase the use of Comet assay in different models from bacteria to man, employing diverse cell types to assess the DNA-damaging potential of chemicals and/or environmental conditions. Sentinel species are the first to be affected by adverse changes in their environment. Determination of DNA damage using the Comet assay in these indicator organisms would thus provide information about the genotoxic potential of their habitat at an early stage. This would allow for intervention strategies to be implemented for prevention or reduction of deleterious health effects in the sentinel species as well as in humans.

Interaction of Comets and the Solar Wind

NASA Technical Reports Server (NTRS)

Wagner, William (Technical Monitor); Raymond, John C.

2004-01-01

The analysis of Comet Kudo-Fujikawa at perihelion was published and picked up by Der Spiegel. Besides a large and rapidly increasing water outgassing rate, we detected a bright tail in doubly ionized carbon. The amount of carbon was greater than could be accounted for by CO photodissociation, and we attribute it to evaporation of organics from dust. A spectacular disconnection event was apparent in the C III tail, and it coincides within the uncertainties to the position of the heliospheric current sheet. The analysis of the sungrazing comet C2001 C2 is in press. It showed evidence for subfragments and for a very long lasting source of neutrals, which we identify as evaporation of pyroxene dust grains. Results were also presented at COSPAR. We are working on observations of another sungrazer, comet C2002 S2, which shows a sudden 2 magnitude drop in optical brightness and an equally sudden recovery. UVCS observations during that time show a steadily increasing outgassing rate. We have derived solar wind densities for both comets, but we are still sorting out the ambiguities involving the fragmentation and optical behavior.

From the Cover: An Investigation of the Genotoxicity and Interference of Gold Nanoparticles in Commonly Used In Vitro Mutagenicity and Genotoxicity Assays.

PubMed

George, Jiya M; Magogotya, Millicent; Vetten, Melissa A; Buys, Antoinette V; Gulumian, Mary

2017-03-01

The suitability of 4 in vitro assays, commonly used for mutagenicity and genotoxicity assessment, was investigated in relation to treatment with 14 nm citrate-stabilized gold nanoparticles (AuNPs). Specifically, the Ames test was conducted without metabolic activation, where no mutagenic effects were observed. High resolution transmission electron microscopy and Cytoviva dark-field image analysis showed that AuNPs did not enter the bacterial cells, thus confirming the unreliability of the Ames test for nanoparticle mutagenicity studies. In addition, the Chinese hamster ovary (CHO) cell line was used for Comet, Chromosome aberration and Micronucleus assays. CHO cells were treated with AuNPs for 20 h at 37 °C. Cytotoxicity was not detected by cell impedance studies even though AuNP uptake was confirmed using Cytoviva image analysis. The DNA damage was statistically significant in treated cells when assessed by the Comet assay. However, minimal and nonstatistically significant chromosomal DNA damage was observed using the chromosome aberration and micronucleus assays. In this study, we showed that false positive results obtained with Comet assay may have been due to the possibility of direct contact between the residual, intracellular AuNPs and DNA during the assay procedure. Therefore, the chromosome aberration and micronucleus assays are better suited to assess the genotoxic effects of nanoparticles due to low probability of such direct contact occurring. Genotoxic effect of 14 and 20 nm citrate-stabilized, as well as, 14 nm PCOOH AuNPs were also investigated using chromosome aberration and micronucleus assays. Based on our acceptance criteria for a positive genotoxic response, none of the AuNPs were found to be genotoxic in either of these assays. © The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Development of a High-Content Orthopoxvirus Infectivity and Neutralization Assays

PubMed Central

Gates, Irina; Olson, Victoria; Smith, Scott; Patel, Nishi; Damon, Inger; Karem, Kevin

2015-01-01

Currently, a number of assays measure Orthopoxvirus neutralization with serum from individuals, vaccinated against smallpox. In addition to the traditional plaque reduction neutralization test (PRNT), newer higher throughput assays are based on neutralization of recombinant vaccinia virus, expressing reporter genes such as β-galactosidase or green fluorescent protein. These methods could not be used to evaluate neutralization of variola virus, since genetic manipulations of this virus are prohibited by international agreements. Currently, PRNT is the assay of choice to measure neutralization of variola virus. However, PRNT assays are time consuming, labor intensive, and require considerable volume of serum sample for testing. Here, we describe the development of a high-throughput, cell-based imaging assay that can be used to measure neutralization, and characterize replication kinetics of various Orthopoxviruses, including variola, vaccinia, monkeypox, and cowpox. PMID:26426117

MODEL-OBSERVATION COMPARISONS OF ELECTRON NUMBER DENSITIES IN THE COMA OF 67P/CHURYUMOV–GERASIMENKO DURING 2015 JANUARY

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vigren, E.; Edberg, N. J. T.; Eriksson, A. I.

2016-09-01

During 2015 January 9–11, at a heliocentric distance of ∼2.58–2.57 au, the ESA Rosetta spacecraft resided at a cometocentric distance of ∼28 km from the nucleus of comet 67P/Churyumov–Gerasimenko, sweeping the terminator at northern latitudes of 43°N–58°N. Measurements by the Rosetta Orbiter Spectrometer for Ion and Neutral Analysis/Comet Pressure Sensor (ROSINA/COPS) provided neutral number densities. We have computed modeled electron number densities using the neutral number densities as input into a Field Free Chemistry Free model, assuming H{sub 2}O dominance and ion-electron pair formation by photoionization only. A good agreement (typically within 25%) is found between the modeled electron numbermore » densities and those observed from measurements by the Mutual Impedance Probe (RPC/MIP) and the Langmuir Probe (RPC/LAP), both being subsystems of the Rosetta Plasma Consortium. This indicates that ions along the nucleus-spacecraft line were strongly coupled to the neutrals, moving radially outward with about the same speed. Such a statement, we propose, can be further tested by observations of H{sub 3}O{sup +}/H{sub 2}O{sup +} number density ratios and associated comparisons with model results.« less

Comparative antioxidant activity of cultivated and wild Vaccinium species investigated by EPR, human neutrophil burst and COMET assay.

PubMed

Braga, P C; Antonacci, R; Wang, Y Y; Lattuada, N; Dal Sasso, M; Marabini, L; Fibiani, M; Lo Scalzo, R

2013-01-01

The Vaccinium (V.) spp. berries are considered a source of antioxidants, mainly belonging to polyphenols, specifically flavonoids and anthocyanins. Wild genotypes generally contain more antioxidants than cultivated counterparts. So, seven different antioxidants assays on extracts from cultivated and wild Vaccinium berries were performed, to evaluate their difference in terms of bioactivity on oxidative protection and minimum dosage to have a significant action. Four cell-free antioxidant assays (ABTS radical scavenging and electronic paramagnetic resonance using Fremy's salt, superoxide anion and hydroxyl radical), and three assays on human cells (two luminol amplified chemiluminescence, LACL, one on DNA damage, COMET) were used to measure the effects of cultivated blueberry (V. corymbosum) and wild bilberry (V. myrtillus) on the differently induced oxidative stress. Concentrations vs activity patterns were obtained by successive dilutions of extracts in order to identify both EC50 and minimum significant activity (MSA). All the assays (except for the hydroxyl radical scavenging) showed a good relationship mainly with anthocyanin and polyphenol content and the significant greater activity of wild Vaccinium extracts. In fact, LACL data gave an EC50 of 11.8 and an MSA of 5.2 g were calculated as fresh weight dosage in cultivated berries, compared with lower doses in wild berries, EC50 of 5.7 g and MSA of 3.4 g. Wild Vaccinium extracts averaged 3.04 and 2.40 fold more activity than cultivated extracts by EC50 and MSA, respectively. COMET assay confirmed the stronger action on DNA protection in wild samples.

Comet assay in reconstructed 3D human epidermal skin models--investigation of intra- and inter-laboratory reproducibility with coded chemicals.

PubMed

Reus, Astrid A; Reisinger, Kerstin; Downs, Thomas R; Carr, Gregory J; Zeller, Andreas; Corvi, Raffaella; Krul, Cyrille A M; Pfuhler, Stefan

2013-11-01

Reconstructed 3D human epidermal skin models are being used increasingly for safety testing of chemicals. Based on EpiDerm™ tissues, an assay was developed in which the tissues were topically exposed to test chemicals for 3h followed by cell isolation and assessment of DNA damage using the comet assay. Inter-laboratory reproducibility of the 3D skin comet assay was initially demonstrated using two model genotoxic carcinogens, methyl methane sulfonate (MMS) and 4-nitroquinoline-n-oxide, and the results showed good concordance among three different laboratories and with in vivo data. In Phase 2 of the project, intra- and inter-laboratory reproducibility was investigated with five coded compounds with different genotoxicity liability tested at three different laboratories. For the genotoxic carcinogens MMS and N-ethyl-N-nitrosourea, all laboratories reported a dose-related and statistically significant increase (P < 0.05) in DNA damage in every experiment. For the genotoxic carcinogen, 2,4-diaminotoluene, the overall result from all laboratories showed a smaller, but significant genotoxic response (P < 0.05). For cyclohexanone (CHN) (non-genotoxic in vitro and in vivo, and non-carcinogenic), an increase compared to the solvent control acetone was observed only in one laboratory. However, the response was not dose related and CHN was judged negative overall, as was p-nitrophenol (p-NP) (genotoxic in vitro but not in vivo and non-carcinogenic), which was the only compound showing clear cytotoxic effects. For p-NP, significant DNA damage generally occurred only at doses that were substantially cytotoxic (>30% cell loss), and the overall response was comparable in all laboratories despite some differences in doses tested. The results of the collaborative study for the coded compounds were generally reproducible among the laboratories involved and intra-laboratory reproducibility was also good. These data indicate that the comet assay in EpiDerm™ skin models is a promising model for the safety assessment of compounds with a dermal route of exposure.

Comet assay in reconstructed 3D human epidermal skin models—investigation of intra- and inter-laboratory reproducibility with coded chemicals

PubMed Central

Pfuhler, Stefan

2013-01-01

Reconstructed 3D human epidermal skin models are being used increasingly for safety testing of chemicals. Based on EpiDerm™ tissues, an assay was developed in which the tissues were topically exposed to test chemicals for 3h followed by cell isolation and assessment of DNA damage using the comet assay. Inter-laboratory reproducibility of the 3D skin comet assay was initially demonstrated using two model genotoxic carcinogens, methyl methane sulfonate (MMS) and 4-nitroquinoline-n-oxide, and the results showed good concordance among three different laboratories and with in vivo data. In Phase 2 of the project, intra- and inter-laboratory reproducibility was investigated with five coded compounds with different genotoxicity liability tested at three different laboratories. For the genotoxic carcinogens MMS and N-ethyl-N-nitrosourea, all laboratories reported a dose-related and statistically significant increase (P < 0.05) in DNA damage in every experiment. For the genotoxic carcinogen, 2,4-diaminotoluene, the overall result from all laboratories showed a smaller, but significant genotoxic response (P < 0.05). For cyclohexanone (CHN) (non-genotoxic in vitro and in vivo, and non-carcinogenic), an increase compared to the solvent control acetone was observed only in one laboratory. However, the response was not dose related and CHN was judged negative overall, as was p-nitrophenol (p-NP) (genotoxic in vitro but not in vivo and non-carcinogenic), which was the only compound showing clear cytotoxic effects. For p-NP, significant DNA damage generally occurred only at doses that were substantially cytotoxic (>30% cell loss), and the overall response was comparable in all laboratories despite some differences in doses tested. The results of the collaborative study for the coded compounds were generally reproducible among the laboratories involved and intra-laboratory reproducibility was also good. These data indicate that the comet assay in EpiDerm™ skin models is a promising model for the safety assessment of compounds with a dermal route of exposure. PMID:24150594

Comet assay: a prognostic tool for DNA integrity assessment in infertile men opting for assisted reproduction.

PubMed

Shamsi, M B; Venkatesh, S; Tanwar, M; Singh, G; Mukherjee, S; Malhotra, N; Kumar, R; Gupta, N P; Mittal, S; Dada, R

2010-05-01

The growing concern on transmission of genetic diseases in assisted reproduction technique (ART) and the lacunae in the conventional semen analysis to accurately predict the semen quality has led to the need for new techniques to identify the best quality sperm that can be used in assisted procreation techniques. This study analyzes the sperm parameters in the context of DNA damage in cytogenetically normal, AZF non deleted infertile men for DNA damage by comet assay. Seventy infertile men and 40 fertile controls were evaluated for the semen quality by conventional semen parameters and the sperms were also analyzed for DNA integrity by comet assay. The patients were classified into oligozoospermic (O), asthenozoospermic (A), teratozoospermic (T), oligoasthenoteratozoospermic (OAT) categories and infertile men with normal semen profile. The extent of DNA damage was assessed by visual scoring method of comets. Idiopathic infertile men with normal semen profile (n=18) according to conventional method and patients with history of spontaneous abortions and normal semen profile (n=10) had high degree of DNA damage (29 and 47% respectively) as compared to fertile controls (7%). The O, A, T and OAT categories of patients had a variably higher DNA damage load as compared to fertile controls. The normal range and threshold for DNA damage as a predictor of male fertility potential and technique which could assess the sperm DNA damage are necessary to lower the trauma of couples experiencing recurrent spontaneous abortion or failure in ART.

Extremely low-frequency electromagnetic fields cause DNA strand breaks in normal cells

PubMed Central

2014-01-01

Background Extremely low frequency electromagnetic fields aren’t considered as a real carcinogenic agent despite the fact that some studies have showed impairment of the DNA integrity in different cells lines. The aim of this study was evaluation of the late effects of a 100 Hz and 5.6 mT electromagnetic field, applied continuously or discontinuously, on the DNA integrity of Vero cells assessed by alkaline Comet assay and by cell cycle analysis. Normal Vero cells were exposed to extremely low frequency electromagnetic fields (100 Hz, 5.6 mT) for 45 minutes. The Comet assay and cell cycle analysis were performed 48 hours after the treatment. Results Exposed samples presented an increase of the number of cells with high damaged DNA as compared with non-exposed cells. Quantitative evaluation of the comet assay showed a significantly (<0.001) increase of the tail lengths, of the quantity of DNA in tail and of Olive tail moments, respectively. Cell cycle analysis showed an increase of the frequency of the cells in S phase, proving the occurrence of single strand breaks. The most probable mechanism of induction of the registered effects is the production of different types of reactive oxygen species. Conclusions The analysis of the registered comet indices and of cell cycle showed that extremely low frequency electromagnetic field of 100 Hz and 5.6 mT had a genotoxic impact on Vero cells. PMID:24401758

DNA damage as a biomarker for assessing the effects of suspended solids on the orange-spotted grouper, Epinephelus coioides.

PubMed

Tse, C Y; Chan, K M; Wong, C K

2010-06-01

In Hong Kong, suspended solids (SS) introduced by dredging and mud disposal activities are a major cause of mass mortality in cage-cultured marine fish. We have used DNA damage in liver cells, as determined by the comet assay, to assess the impact of SS on the orange-spotted grouper Epinephelus coioides. Seabed sediments were collected from a heavily polluted site in Victoria Harbor and two less polluted sites in Port Shelter and Mirs Bay. Sediments from Victoria Harbor contained higher levels of copper (Cu) and polycyclic aromatic hydrocarbons (PAHs) than those from the other sites. In a 10-day experiment, SS from all three sites induced significant increase in comet tail length, but not in percentage (%) tail DNA. In a 20-day experiment, fish exposed to polluted SS from Victoria Harbor exhibited a significant increase in comet tail length after 5 days and % tail DNA after 10 days. After a 10-day recovery period, however, DNA damage was reduced as tail length and % tail DNA returned to control levels. These results suggest that DNA damage measured by the comet assay is a highly sensitive biomarker for assessing the genotoxic effects of SS to marine fish.

In-situ investigations of the ionosphere of comet 67P

NASA Astrophysics Data System (ADS)

Eriksson, A. I.; Edberg, N. J. T.; Odelstad, E.; Vigren, E.; Engelhardt, I.; Henri, P.; Lebreton, J.-P.; Galand, M.; Carr, C. M.; Koenders, C.; Nilsson, H.; Broiles, T.; Rubin, M.

2015-10-01

Since arrival of Rosetta at its target comet 67P/Churyumov-Gerasimenko in August 2014, the plasma environment has been dominated by ionized gas emanating from the comet nucleus rather than by solar wind plasma. This was evident early on from the strong modulation seen with Rosetta's position in a reference frame fixed to the rotating nucleus, with higher plasma densities observed when the spacecraft is above the neck region and when the comet exposes maximum area to the sun. In this respect, Rosetta is inside the comet ionosphere, providing excellent in situ investigation opportunities for the instruments of the Rosetta Plasma Consortium (RPC). In contrast to the often modelled scenario for a very active comet, the Langmuir probe instrument (RPC-LAP) finds electron temperatures mainly in the range of tens of thousand kelvin around this less active comet. This can be attributed to the lower density of neutral gas, meaning little cooling of recently produced electrons. A side effect of this is that the spacecraft charges negatively when within about 100 km from the nucleus. Interesting in itself, this also may point to similar charging for dust grains in the coma, with implications for the detection of the smallest particles and possibly for processes like electrostatic fragmentation. The inner coma also proves to be very dynamic, with large variations not only with latitude and longitude in a comet frame, but also with the solar wind and various wave phenomena.

Use of in vitro assays to assess the potential cytotoxic, genotoxic and antigenotoxic effects of vanillic and cinnamic acid.

PubMed

Taner, Gökçe; Özkan Vardar, Deniz; Aydin, Sevtap; Aytaç, Zeki; Başaran, Ahmet; Başaran, Nurşen

2017-04-01

Vanillic acid (VA) found in vanilla and cinnamic acid (CA) the precursor of flavonoids and found in cinnamon oil, are natural plant phenolic acids which are secondary aromatic plant products suggested to possess many physiological and pharmacological functions. In vitro and in vivo experiments have shown that phenolic acids exhibit powerful effects on biological responses by scavenging free radicals and eliciting antioxidant capacity. In the present study, we investigated the antioxidant capacity of VA and CA by the trolox equivalent antioxidant capacity (TEAC) assay, cytotoxicity by neutral red uptake (NRU) assay in Chinese Hamster Ovary (CHO) cells and also the genotoxic and antigenotoxic effects of these phenolic acids using the cytokinesis-blocked micronucleus (CBMN) and the alkaline comet assays in human peripheral blood lymphocytes. At all tested concentrations, VA (0.17-67.2 μg/ml) showed antioxidant activity but CA (0.15-59.2 μg/ml) did not show antioxidant activity against 2,2-azino-bis (3-ethylbenz-thiazoline-6-sulphonic acid) (ABTS). VA (0.84, 4.2, 8.4, 16.8, 84 and 168 μg/ml) and CA (0.74, 3.7, 7.4, 14.8, 74, 148 μg/ml) did not have cytotoxic and genotoxic effects alone at the studied concentrations as compared with the controls. Both VA and CA seem to decrease DNA damage induced by H 2 O 2 in human lymphocytes.

Laboratory simulation of charge exchange-produced X-ray emission from comets.

PubMed

Beiersdorfer, P; Boyce, K R; Brown, G V; Chen, H; Kahn, S M; Kelley, R L; May, M; Olson, R E; Porter, F S; Stahle, C K; Tillotson, W A

2003-06-06

In laboratory experiments using the engineering spare microcalorimeter detector from the ASTRO-E satellite mission, we recorded the x-ray emission of highly charged ions of carbon, nitrogen, and oxygen, which simulates charge exchange reactions between heavy ions in the solar wind and neutral gases in cometary comae. The spectra are complex and do not readily match predictions. We developed a charge exchange emission model that successfully reproduces the soft x-ray spectrum of comet Linear C/1999 S4, observed with the Chandra X-ray Observatory.

Identification of irradiated refrigerated pork with the DNA comet assay

NASA Astrophysics Data System (ADS)

Araújo, M. M.; Marin-Huachaca, N. S.; Mancini-Filho, J.; Delincée, H.; Villavicencio, A. L. C. H.

2004-09-01

Food irradiation can contribute to a safer and more plentiful food supply by inactivating pathogens, eradicating pests and by extending shelf-life. Particularly in the case of pork meat, this process could be a useful way to inactivate harmful parasites such as Trichinella and Taenia solium. Ionizing radiation causes damage to the DNA of the cells (e.g. strand breaks), which can be used to detect irradiated food. Microelectrophoresis of single cells (``Comet Assay'') is a simple and rapid test for DNA damage and can be used over a wide dose range and for a variety of products. Refrigerated pork meat was irradiated with a 60Co source, Gammacell 220 (A.E.C.L.) installed in IPEN (Sa~o Paulo, Brazil). The doses given were 0, 1.5, 3.0 and 4.5kGy for refrigerated samples. Immediately after irradiation the samples were returned to the refrigerator (6°C). Samples were kept in the refrigerator after irradiation. Pork meat was analyzed 1, 8 and 10 days after irradiation using the DNA ``Comet Assay''. This method showed to be an inexpensive and rapid technique for qualitative detection of irradiation treatment.

Evaluation of basal DNA damage and oxidative stress in Wistar rat leukocytes after exposure to microwave radiation.

PubMed

Garaj-Vrhovac, Vera; Gajski, Goran; Trosić, Ivancica; Pavicić, Ivan

2009-05-17

The aim of this study was to assess whether microwave-induced DNA damage is basal or it is also generated through reactive oxygen species (ROS) formation. After having irradiated Wistar rats with 915MHz microwave radiation, we assessed different DNA alterations in peripheral leukocytes using standard and formamidopyrimidine DNA-glycosylase (Fpg)-modified comet assay. The first is a sensitive tool for detecting primary DNA damage, and the second is much more specific for detecting oxidative damage. The animals were irradiated for 1h a day for 2 weeks at a field power density of 2.4W/m(2), and the whole-body average specific absorption rate (SAR) of 0.6W/kg. Both the standard and the Fpg-modified comet assay detected increased DNA damage in blood leukocytes of the exposed rats. The significant increase in Fpg-detected DNA damage in the exposed rats suggests that oxidative stress is likely to be responsible. DNA damage detected by the standard comet assay indicates that some other mechanisms may also be involved. In addition, both methods served proved sensitive enough to measure basal and oxidative DNA damage after long-term exposure to 915MHz microwave radiation in vivo.

In vitro assessment of genotoxic effects of electric arc furnace dust on human lymphocytes using the alkaline comet assay.

PubMed

Garaj-Vrhovac, Vera; Orescanin, Visnja; Ruk, Damir; Gajski, Goran

2009-02-15

In vitro genotoxic effects of leachates of electric arc furnace dust (EAFD) on human peripheral lymphocytes, assessed prior and following the treatment with a strong alkaline solution were investigated using the alkaline comet assay. Prior and following the treatment, lymphocytes were incubated with leachate of EAFD for 6 and 24 hours at 37 degrees C. Negative controls were also included. Mean values of the tail lengths established in the samples treated with the leachate stemming from the original dust for 6 and 24 hours, were 15.70 microm and 16.78 microm, respectively, as compared to 12.33 microm found in the control sample. Slight, but significant increase in the tail length was also found with the dust treated with a strong alkaline solution (13.37 microm and 13.60 microm). In case of high heavy metal concentrations (the extract of the original furnace dust), the incubation period was revealed to be of significance as well. The obtained results lead to the conclusion that alkaline comet assay could be used as a rapid, sensitive and low-cost tool when assessing genotoxicity of various waste materials, such as leachates of the electric arc furnace dust.

Sister chromatid exchange rate and alkaline comet assay scores in patients with ovarian cancer.

PubMed

Baltaci, Volkan; Kayikçioğlu, Fulya; Alpas, Idil; Zeyneloğlu, Hulusi; Haberal, Ali

2002-01-01

Sister chromatid exchange (SCE) frequencies were studied in patients with different types of ovarian malignancies and in healthy volunteers. The level of DNA damage in patients with ovarian malignancy and control subjects has also been studied by alkaline single cell gel electrophoresis (SCGE), also known as the comet assay. Peripheral blood was collected from 30 patients after histological confirmation of malignancy and 20 healthy female volunteers. The cells were evaluated according to their grade of damage. We found that the sister chromatid exchange frequencies of cancer cases were significantly greater than that of controls (P < 0.001). The frequency of exchange in chromosomal groups A, B, and C, which include chromosomes 1-12, was higher than that of the other chromosomal groups in both groups. Comparison of the results of the alkaline comet assay in patient and control subjects showed a significant difference in the number of damaged cells. The frequency of limited migrated and extensive migrated cells in the women with ovarian malignancies was higher than that of control women (P < 0.001). SCE and SCGE can be used successfully to monitor DNA damage in women with ovarian cancer.

Assaying Cellular Viability Using the Neutral Red Uptake Assay.

PubMed

Ates, Gamze; Vanhaecke, Tamara; Rogiers, Vera; Rodrigues, Robim M

2017-01-01

The neutral red uptake assay is a cell viability assay that allows in vitro quantification of xenobiotic-induced cytotoxicity. The assay relies on the ability of living cells to incorporate and bind neutral red, a weak cationic dye, in lysosomes. As such, cytotoxicity is expressed as a concentration-dependent reduction of the uptake of neutral red after exposure to the xenobiotic under investigation. The neutral red uptake assay is mainly used for hazard assessment in in vitro toxicology applications. This method has also been introduced in regulatory recommendations as part of 3T3-NRU-phototoxicity-assay, which was regulatory accepted in all EU member states in 2000 and in the OECD member states in 2004 as a test guideline (TG 432). The present protocol describes the neutral red uptake assay using the human hepatoma cell line HepG2, which is often employed as an alternative in vitro model for human hepatocytes. As an example, the cytotoxicity of acetaminophen and acetyl salicylic acid is assessed.

DNA Protection against Oxidative Damage Using the Hydroalcoholic Extract of Garcinia mangostana and Alpha-Mangostin.

PubMed

Carvalho-Silva, Ronaldo; Pereira, Alanna Cibelle Fernandes; Dos Santos Alves, Rúbens Prince; Guecheva, Temenouga N; Henriques, João A P; Brendel, Martin; Pungartnik, Cristina; Rios-Santos, Fabrício

2016-01-01

Garcinia mangostana, popularly known as "mangosteen fruit," originates from Southeast Asia and came to Brazil about 80 years ago where it mainly grows in the states of Pará and Bahia. Although mangosteen or its extracts have been used for ages in Asian folk medicine, data on its potential genotoxicity is missing. We, therefore, evaluated genotoxicity/mutagenicity of hydroethanolic mangosteen extract [HEGM, 10 to 640 μg/mL] in established test assays (Comet assay, micronucleus test, and Salmonella/microsome test). In the Comet assay, HEGM-exposed human leukocytes showed no DNA damage. No significant HEGM-induced mutation in TA98 and TA100 strains of Salmonella typhimurium (with or without metabolic activation) was observed and HEGM-exposed human lymphocytes had no increase of micronuclei. However, HEGM suggested exposure concentration-dependent antigenotoxic potential in leukocytes and antioxidant potential in the yeast Saccharomyces cerevisiae. HEGM preloading effectively protected against H2O2-induced DNA damage in leukocytes (Comet assay). Preloading of yeast with HEGM for up to 4 h significantly protected the cells from lethality of chronic H2O2-exposure, as expressed in better survival. Absence of genotoxicity and demonstration of an antigenotoxic and antioxidant potential suggest that HEGM or some substances contained in it may hold promise for pharmaceutical or nutraceutical application.

DNA Protection against Oxidative Damage Using the Hydroalcoholic Extract of Garcinia mangostana and Alpha-Mangostin

PubMed Central

Carvalho-Silva, Ronaldo; Pereira, Alanna Cibelle Fernandes; dos Santos Alves, Rúbens Prince; Guecheva, Temenouga N.; Henriques, João A. P.; Brendel, Martin; Rios-Santos, Fabrício

2016-01-01

Garcinia mangostana, popularly known as “mangosteen fruit,” originates from Southeast Asia and came to Brazil about 80 years ago where it mainly grows in the states of Pará and Bahia. Although mangosteen or its extracts have been used for ages in Asian folk medicine, data on its potential genotoxicity is missing. We, therefore, evaluated genotoxicity/mutagenicity of hydroethanolic mangosteen extract [HEGM, 10 to 640 μg/mL] in established test assays (Comet assay, micronucleus test, and Salmonella/microsome test). In the Comet assay, HEGM-exposed human leukocytes showed no DNA damage. No significant HEGM-induced mutation in TA98 and TA100 strains of Salmonella typhimurium (with or without metabolic activation) was observed and HEGM-exposed human lymphocytes had no increase of micronuclei. However, HEGM suggested exposure concentration-dependent antigenotoxic potential in leukocytes and antioxidant potential in the yeast Saccharomyces cerevisiae. HEGM preloading effectively protected against H2O2-induced DNA damage in leukocytes (Comet assay). Preloading of yeast with HEGM for up to 4 h significantly protected the cells from lethality of chronic H2O2-exposure, as expressed in better survival. Absence of genotoxicity and demonstration of an antigenotoxic and antioxidant potential suggest that HEGM or some substances contained in it may hold promise for pharmaceutical or nutraceutical application. PMID:27042187

Three component plasma electron distribution in the intermediate ionized coma of Comet Giacobini-Zinner

NASA Astrophysics Data System (ADS)

Zwickl, R. D.; Baker, D. N.; Bame, S. J.; Feldman, W. C.; Fuselier, S. A.; Huebner, W. F.; McComas, D. J.; Young, D. T.

1986-04-01

The observation of three distinct components of the electron distribution function measured in the intermediate ionized coma (IIC) and plasma tail of Comet Giacobini-Zinner is reported. It is believed that the cold component represents electrons produced close to the comet nucleus by ionization of cometary matter and subsequent cooling by Coulomb collisions. The second component also appears to be composed of electrons produced by photoionization of cometary neutrals, but sufficiently far from the nucleus that the distributions are largely unaffected by Coulomb interactions. The hot component is probably a population of electrons originating in the solar wind. Throughout the IIC, the electrostatic potential of the spacecraft was very low (less than 0.8 eV), implying that ICE generated very little impact-produced plasma during its passage.

Attenuation of the DNA Damage Response by Transforming Growth Factor-Beta Inhibitors Enhances Radiation Sensitivity of Non–Small-Cell Lung Cancer Cells In Vitro and In Vivo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Du, Shisuo; Bouquet, Sophie; Lo, Chen-Hao

2015-01-01

Purpose: To determine whether transforming growth factor (TGF)-β inhibition increases the response to radiation therapy in human and mouse non–small-cell lung carcinoma (NSCLC) cells in vitro and in vivo. Methods and Materials: TGF-β–mediated growth response and pathway activation were examined in human NSCLC NCI-H1299, NCI-H292, and A549 cell lines and murine Lewis lung cancer (LLC) cells. Cells were treated in vitro with LY364947, a small-molecule inhibitor of the TGF-β type 1 receptor kinase, or with the pan-isoform TGF-β neutralizing monoclonal antibody 1D11 before radiation exposure. The DNA damage response was assessed by ataxia telangiectasia mutated (ATM) or Trp53 protein phosphorylation, γH2AX foci formation,more » or comet assay in irradiated cells. Radiation sensitivity was determined by clonogenic assay. Mice bearing syngeneic subcutaneous LLC tumors were treated with 5 fractions of 6 Gy and/or neutralizing or control antibody. Results: The NCI-H1299, A549, and LLC NSCLC cell lines pretreated with LY364947 before radiation exposure exhibited compromised DNA damage response, indicated by decreased ATM and p53 phosphorylation, reduced γH2AX foci, and increased radiosensitivity. The NCI-H292 cells were unresponsive. Transforming growth factor-β signaling inhibition in irradiated LLC cells resulted in unresolved DNA damage. Subcutaneous LLC tumors in mice treated with TGF-β neutralizing antibody exhibited fewer γH2AX foci after irradiation and significantly greater tumor growth delay in combination with fractionated radiation. Conclusions: Inhibition of TGF-β before radiation attenuated DNA damage recognition and increased radiosensitivity in most NSCLC cells in vitro and promoted radiation-induced tumor control in vivo. These data support the rationale for concurrent TGF-β inhibition and RT to provide therapeutic benefit in NSCLC.« less

Halogens as tracers of protosolar nebula material in comet 67P/Churyumov-Gerasimenko

NASA Astrophysics Data System (ADS)

Dhooghe, Frederik; De Keyser, Johan; Altwegg, Kathrin; Briois, Christelle; Balsiger, Hans; Berthelier, Jean-Jacques; Calmonte, Ursina; Cessateur, Gaël; Combi, Michael R.; Equeter, Eddy; Fiethe, Björn; Fray, Nicolas; Fuselier, Stephen; Gasc, Sébastien; Gibbons, Andrew; Gombosi, Tamas; Gunell, Herbert; Hässig, Myrtha; Hilchenbach, Martin; Le Roy, Léna; Maggiolo, Romain; Mall, Urs; Marty, Bernard; Neefs, Eddy; Rème, Henri; Rubin, Martin; Sémon, Thierry; Tzou, Chia-Yu; Wurz, Peter

2017-12-01

We report the first in situ detection of halogens in a cometary coma, that of 67P/Churyumov-Gerasimenko. Neutral gas mass spectra collected by the European Space Agency's Rosetta spacecraft during four periods of interest from the first comet encounter up to perihelion indicate that the main halogen-bearing compounds are HF, HCl and HBr. The bulk elemental abundances relative to oxygen are ∼8.9 × 10-5 for F/O, ∼1.2 × 10-4 for Cl/O and ∼2.5 × 10-6 for Br/O, for the volatile fraction of the comet. The cometary isotopic ratios for 37Cl/35Cl and 81Br/79Br match the Solar system values within the error margins. The observations point to an origin of the hydrogen halides in molecular cloud chemistry, with frozen hydrogen halides on dust grains, and a subsequent incorporation into comets as the cloud condensed and the Solar system formed.

Prediction of cellular radiosensitivity from DNA damage induced by gamma-rays and carbon ion irradiation in canine tumor cells.

PubMed

Wada, Seiichi; Van Khoa, Tran; Kobayashi, Yasuhiko; Funayama, Tomoo; Ogihara, Kikumi; Ueno, Shunji; Ito, Nobuhiko

2005-11-01

Diseases of companion animals are shifting from infectious diseases to neoplasms (cancer), and since radiation therapy is one of the effective choices available for cancer treatment, the application of radiotherapy in veterinary medicine is likely to increase. However tumor tissues have different radiosensitivities, and therefore it is important to determine the intrinsic radiosensitivity of tumors in individual patients in advance of radiotherapy. We have studied the relationship between the surviving cell fraction measured by a clonogenic assay and DNA double strand breaks detected by a comet assay under neutral conditions in three canine tumor cell lines, after gamma-ray and carbon ion irradiation. In all the cell lines, cell death assessed by the clonogenic assay was much higher following irradiation with carbon ions than with gamma-rays. The initial and residual (4 hr) DNA damage due to gamma-ray and carbon ion irradiation were higher in a radiosensitive cell line than in a radioresistant cell line. The surviving cell fraction at 2 Gy (SF2) showed a tendency for correlation with both the initial and residual DNA damage. In particular, the residual damage per Gy was significantly correlated with SF2, regardless of the type of radiation. This indicates that cellular radiosensitivity can be predicted by detection of radiation-induced residual DNA damage.

PREDICTION OF FORBIDDEN ULTRAVIOLET AND VISIBLE EMISSIONS IN COMET 67P/CHURYUMOV–GERASIMENKO

DOE Office of Scientific and Technical Information (OSTI.GOV)

Raghuram, Susarla; Galand, Marina; Bhardwaj, Anil, E-mail: raghuramsusarla@gmail.com

Remote observation of spectroscopic emissions is a potential tool for the identification and quantification of various species in comets. The CO Cameron band (to trace CO{sub 2}) and atomic oxygen emissions (to trace H{sub 2}O and/or CO{sub 2}, CO) have been used to probe neutral composition in the cometary coma. Using a coupled-chemistry-emission model, various excitation processes controlling the CO Cameron band and different atomic oxygen and atomic carbon emissions have been modeled in comet 67P/Churyumov–Gerasimenko at 1.29 AU (perihelion) and at 3 AU heliocentric distances, which is being explored by ESA's Rosetta mission. The intensities of the CO Cameronmore » band, atomic oxygen, and atomic carbon emission lines as a function of projected distance are calculated for different CO and CO{sub 2} volume mixing ratios relative to water. Contributions of different excitation processes controlling these emissions are quantified. We assess how CO{sub 2} and/or CO volume mixing ratios with respect to H{sub 2}O can be derived based on the observed intensities of the CO Cameron band, atomic oxygen, and atomic carbon emission lines. The results presented in this work serve as baseline calculations to understand the behavior of low out-gassing cometary coma and compare them with the higher gas production rate cases (e.g., comet Halley). Quantitative analysis of different excitation processes governing the spectroscopic emissions is essential to study the chemistry of inner coma and to derive neutral gas composition.« less

VizieR Online Data Catalog: X-ray line ratios for diverse ion collisions (Mullen+, 2017)

NASA Astrophysics Data System (ADS)

Mullen, P. D.; Cumbee, R. S.; Lyons, D.; Gu, L.; Kaastra, J.; Shelton, R. L.; Stancil, P. C.

2018-03-01

Charge exchange (CX) has emerged in X-ray emission modeling as a significant process that must be considered in many astrophysical environments- particularly comets. Comets host an interaction between solar wind ions and cometary neutrals to promote solar wind charge exchange (SWCX). X-ray observatories provide astronomers and astrophysicists with data for many X-ray emitting comets that are impossible to accurately model without reliable CX data. Here, we utilize a streamlined set of computer programs that incorporate the multi-channel Landau-Zener theory and a cascade model for X-ray emission to generate cross sections and X-ray line ratios for a variety of bare and non-bare ion single electron capture (SEC) collisions. Namely, we consider collisions between the solar wind constituent bare and H-like ions of C, N, O, Ne, Na, Mg, Al, and Si and the cometary neutrals H2O, CO, CO2, OH, and O. To exemplify the application of this data, we model the X-ray emission of Comet C/2000 WM1 (linear) using the CX package in SPEX and find excellent agreement with observations made with the XMM-Newton RGS detector. Our analyses show that the X-ray intensity is dominated by SWCX with H, while H2O plays a secondary role. This is the first time, to our knowledge, that CX cross sections have been implemented into a X-ray spectral fitting package to determine the H to H2O ratio in cometary atmospheres. The CX data sets are incorporated into the modeling packages SPEX and Kronos. (1 data file).

An empirical model of H2O, CO2 and CO coma distributions and production rates for comet 67P/Churyumov-Gerasimenko based on ROSINA/DFMS measurements and AMPS-DSMC simulations

NASA Astrophysics Data System (ADS)

Hansen, Kenneth C.; Altwegg, Kathrin; Bieler, Andre; Berthelier, Jean-Jacques; Calmonte, Ursina; Combi, Michael R.; De Keyser, Johan; Fiethe, Björn; Fougere, Nicolas; Fuselier, Stephen; Gombosi, T. I.; Hässig, Myrtha; Huang, Zhenguang; Le Roy, Léna; Rubin, Martin; Tenishev, Valeriy; Toth, Gabor; Tzou, Chia-Yu; ROSINA Team

2016-10-01

We have previously used results from the AMPS DSMC (Adaptive Mesh Particle Simulator Direct Simulation Monte Carlo) model to create an empirical model of the near comet water (H2O) coma of comet 67P/Churyumov-Gerasimenko. In this work we create additional empirical models for the coma distributions of CO2 and CO. The AMPS simulations are based on ROSINA DFMS (Rosetta Orbiter Spectrometer for Ion and Neutral Analysis, Double Focusing Mass Spectrometer) data taken over the entire timespan of the Rosetta mission. The empirical model is created using AMPS DSMC results which are extracted from simulations at a range of radial distances, rotation phases and heliocentric distances. The simulation results are then averaged over a comet rotation and fitted to an empirical model distribution. Model coefficients are then fitted to piecewise-linear functions of heliocentric distance. The final product is an empirical model of the coma distribution which is a function of heliocentric distance, radial distance, and sun-fixed longitude and latitude angles. The model clearly mimics the behavior of water shifting production from North to South across the inbound equinox while the CO2 production is always in the South.The empirical model can be used to de-trend the spacecraft motion from the ROSINA COPS and DFMS data. The ROSINA instrument measures the neutral coma density at a single point and the measured value is influenced by the location of the spacecraft relative to the comet and the comet-sun line. Using the empirical coma model we can correct for the position of the spacecraft and compute a total production rate based on single point measurements. In this presentation we will present the coma production rates as a function of heliocentric distance for the entire Rosetta mission.This work was supported by contracts JPL#1266313 and JPL#1266314 from the US Rosetta Project and NASA grant NNX14AG84G from the Planetary Atmospheres Program.

International network for comparison of HIV neutralization assays: the NeutNet report.

PubMed

Fenyö, Eva Maria; Heath, Alan; Dispinseri, Stefania; Holmes, Harvey; Lusso, Paolo; Zolla-Pazner, Susan; Donners, Helen; Heyndrickx, Leo; Alcami, Jose; Bongertz, Vera; Jassoy, Christian; Malnati, Mauro; Montefiori, David; Moog, Christiane; Morris, Lynn; Osmanov, Saladin; Polonis, Victoria; Sattentau, Quentin; Schuitemaker, Hanneke; Sutthent, Ruengpung; Wrin, Terri; Scarlatti, Gabriella

2009-01-01

Neutralizing antibody assessments play a central role in human immunodeficiency virus type-1 (HIV-1) vaccine development but it is unclear which assay, or combination of assays, will provide reliable measures of correlates of protection. To address this, an international collaboration (NeutNet) involving 18 independent participants was organized to compare different assays. Each laboratory evaluated four neutralizing reagents (TriMab, 447-52D, 4E10, sCD4) at a given range of concentrations against a panel of 11 viruses representing a wide range of genetic subtypes and phenotypes. A total of 16 different assays were compared. The assays utilized either uncloned virus produced in peripheral blood mononuclear cells (PBMCs) (virus infectivity assays, VI assays), or their Env-pseudotyped (gp160) derivatives produced in 293T cells (PSV assays) from molecular clones or uncloned virus. Target cells included PBMC and genetically-engineered cell lines in either a single- or multiple-cycle infection format. Infection was quantified by using a range of assay read-outs that included extracellular or intracellular p24 antigen detection, RNA quantification and luciferase and beta-galactosidase reporter gene expression. PSV assays were generally more sensitive than VI assays, but there were important differences according to the virus and inhibitor used. For example, for TriMab, the mean IC50 was always lower in PSV than in VI assays. However, with 4E10 or sCD4 some viruses were neutralized with a lower IC50 in VI assays than in the PSV assays. Inter-laboratory concordance was slightly better for PSV than for VI assays with some viruses, but for other viruses agreement between laboratories was limited and depended on both the virus and the neutralizing reagent. The NeutNet project demonstrated clear differences in assay sensitivity that were dependent on both the neutralizing reagent and the virus. No single assay was capable of detecting the entire spectrum of neutralizing activities. Since it is not known which in vitro assay correlates with in vivo protection, a range of neutralization assays is recommended for vaccine evaluation.

International Network for Comparison of HIV Neutralization Assays: The NeutNet Report

PubMed Central

Fenyö, Eva Maria; Heath, Alan; Dispinseri, Stefania; Holmes, Harvey; Lusso, Paolo; Zolla-Pazner, Susan; Donners, Helen; Heyndrickx, Leo; Alcami, Jose; Bongertz, Vera; Jassoy, Christian; Malnati, Mauro; Montefiori, David; Moog, Christiane; Morris, Lynn; Osmanov, Saladin; Polonis, Victoria; Sattentau, Quentin; Schuitemaker, Hanneke; Sutthent, Ruengpung; Wrin, Terri; Scarlatti, Gabriella

2009-01-01

Background Neutralizing antibody assessments play a central role in human immunodeficiency virus type-1 (HIV-1) vaccine development but it is unclear which assay, or combination of assays, will provide reliable measures of correlates of protection. To address this, an international collaboration (NeutNet) involving 18 independent participants was organized to compare different assays. Methods Each laboratory evaluated four neutralizing reagents (TriMab, 447-52D, 4E10, sCD4) at a given range of concentrations against a panel of 11 viruses representing a wide range of genetic subtypes and phenotypes. A total of 16 different assays were compared. The assays utilized either uncloned virus produced in peripheral blood mononuclear cells (PBMCs) (virus infectivity assays, VI assays), or their Env-pseudotyped (gp160) derivatives produced in 293T cells (PSV assays) from molecular clones or uncloned virus. Target cells included PBMC and genetically-engineered cell lines in either a single- or multiple-cycle infection format. Infection was quantified by using a range of assay read-outs that included extracellular or intracellular p24 antigen detection, RNA quantification and luciferase and beta-galactosidase reporter gene expression. Findings PSV assays were generally more sensitive than VI assays, but there were important differences according to the virus and inhibitor used. For example, for TriMab, the mean IC50 was always lower in PSV than in VI assays. However, with 4E10 or sCD4 some viruses were neutralized with a lower IC50 in VI assays than in the PSV assays. Inter-laboratory concordance was slightly better for PSV than for VI assays with some viruses, but for other viruses agreement between laboratories was limited and depended on both the virus and the neutralizing reagent. Conclusions The NeutNet project demonstrated clear differences in assay sensitivity that were dependent on both the neutralizing reagent and the virus. No single assay was capable of detecting the entire spectrum of neutralizing activities. Since it is not known which in vitro assay correlates with in vivo protection, a range of neutralization assays is recommended for vaccine evaluation. PMID:19229336

Suprathermal electron environment of comet 67P/Churyumov-Gerasimenko: Observations from the Rosetta Ion and Electron Sensor

NASA Astrophysics Data System (ADS)

Clark, G.; Broiles, T. W.; Burch, J. L.; Collinson, G. A.; Cravens, T.; Frahm, R. A.; Goldstein, J.; Goldstein, R.; Mandt, K.; Mokashi, P.; Samara, M.; Pollock, C. J.

2015-11-01

Context. The Rosetta spacecraft is currently escorting comet 67P/Churyumov-Gerasimenko until its perihelion approach at 1.2 AU. This mission has provided unprecedented views into the interaction of the solar wind and the comet as a function of heliocentric distance. Aims: We study the interaction of the solar wind and comet at large heliocentric distances (>2 AU) using data from the Rosetta Plasma Consortium Ion and Electron Sensor (RPC-IES). From this we gain insight into the suprathermal electron distribution, which plays an important role in electron-neutral chemistry and dust grain charging. Methods: Electron velocity distribution functions observed by IES fit to functions used to previously characterize the suprathermal electrons at comets and interplanetary shocks. We used the fitting results and searched for trends as a function of cometocentric and heliocentric distance. Results: We find that interaction of the solar wind with this comet is highly turbulent and stronger than expected based on historical studies, especially for this weakly outgassing comet. The presence of highly dynamical suprathermal electrons is consistent with observations of comets (e.g., Giacobinni-Zinner, Grigg-Skjellerup) near 1 AU with higher outgassing rates. However, comet 67P/Churyumov-Gerasimenko is much farther from the Sun and appears to lack an upstream bow shock. Conclusions: The mass loading process, which likely is the cause of these processes, plays a stronger role at large distances from the Sun than previously expected. We discuss the possible mechanisms that most likely are responsible for this acceleration: heating by waves generated by the pick-up ion instability, and the admixture of cometary photoelectrons.

Pharmaceutical wastewater being composite mixture of environmental pollutants may be associated with mutagenicity and genotoxicity.

PubMed

Sharif, Ali; Ashraf, Muhammad; Anjum, Aftab Ahmed; Javeed, Aqeel; Altaf, Imran; Akhtar, Muhammad Furqan; Abbas, Mateen; Akhtar, Bushra; Saleem, Ammara

2016-02-01

Pharmaceutical industries are amongst the foremost contributor to industrial waste. Ecological well-being is endangered owing to its facile discharge. In the present study, heavy metals and organic contaminants in waste water were characterized using atomic absorption spectrophotometer and GC-MS, respectively. Mutagenicity and genotoxic potential of pharmaceutical waste water were investigated through bacterial reverse mutation assay and in vitro comet assay, respectively. Ames test and comet assay of first sample were carried out at concentrations of 100, 50, 25, 12.5, 6.25 % v/v effluent with distilled water. Chromium (Cr), lead (Pb), arsenic (As), and cadmium (Cd) were found in high concentrations as compared to WHO- and EPA-recommended maximum limits. Arsenic was found to be the most abundant metal and its maximum concentration was 0.8 mg.L(-1). GC-MS revealed the presence of lignocaine, digitoxin, trimethoprim, caffeine, and vitamin E in waste water. Dose-dependent decrease in mutagenic index was observed in both strains. Substantial increase in mutagenicity was observed for TA-100, when assay was done by incorporating an enzyme activation system, whereas a slight increase was detected for TA-102. In vitro comet assay of waste water exhibited decrease in damage index and percentage fragmentation with the increase in dilution of waste water. Tail length also decreased with an increase in the dilution factor of waste water. These findings suggest that pharmaceutical waste water being a mix of different heavy metals and organic contaminants may have a potent mutagenic and genotoxic effect on exposed living organisms.

Assessment of status of three water bodies in Serbia based on tissue metal and metalloid concentration (ICP-OES) and genotoxicity (comet assay).

PubMed

Sunjog, Karolina; Kolarević, Stoimir; Kračun-Kolarević, Margareta; Višnjić-Jeftić, Željka; Skorić, Stefan; Gačić, Zoran; Lenhardt, Mirjana; Vasić, Nebojša; Vuković-Gačić, Branka

2016-06-01

Metals and metalloids are natural components of the biosphere, which are not produced per se by human beings, but whose form and distribution can be affected by human activities. Like all substances, they are a contaminant if present in excess compared to background levels and/or in a form that would not normally occur in the environment. Samples of liver, gills, gonads and muscle from European chub, Squalius cephalus, were analyzed for Al, As, B, Ba, Cr, Cu, Fe, Hg, Mn, Mo, Sr and Zn using inductively coupled plasma optical emission spectrometry (ICP-OES) to highlight the importance of tissue selection in monitoring research. The comet assay or single cell gel electrophoresis (SCGE) was selected as an in vivo genotoxicity assay, a rapid and sensitive method for measuring genotoxic effects in blood, liver and gills of the European chub. Microscopic images of comets were scored using Comet IV Computer Software (Perceptive Instruments, UK). The objective of our study was to investigate two reservoirs, Zlatar and Garasi, and one river, Pestan by: (i) determining and comparing metal and metalloid concentrations in sediment, water and tissues of European chub: liver, gills, muscle and gonads (ii) comparing these findings with genotoxicity of water expressed through DNA damage of fish tissues. A clear link between the level of metals in water, sediment and tissues and between metal and genotoxicity levels at examined sites was not found. This suggests that other xenobiotics (possibly the organic compounds), contribute to DNA damage. Copyright © 2016 Elsevier Ltd. All rights reserved.

Multi-fluid model of a sun-grazing comet in the rapidly ionizing, magnetized low corona

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jia, Y.-D.; Russell, C. T.; Liu, W.

2014-11-20

Two Sun-grazing comets were recently imaged in the low solar corona by space telescopes in unprecedented detail, revealing a wide range of new phenomena. This sparked growing interest in the interaction of comets with the coronal plasma and magnetic field and their diagnostic potential as solar probes. However, interpretation of such rich observational data requires profound understanding of relevant physical processes in an unexplored regime. Here advanced numerical modeling can provide critical clues. To this end, we present a prototype, multi-fluid, magnetohydrodynamic model of a steady-state comet in the low solar corona. These simulation results are compared with previously modeledmore » comets in the solar wind environment. By inspecting their projection and column densities, we find a dominance of O{sup 6+} ions in the cometary tail, which can explain the observed extreme ultraviolet emission. The tail is found to be comparable to recent EUV images of these comets. In addition, the comet tail appears wider when the observer's line of sight is perpendicular rather than parallel to the local magnetic field. This is opposite to the trend in the interplanetary space permeated in the solar wind, because the ratio between dynamic pressure and magnetic pressure is an order of magnitude smaller than at 1 AU. On the other hand, we find that iron ions in the comet head build up to a density comparable to that of oxygen ions, but are unlikely to form a visible tail because of the shorter mean free paths of the neutrals.« less

Rapid communications: antiperspirant induced DNA damage in canine cells by comet assay.

PubMed

Yiu, Gloria

2004-01-01

Abstract Millions of people around the world use antiperspirants to decrease or eliminate body odors. Most antiperspirants contain aluminum zirconium or another form of aluminum as its active ingredient. The present investigation applied Comet assay to detect if Secret Platinum for women, Old Spice for men, or Crystal Natural produced DNA damage in Madin-Darby canine kidney cells (MDCKII). This study has shown that antiperspirants cause DNA damage on a single-cell level. Additionally, our data showed us that in general, Secret Platinum for women and Old Spice for men, produced equivalent damage. Crystal Natural, marketed as being safer or less damaging, induced the most extensive damage of all three antiperspirants tested.

Detection of ozone-induced DNA single strand breaks in murine bronchoalveolar lavage cells acutely exposed in vivo.

PubMed

Haney, J T; Connor, T H; Li, L

1999-04-01

Single-strand breaks (SSBs) in DNA have been used a biomarker of oxidative damage. The comet assay, also known as single-cell gel electrophoresis, was used to investigate the ability of ozone (O(3)) to induce DNA SSBs in murine bronchoalveolar lavage (BAL) cells. The comet assay is more sensitive than other techniques currently utilized for detecting SSBs and requires fewer cells. In the present study, 3 mice were exposed for 3 h to 0.25 ppm of O(3), and 3 to 0.5 ppm of O(3) for 3 h. Two air-exposed mice served as negative controls. All mice were euthanized 3 h after exposure, at which time BAL cells were recovered from the lungs and stained with ethidium bromide. BAL cells recovered from an air-exposed mouse were exposed to various concentrations of H(2)O(2) in vitro for 1 h at 4 degrees C. Excluding cells from the H(2)O(2) group (n = 25), 50 randomly selected BAL cells were graded by comet tail length into 1 of 4 categories: no damage (0 mm), low damage (1-10 mm), medium damage (11-30 mm), and high damage (31 + mm). The nonparametric Wilcoxon rank-sum test was used for statistical analysis, and p values lower than .05 were considered significant. The H(2)O(2) and the 0.25 and 0.5 ppm O3 groups showed statistically significant increases in DNA SSBs as compared to air-exposed controls. The results of this study indicate that (1) O(3) induces DNA strand breaks in murine BAL cells at 0.25 and 0.5 ppm, as evidenced by statistically significant increases in the length of comet tails for O(3)-exposed groups, and (2) the comet assay can be used to assess O(3)-induced SSBs for in vivo exposures. Therefore, it has the potential as a biomarker for in vivo oxidant exposures.

Diamagnetic region(s): structure of the unmagnetized plasma around Comet 67P/CG

NASA Astrophysics Data System (ADS)

Henri, P.; Vallières, X.; Hajra, R.; Goetz, C.; Richter, I.; Glassmeier, K.-H.; Galand, M.; Rubin, M.; Eriksson, A. I.; Nemeth, Z.; Vigren, E.; Beth, A.; Burch, J. L.; Carr, C.; Nilsson, H.; Tsurutani, B.; Wattieaux, G.

2017-07-01

The ESA's comet chaser Rosetta has monitored the evolution of the ionized atmosphere of comet 67P/Churyumov-Gerasimenko (67P/CG) and its interaction with the solar wind, during more than 2 yr. Around perihelion, while the cometary outgassing rate was highest, Rosetta crossed hundreds of unmagnetized regions, but did not seem to have crossed a large-scale diamagnetic cavity as anticipated. Using in situ Rosetta observations, we characterize the structure of the unmagnetized plasma found around comet 67P/CG. Plasma density measurements from RPC-MIP are analysed in the unmagnetized regions identified with RPC-MAG. The plasma observations are discussed in the context of the cometary escaping neutral atmosphere, observed by ROSINA/COPS. The plasma density in the different diamagnetic regions crossed by Rosetta ranges from ˜100 to ˜1500 cm-3. They exhibit a remarkably systematic behaviour that essentially depends on the comet activity and the cometary ionosphere expansion. An effective total ionization frequency is obtained from in situ observations during the high outgassing activity phase of comet 67P/CG. Although several diamagnetic regions have been crossed over a large range of distances to the comet nucleus (from 50 to 400 km) and to the Sun (1.25-2.4 au), in situ observations give strong evidence for a single diamagnetic region, located close to the electron exobase. Moreover, the observations are consistent with an unstable contact surface that can locally extend up to about 10 times the electron exobase.

Fluorescence Adherence Inhibition Assay: A Novel Functional Assessment of Blocking Virus Attachment by Vaccine-Induced Antibodies

PubMed Central

Asati, Atul; Kachurina, Olga; Karol, Alex; Dhir, Vipra; Nguyen, Michael; Parkhill, Robert; Kouiavskaia, Diana; Chumakov, Konstantin; Warren, William; Kachurin, Anatoly

2016-01-01

Neutralizing antibodies induced by vaccination or natural infection play a critically important role in protection against the viral diseases. In general, neutralization of the viral infection occurs via two major pathways: pre- and post-attachment modes, the first being the most important for such infections as influenza and polio, the latter being significant for filoviruses. Neutralizing capacity of antibodies is typically evaluated by virus neutralization assays that assess reduction of viral infectivity to the target cells in the presence of functional antibodies. Plaque reduction neutralization test, microneutralization and immunofluorescent assays are often used as gold standard virus neutralization assays. However, these methods are associated with several important prerequisites such as use of live virus requiring safety precautions, tedious evaluation procedure and long assessment time. Hence, there is a need for a robust, inexpensive high throughput functional assay that can be performed rapidly using inactivated virus, without extensive safety precautions. Herein, we report a novel high throughput Fluorescence Adherence Inhibition assay (fADI) using inactivated virus labeled with fluorescent secondary antibodies virus and Vero cells or erythrocytes as targets. It requires only few hours to assess pre-attachment neutralizing capacity of donor sera. fADI assay was tested successfully on donors immunized with polio, yellow fever and influenza vaccines. To further simplify and improve the throughput of the assay, we have developed a mathematical approach for calculating the 50% titers from a single sample dilution, without the need to analyze multi-point titration curves. Assessment of pre- and post-vaccination human sera from subjects immunized with IPOL®, YF-VAX® and 2013–2014 Fluzone® vaccines demonstrated high efficiency of the assay. The results correlated very well with microneutralization assay performed independently by the FDA Center of Biologics Evaluation and Research, with plaque reduction neutralization test performed by Focus Diagnostics, and with hemaglutination inhibition assay performed in-house at Sanofi Pasteur. Taken together, fADI assay appears to be a useful high throughput functional immunoassay for assessment of antibody-related neutralization of the viral infections for which pre-attachment neutralization pathway is predominant, such as polio, influenza, yellow fever and dengue. PMID:26863313

Radio-protective effect of cinnamic acid, a phenolic phytochemical, on genomic instability induced by X-rays in human blood lymphocytes in vitro.

PubMed

Cinkilic, Nilufer; Tüzün, Ece; Çetintaş, Sibel Kahraman; Vatan, Özgür; Yılmaz, Dilek; Çavaş, Tolga; Tunç, Sema; Özkan, Lütfi; Bilaloğlu, Rahmi

2014-08-01

The present study was designed to determine the protective activity of cinnamic acid against induction by X-rays of genomic instability in normal human blood lymphocytes. This radio-protective activity was assessed by use of the cytokinesis-block micronucleus test and the alkaline comet assay, with human blood lymphocytes isolated from two healthy donors. A Siemens Mevatron MD2 (Siemens AG, USA, 1994) linear accelerator was used for the irradiation with 1 or 2 Gy. Treatment of the lymphocytes with cinnamic acid prior to irradiation reduced the number of micronuclei when compared with that in control samples. Treatment with cinnamic acid without irradiation did not increase the number of micronuclei and did not show a cytostatic effect in the lymphocytes. The results of the alkaline comet assay revealed that cinnamic acid reduces the DNA damage induced by X-rays, showing a significant radio-protective effect. Cinnamic acid decreased the frequency of irradiation-induced micronuclei by 16-55% and reduced DNA breakage by 17-50%, as determined by the alkaline comet assay. Cinnamic acid may thus act as a radio-protective compound, and future studies may focus on elucidating the mechanism by which cinnamic acid offers radioprotection. Copyright © 2014 Elsevier B.V. All rights reserved.

Occupational risk assessment of paint industry workers

PubMed Central

de Oliveira, Hugo M.; Dagostim, Gracilene P.; da Silva, Arielle Mota; Tavares, Priscila; da Rosa, Luiz A. Z. C.; de Andrade, Vanessa M.

2011-01-01

Background: Thousands of chemical compounds are used in paint products, like pigments, extenders, binders, additives, and solvents (toluene, xylene, ketones, alcohols, esters, and glycol ethers). Paint manufacture workers are potentially exposed to the chemicals present in paint products although the patterns and levels of exposure to individual agents may differ from those of painters. The aim of the present study was to evaluate genome damage induced in peripheral blood lymphocytes and oral mucosa cells of paint industry workers. Materials and Methods: Genotoxicity was evaluated using the alkaline Comet assay in blood lymphocytes and oral mucosa cells, and the Micronucleus test in oral mucosa cells. For the micronucleus test in exfoliated buccal cells, no significant difference was detected between the control and paint industry workers. Results: The Comet assay in epithelia buccal cells showed that the damage index (DI) and damage frequency (DF) observed in the exposed group were significantly higher relative to the control group (P≤0.05). In the same way, the Comet assay data in peripheral blood leukocytes showed that both analysis parameters (DI and DF) were significantly greater than that for the control group (P≤0.05). Conclusions: Chronic occupational exposure to paints may lead to a slightly increased risk of genetic damage among paint industry workers. PMID:22223950

Evaluation of cytogenetic and DNA damage in human lymphocytes treated with adrenaline in vitro.

PubMed

Djelić, Ninoslav; Radaković, Milena; Spremo-Potparević, Biljana; Zivković, Lada; Bajić, Vladan; Stevanović, Jevrosima; Stanimirović, Zoran

2015-02-01

Catechol groups can be involved in redox cycling accompanied by generation of reactive oxygen species (ROS) which may lead to oxidative damage of cellular macromolecules including DNA. The objective of this investigation was to evaluate possible genotoxic effects of a natural catecholamine adrenaline in cultured human lymphocytes using cytogenetic (sister chromatid exchange and micronuclei) and the single cell gel electrophoresis (Comet) assay. In cytogenetic tests, six experimental concentrations of adrenaline were used in a range from 0.01-500 μM. There were no indications of genotoxic effects of adrenaline in sister chromatid exchange and micronucleus tests. However, at four highest concentrations of adrenaline (5 μM, 50 μM, 150 μM and 300 μM) we observed a decreased mitotic index and cell-cycle delay. In addition, in the Comet assay we used adrenaline in a range from 0.0005-500 μM, at two treatment times: 15 min or 60 min. In contrast to cytogenetic analysis, there was a dose-dependent increase of DNA damage detected in the Comet assay. These effects were significantly reduced by concomitant treatment with quercetin or catalase. Therefore, the obtained results indicate that adrenaline may exhibit genotoxic effects in cultured human lymphocytes, most likely due to production of reactive oxygen species. Copyright © 2014 Elsevier Ltd. All rights reserved.

Chemical composition and genotoxicity assessment of sanitary landfill leachate from Rovinj, Croatia.

PubMed

Gajski, Goran; Oreščanin, Višnja; Garaj-Vrhovac, Vera

2012-04-01

Chemical analysis and an in vitro approach were performed to assess elemental composition and genotoxic effects of the samples of landfill leachate taken from Lokva Vidotto sanitary landfill the official landfill for Rovinj town, Croatia. Two samples of landfill leachate were collected and analyzed in order to evaluate macro, micro and trace elements by atomic absorption spectroscopy, energy dispersive X-ray spectrometry and colorimetry. Genotoxicity of sanitary landfill leachate was evaluated in human lymphocytes by the use of the micronucleus test and comet assay. Samples were characterized with relatively low concentrations of heavy metals while organic component level exceeded upper permissible limit up to 39 times. Observed genotoxic effects should be connected with high concentrations of ammonia nitrogen, which exceeded permissible limit up to 180 times. Leachate samples of both sanitary landfills increased the frequency of micronuclei, nucleoplasmic bridges and nuclear buds. Increase of DNA damage in human lymphocytes was also detected by virtue of measuring comet assay parameters. All parameters showed statistically significant difference compared to negative control. Increased micronucleus and comet assay parameters indicate that both samples of sanitary landfill leachate are genotoxic and could pose environmental and human health risk if discharged to an aquatic environment. Copyright © 2011 Elsevier Inc. All rights reserved.

Role of Macronutrients and Micronutrients in DNA Damage: Results From a Food Frequency Questionnaire.

PubMed

Ladeira, Carina; Carolino, Elisabete; Gomes, Manuel C; Brito, Miguel

2017-01-01

The links between diet and genomic instability have been under investigation for several decades, and evidence suggests a significant causal or preventive role for various dietary factors. This study investigates the influence of macronutrients (calories, protein, and glucides) and micronutrients, such as vitamins and minerals, as assessed by a food frequency questionnaire, on genotoxicity biomarkers measured by cytokinesis-blocked micronucleus assay and comet assay. The results found significant positive and negative correlations. Micronucleus frequency tends to increase with higher intake of caffeine, calcium, magnesium, zinc, and protein ( P < .05, Spearman correlation). Calorie and omega-6 intakes are negatively correlated with DNA damage measured by the comet assay. These results are somewhat controversial because some of the correlations found are contrary to dominant views in the literature; however, we suggest that unraveling the association between diet and genetic instability requires a much better understanding of the modulating role of macronutrients and micronutrients.

The Zodiacal Cloud Model applied to the Martian atmosphere. Diurnal variations in meteoric ion layers

NASA Astrophysics Data System (ADS)

Carrillo-Sánchez, J. D.; Plane, J. M. C.; Withers, P.; Fallows, K.; Nesvorny, D.; Pokorný, P.

2016-12-01

Sporadic metal layers have been detected in the Martian atmosphere by radio occultation measurements using the Mars Express Orbiter and Mars Global Surveyor spacecraft. More recently, metallic ion layers produced by the meteor storm event following the close encounter between Comet Siding Spring (C/2013 A1) and Mars were identified by the Imaging UltraViolet Spectrograph (IUVS) and the Neutral Gas and Ion Mass Spectrometer (NGIMS) on the Mars Atmosphere and Volatile EvolutioN (MAVEN) spacecraft. Work is now in progress to detect the background metal layers produced by the influx of sporadic meteors. In this study we predict the likely appearance of these layers. The Zodiacal Dust Cloud (ZDC) model for particle populations released by asteroids (AST), and dust grains from Jupiter Family Comets (JFCs) and Halley-Type Comets (HTCs) has been combined with a Monte Carlo sampling method and the Chemical ABlation MODel (CABMOD) to predict the ablation rates of Na, K, Fe, Si, Mg, Ca and Al above 40 km altitude in the Martian atmosphere. CABMOD considers the standard treatment of meteor physics, including the balance of frictional heating by radiative losses and the absorption of heat energy through temperature increases, melting phase transitions and vaporization, as well as sputtering by inelastic collisions with the air molecules. The vertical injection profiles are input into the Leeds 1-D Mars atmospheric model which includes photo-ionization, and gas-phase ion-molecule and neutral chemistry, in order to explore the evolution of the resulting metallic ions and atoms. We conclude that the dominant contributor in the Martian's atmosphere is the JFCs over other sources. Finally, we explore the changes of the neutral and ionized Na, Mg and Fe layers over a diurnal cycle.

Vertical structure of the near-surface expanding ionosphere of comet 67P probed by Rosetta

NASA Astrophysics Data System (ADS)

Heritier, K. L.; Henri, P.; Vallières, X.; Galand, M.; Odelstad, E.; Eriksson, A. I.; Johansson, F. L.; Altwegg, K.; Behar, E.; Beth, A.; Broiles, T. W.; Burch, J. L.; Carr, C. M.; Cupido, E.; Nilsson, H.; Rubin, M.; Vigren, E.

2017-07-01

The plasma environment has been measured for the first time near the surface of a comet. This unique data set has been acquired at 67P/Churyumov-Gerasimenko during ESA/Rosetta spacecraft's final descent on 2016 September 30. The heliocentric distance was 3.8 au and the comet was weakly outgassing. Electron density was continuously measured with Rosetta Plasma Consortium (RPC)-Mutual Impedance Probe (MIP) and RPC-LAngmuir Probe (LAP) during the descent from a cometocentric distance of 20 km down to the surface. Data set from both instruments have been cross-calibrated for redundancy and accuracy. To analyse this data set, we have developed a model driven by Rosetta Orbiter Spectrometer for Ion and Neutral Analysis-COmetary Pressure Sensor total neutral density. The two ionization sources considered are solar extreme ultraviolet radiation and energetic electrons. The latter are estimated from the RPC-Ion and Electron Sensor (IES) and corrected for the spacecraft potential probed by RPC-LAP. We have compared the results of the model to the electron densities measured by RPC-MIP and RPC-LAP at the location of the spacecraft. We find good agreement between observed and modelled electron densities. The energetic electrons have access to the surface of the nucleus and contribute as the main ionization source. As predicted, the measurements exhibit a peak in the ionospheric density close to the surface. The location and magnitude of the peak are estimated analytically. The measured ionospheric densities cannot be explained with a constant outflow velocity model. The use of a neutral model with an expanding outflow is critical to explain the plasma observations.

Pickup Ions in the Plasma Environments of Mars, Comets, and Enceladus

NASA Astrophysics Data System (ADS)

Cravens, T.; Rahmati, A.; Sakai, S.; Madanian, H.; Larson, D. E.; Lillis, R. J.; Halekas, J. S.; Goldstein, R.; Burch, J. L.; Clark, G. B.; Jakosky, B. M.

2015-12-01

Ions created within a flowing plasma by ionization of neutrals respond to the electric and magnetic fields associated with the flow becoming what are called pick-up ions (PUI). PUI play an important role in many solar system plasma environments and affect the energy and momentum balance of the plasma flow. PUI have been observed during several recent space missions and PUI data will be compared and interpreted using models. Pick-up oxygen ions were observed in the solar wind upstream of Mars by the Solar Energetic Particle (SEP) and Solar Wind Ion Analyzer (SWIA) instruments on NASA's MAVEN (Mars Atmosphere and Volatile EvolutioN) spacecraft. The pick-up oxygen ions are created when atoms in the hot corona are ionized by solar radiation and charge exchange with solar wind protons. The ion fluxes measured by SEP can constrain the oxygen escape rate from Mars. PUI were also been detected at distances of 10 - 100 km from the nucleus of comet 67P/Churyumov- Gerasimenko (67P/CG) by plasma instruments (IES and ICA) onboard the Rosetta Orbiter when the comet was at 3 AU. The newly-born cometary ions are accelerated by the solar wind motional electric field but remain un-magnetized, as suggested by pre-encounter models (Rubin et al., 2014). The inner magnetosphere of Saturn and the water plume of the icy satellite Enceladus provide a third example of PUI. H2O+ ions created by ionization of neutral water producing ions that are picked-up by the co-rotating magnetospheric plasma flow. These ions then undergo a complex interaction with the plume gas including collisions that convert most H2O+ ions to H3O+, as measured by the Ion and Neutral Mass Spectrometer (INMS) onboard the Cassini spacecraft.

Absence of genotoxic effects of the chalcone (E)-1-(2-hydroxyphenyl)-3-(4-methylphenyl)-prop-2-en-1-one) and its potential chemoprevention against DNA damage using in vitro and in vivo assays

PubMed Central

2017-01-01

The chalcone (E)-1-(2-hydroxyphenyl)-3-(4-methylphenyl)-prop-2-en-1-one), or 2HMC, displays antileishmanial, antimalarial, and antioxidant activities. The aim of this study was to investigate the cytotoxic, genotoxic, mutagenic, and protective effects of 2HMC using the Ames mutagenicity test, the mouse bone marrow micronucleus test, and the comet assay in mice. In the assessment using the Ames test, 2HMC did not increase the number of His+ revertants in Salmonella typhimurium strains, demonstrating lack of mutagenicity. 2HMC showed no significant increase in micronucleated polychromatic erythrocyte frequency (MNPCE) in the micronucleus test, or in DNA strand breaks using the comet assay, evidencing absence of genotoxicity. Regarding cytotoxicity, 2HMC exhibited moderate cytotoxicity in mouse bone marrow cells by micronucleus test. 2HMC showed antimutagenic action in co-administration with the positive controls, sodium azide (SA) and 4-nitroquinoline-1-oxide (4NQO), in the Ames test. Co-administered and mainly pre-administered with cyclophosphamide (CPA), 2HMC caused a decrease in the frequency of MNPCE using the micronucleus test and in DNA strand breaks using the comet assay. Thus, 2HMC exhibited antimutagenic and antigenotoxic effects, displaying a DNA-protective effect against CPA, SA, and 4NQO carcinogens. In conclusion, 2HMC presented antimutagenic, antigenotoxic and moderate cytotoxic effects; therefore it is a promising molecule for cancer prevention. PMID:28207781

Absence of genotoxic effects of the chalcone (E)-1-(2-hydroxyphenyl)-3-(4-methylphenyl)-prop-2-en-1-one) and its potential chemoprevention against DNA damage using in vitro and in vivo assays.

PubMed

Lima, Débora Cristina da Silva; Vale, Camila Regina do; Véras, Jefferson Hollanda; Bernardes, Aline; Pérez, Caridad Noda; Chen-Chen, Lee

2017-01-01

The chalcone (E)-1-(2-hydroxyphenyl)-3-(4-methylphenyl)-prop-2-en-1-one), or 2HMC, displays antileishmanial, antimalarial, and antioxidant activities. The aim of this study was to investigate the cytotoxic, genotoxic, mutagenic, and protective effects of 2HMC using the Ames mutagenicity test, the mouse bone marrow micronucleus test, and the comet assay in mice. In the assessment using the Ames test, 2HMC did not increase the number of His+ revertants in Salmonella typhimurium strains, demonstrating lack of mutagenicity. 2HMC showed no significant increase in micronucleated polychromatic erythrocyte frequency (MNPCE) in the micronucleus test, or in DNA strand breaks using the comet assay, evidencing absence of genotoxicity. Regarding cytotoxicity, 2HMC exhibited moderate cytotoxicity in mouse bone marrow cells by micronucleus test. 2HMC showed antimutagenic action in co-administration with the positive controls, sodium azide (SA) and 4-nitroquinoline-1-oxide (4NQO), in the Ames test. Co-administered and mainly pre-administered with cyclophosphamide (CPA), 2HMC caused a decrease in the frequency of MNPCE using the micronucleus test and in DNA strand breaks using the comet assay. Thus, 2HMC exhibited antimutagenic and antigenotoxic effects, displaying a DNA-protective effect against CPA, SA, and 4NQO carcinogens. In conclusion, 2HMC presented antimutagenic, antigenotoxic and moderate cytotoxic effects; therefore it is a promising molecule for cancer prevention.

Cytogenetic status of human lymphocytes after exposure to low concentrations of p,p'-DDT, and its metabolites (p,p'-DDE, and p,p'-DDD) in vitro.

PubMed

Gerić, Marko; Ceraj-Cerić, Nikolina; Gajski, Goran; Vasilić, Želimira; Capuder, Željka; Garaj-Vrhovac, Vera

2012-06-01

Despite that the use of DDT has been restricted for more than 40 years to malaria affected areas, low doses of this pesticide and its metabolites DDE and DDD can be found in the environment around the world. Although it has been shown that these pollutants induce cell and DNA damage, the mechanisms of their cytogenotoxic activity remains largely unknown. This study looks into their possible genotoxic effects, at doses that can be found in body fluids, on human lymphocytes using the cytokinesis-block micronucleus assay and the comet assay. After exposure for 1, 6, and 24 h compounds p,p'-DDT (0.1 μg mL(-1)), p,p'-DDE (4.1 μg mL(-1)), and p,p'-DDD (3.9 μg mL(-1)) showed increase in DNA damage. The most significant results were observed at exposure period of 24 h where number of micronucleated cells increased from control 2.5±0.71 to 23.5±3.54, 13.5±0.71, and 16.5±6.36 for DDT, DDE, and DDD, respectively. Similar effect was observed using comet test where the percentage of DNA in comets tail increased from control 1.81±0.16 to 17.24±0.55, 11.21±0.56 and 9.28±0.50 for each compound, respectively. At the same time Fpg-comet assay failed to report induction of oxidative DNA damage of these pollutants. Additionally, the type of cell death was determined using diffusion assay and necrosis dominated. Our findings suggest that even at low concentrations, these pesticides could induce cytogenetic damage to human peripheral blood lymphocytes and in that manner have the impact on human health as well. Copyright © 2012 Elsevier Ltd. All rights reserved.

Monitoring of DNA breakage in embryonic stages of the African catfish Clarias gariepinus (Burchell, 1822) after exposure to lead nitrate using alkaline comet assay.

PubMed

Osman, Alaa G M; Mekkawy, Imam A; Verreth, Johan; Wuertz, Sven; Kloas, Werner; Kirschbaum, Frank

2008-12-01

Increasing lead contamination in Egyptian ecosystems and high lead concentrations in food items have raised concern for human health and stimulated studies on monitoring ecotoxicological impact of lead-caused genotoxicity. In this work, the alkaline comet assay was modified for monitoring DNA strand breakage in sensitive early life stages of the African catfish Clarias gariepinus. Following exposure to 100, 300, and 500 microg/L lead nitrate, DNA strand breakage was quantified in embryos at 30, 48, 96, 144, and 168 h post-fertilization (PFS). For quantitative analysis, four commonly used parameters (tail % DNA, %TDNA; head % DNA, %HDNA; tail length, TL; tail moment, TM) were analyzed in 96 nuclei (in triplicates) at each sampling point. The parameter %TDNA revealed highest resolution and lowest variation. A strong correlation between lead concentration, time of exposure, and DNA strand breakage was observed. Here, genotoxicity detected by comet assay preceded the manifested malformations assessed with conventional histology. Qualitative evaluation was carried out using five categories are as follows: undamaged (%TDNA < or = 10%), low damaged (10% < %TDNA < or = 25%), median damaged (25 < %TDNA < or = 50%), highly damaged (50 < %TDNA < or = 75%), and extremely damaged (%TDNA > 75%) nuclei confirming a dose and time-dependent shift towards increased frequencies of highly and extremely damaged nuclei. A protective capacity provided by a hardened chorion is a an interesting finding in this study as DNA damage in the prehatching stages 30 h-PFS and 48 h-PFS was low in all treatments (qualitative and quantitative analyses). These results clearly show that the comet assay is a sensitive tool for the detection of genotoxicity in vulnerable early life stages of the African catfish and is a method more sensitive than histological parameters for monitoring genotoxic effects. 2008 Wiley Periodicals, Inc.

A novel reporter system for neutralizing and enhancing antibody assay against dengue virus.

PubMed

Song, Ke-Yu; Zhao, Hui; Jiang, Zhen-You; Li, Xiao-Feng; Deng, Yong-Qiang; Jiang, Tao; Zhu, Shun-Ya; Shi, Pei-Yong; Zhang, Bo; Zhang, Fu-Chun; Qin, E-De; Qin, Cheng-Feng

2014-02-18

Dengue virus (DENV) still poses a global public health threat, and no vaccine or antiviral therapy is currently available. Antibody plays distinct roles in controlling DENV infections. Neutralizing antibody is protective against DENV infection, whereas sub-neutralizing concentration of antibody can increase DENV infection, termed antibody-dependent enhancement (ADE). Plaque-based assay represents the most widely accepted method measuring neutralizing or enhancing antibodies. In this study, a novel reporter virus-based system was developed for measuring neutralization and ADE activity. A stable Renilla luciferase reporter DENV (Luc-DENV) that can produce robust luciferase signals in BHK-21 and K562 cells were used to establish the assay and validated against traditional plaque-based assay. Luciferase value analysis using various known DENV-specific monoclonal antibodies showed good repeatability and a well linear correlation with conventional plaque-based assays. The newly developed assay was finally validated with clinical samples from infected animals and individuals. This reporter virus-based assay for neutralizing and enhancing antibody evaluation is rapid, lower cost, and high throughput, and will be helpful for laboratory detection and epidemiological investigation for DENV antibodies.

Development of a Test Method for the Evaluation of DNA Damage in Mouse Spermatogonial Stem Cells

PubMed Central

Jeon, Hye Lyun; Yi, Jung-Sun; Kim, Tae Sung; Oh, Youkyung; Lee, Hye Jeong; Lee, Minseong; Bang, Jin Seok; Ko, Kinarm; Ahn, Il Young; Ko, Kyungyuk; Kim, Joohwan; Park, Hye-Kyung; Lee, Jong Kwon; Sohn, Soo Jung

2017-01-01

Although alternative test methods based on the 3Rs (Replacement, Reduction, Refinement) are being developed to replace animal testing in reproductive and developmental toxicology, they are still in an early stage. Consequently, we aimed to develop alternative test methods in male animals using mouse spermatogonial stem cells (mSSCs). Here, we modified the OECD TG 489 and optimized the in vitro comet assay in our previous study. This study aimed to verify the validity of in vitro tests involving mSSCs by comparing their results with those of in vivo tests using C57BL/6 mice by gavage. We selected hydroxyurea (HU), which is known to chemically induce male reproductive toxicity. The 50% inhibitory concentration (IC50) value of HU was 0.9 mM, as determined by the MTT assay. In the in vitro comet assay, % tail DNA and Olive tail moment (OTM) after HU administration increased significantly, compared to the control. Annexin V, PI staining and TUNEL assays showed that HU caused apoptosis in mSSCs. In order to compare in vitro tests with in vivo tests, the same substances were administered to male C57BL/6 mice. Reproductive toxicity was observed at 25, 50, 100, and 200 mg/kg/day as measured by clinical measures of reduction in sperm motility and testicular weight. The comet assay, DCFH-DA assay, H&E staining, and TUNEL assay were also performed. The results of the test with C57BL/6 mice were similar to those with mSSCs for HU treatment. Finally, linear regression analysis showed a strong positive correlation between results of in vitro tests and those of in vivo. In conclusion, the present study is the first to demonstrate the effect of HU-induced DNA damage, ROS formation, and apoptosis in mSSCs. Further, the results of the current study suggest that mSSCs could be a useful model to predict male reproductive toxicity. PMID:28443181

Solar electric propulsion thruster interactions with solar arrays

NASA Technical Reports Server (NTRS)

Parks, D. E.; Katz, I.

1977-01-01

The effect of interactions of spacecraft-generated and naturally occurring plasmas with high voltage solar array components on an advanced solar electric propulsion system proposed for the Halley's Comet rendezvous mission was investigated. The spacecraft-generated plasma consists of mercury ions and neutralizing electrons resulting from the operation of ion thrusters (the charge-exchange plasma) and associated hollow cathode neutralizers. Quantitative results are given for the parasitic currents and power coupled into solar arrays with voltage fixed as a function of position on the array.

Analysis of variola and vaccinia virus neutralization assays for smallpox vaccines.

PubMed

Hughes, Christine M; Newman, Frances K; Davidson, Whitni B; Olson, Victoria A; Smith, Scott K; Holman, Robert C; Yan, Lihan; Frey, Sharon E; Belshe, Robert B; Karem, Kevin L; Damon, Inger K

2012-07-01

Possible smallpox reemergence drives research for third-generation vaccines that effectively neutralize variola virus. A comparison of neutralization assays using different substrates, variola and vaccinia (Dryvax and modified vaccinia Ankara [MVA]), showed significantly different 90% neutralization titers; Dryvax underestimated while MVA overestimated variola neutralization. Third-generation vaccines may rely upon neutralization as a correlate of protection.

Ion Acoustic Waves Observed at Comet 67P/Churyumov-Gerasimenko

NASA Astrophysics Data System (ADS)

Gunell, H.; Nilsson, H.; Hamrin, M.; Eriksson, A.; Maggiolo, R.; Pierre, H.; Altwegg, K.; Tzou, C. Y.; Rubin, M.; Glassmeier, K. H.; Stenberg Wieser, G.; Wedlund, C. S.; De Keyser, J.; Dhooghe, F.; Cessateur, G.; Gibbons, A.

2016-12-01

We present observations of ion acoustic waves at Comet 67P/Churyumov-Gerasimenko performed on 20 January 2015 when the Rosetta spacecraft was located near the terminator, 28 km from the nucleus of the comet. At the time of the observations the activity of the comet was still low. We use distribution functions obtained by the Ion Composition Analyser of the Rosetta Plasma Consortium (RPC-ICA) and electron temperature estimatesfrom the Langmuir Probes (RPC-LAP) to compute dispersion relations for waves on the ion timescale, and compare the results to spectra obtained by RPC-LAP. The peaks of the wave spectra appear at frequencies near 500 Hz. We perform cross-calibrations between RPC-ICA, RPC-LAP, and the Mutual Impedance Probe (RPC-MIP). Matching the dispersion relations to the wave observations helps us to form an estimate of the plasma density. At times when there is significant wave activity the water ion distribution is constituted by a cold (0.01 eV) population of locally produced ions and a thin tail of ions that have been accelerated by an electric field. The tail is approximately unidirectional, covering a wide velocity range, and centred at 20km/s in the spacecraft frame. At other times a warm (approximately 1 eV), mainly isotropic, ion population renders the ion acoustic mode heavily damped, and no waves are observed. Observations of the neutral density by the ROSINA COPS instrument indicate that frictional heating by the radial neutral flow contributes to this warm ion population. This work was supported by the Belgian Science Policy Office through the Solar-Terrestrial Centre of Excellence and by PRODEX/ROSETTA/ROSINA PEA 4000107705.

Chicken Fetal Liver DNA Damage and Adduct Formation by Activation-Dependent DNA-Reactive Carcinogens and Related Compounds of Several Structural Classes

PubMed Central

Williams, Gary M.; Duan, Jian-Dong; Brunnemann, Klaus D.; Iatropoulos, Michael J.; Vock, Esther; Deschl, Ulrich

2014-01-01

The chicken egg genotoxicity assay (CEGA), which utilizes the liver of an intact and aseptic embryo-fetal test organism, was evaluated using four activation-dependent DNA-reactive carcinogens and four structurally related less potent carcinogens or non-carcinogens. In the assay, three daily doses of test substances were administered to eggs containing 9–11-day-old fetuses and the fetal livers were assessed for two endpoints, DNA breaks using the alkaline single cell gel electrophoresis (comet) assay and DNA adducts using the 32P-nucleotide postlabeling (NPL) assay. The effects of four carcinogens of different structures requiring distinct pathways of bioactivation, i.e., 2-acetylaminofluorene (AAF), aflatoxin B1 (AFB1), benzo[a]pyrene (B[a]P), and diethylnitrosamine (DEN), were compared with structurally related non-carcinogens fluorene (FLU) and benzo[e]pyrene (B[e]P) or weak carcinogens, aflatoxin B2 (AFB2) and N-nitrosodiethanolamine (NDELA). The four carcinogens all produced DNA breaks at microgram or low milligram total doses, whereas less potent carcinogens and non-carcinogens yielded borderline or negative results, respectively, at higher doses. AAF and B[a]P produced DNA adducts, whereas none was found with the related comparators FLU or B[e]P, consistent with comet results. DEN and NDELA were also negative for adducts, as expected in the case of DEN for an alkylating agent in the standard NPL assay. Also, AFB1 and AFB2 were negative in NPL, as expected, due to the nature of ring opened aflatoxin adducts, which are resistant to enzymatic digestion. Thus, the CEGA, using comet and NPL, is capable of detection of the genotoxicity of diverse DNA-reactive carcinogens, while not yielding false positives for non-carcinogens. PMID:24973097

Prebiotic chemicals-amino acid and phosphorus-in the coma of comet 67P/Churyumov-Gerasimenko.

PubMed

Altwegg, Kathrin; Balsiger, Hans; Bar-Nun, Akiva; Berthelier, Jean-Jacques; Bieler, Andre; Bochsler, Peter; Briois, Christelle; Calmonte, Ursina; Combi, Michael R; Cottin, Hervé; De Keyser, Johan; Dhooghe, Frederik; Fiethe, Bjorn; Fuselier, Stephen A; Gasc, Sébastien; Gombosi, Tamas I; Hansen, Kenneth C; Haessig, Myrtha; Jäckel, Annette; Kopp, Ernest; Korth, Axel; Le Roy, Lena; Mall, Urs; Marty, Bernard; Mousis, Olivier; Owen, Tobias; Rème, Henri; Rubin, Martin; Sémon, Thierry; Tzou, Chia-Yu; Hunter Waite, James; Wurz, Peter

2016-05-01

The importance of comets for the origin of life on Earth has been advocated for many decades. Amino acids are key ingredients in chemistry, leading to life as we know it. Many primitive meteorites contain amino acids, and it is generally believed that these are formed by aqueous alterations. In the collector aerogel and foil samples of the Stardust mission after the flyby at comet Wild 2, the simplest form of amino acids, glycine, has been found together with precursor molecules methylamine and ethylamine. Because of contamination issues of the samples, a cometary origin was deduced from the (13)C isotopic signature. We report the presence of volatile glycine accompanied by methylamine and ethylamine in the coma of 67P/Churyumov-Gerasimenko measured by the ROSINA (Rosetta Orbiter Spectrometer for Ion and Neutral Analysis) mass spectrometer, confirming the Stardust results. Together with the detection of phosphorus and a multitude of organic molecules, this result demonstrates that comets could have played a crucial role in the emergence of life on Earth.

Prebiotic chemicals—amino acid and phosphorus—in the coma of comet 67P/Churyumov-Gerasimenko

PubMed Central

Altwegg, Kathrin; Balsiger, Hans; Bar-Nun, Akiva; Berthelier, Jean-Jacques; Bieler, Andre; Bochsler, Peter; Briois, Christelle; Calmonte, Ursina; Combi, Michael R.; Cottin, Hervé; De Keyser, Johan; Dhooghe, Frederik; Fiethe, Bjorn; Fuselier, Stephen A.; Gasc, Sébastien; Gombosi, Tamas I.; Hansen, Kenneth C.; Haessig, Myrtha; Jäckel, Annette; Kopp, Ernest; Korth, Axel; Le Roy, Lena; Mall, Urs; Marty, Bernard; Mousis, Olivier; Owen, Tobias; Rème, Henri; Rubin, Martin; Sémon, Thierry; Tzou, Chia-Yu; Hunter Waite, James; Wurz, Peter

2016-01-01

The importance of comets for the origin of life on Earth has been advocated for many decades. Amino acids are key ingredients in chemistry, leading to life as we know it. Many primitive meteorites contain amino acids, and it is generally believed that these are formed by aqueous alterations. In the collector aerogel and foil samples of the Stardust mission after the flyby at comet Wild 2, the simplest form of amino acids, glycine, has been found together with precursor molecules methylamine and ethylamine. Because of contamination issues of the samples, a cometary origin was deduced from the 13C isotopic signature. We report the presence of volatile glycine accompanied by methylamine and ethylamine in the coma of 67P/Churyumov-Gerasimenko measured by the ROSINA (Rosetta Orbiter Spectrometer for Ion and Neutral Analysis) mass spectrometer, confirming the Stardust results. Together with the detection of phosphorus and a multitude of organic molecules, this result demonstrates that comets could have played a crucial role in the emergence of life on Earth. PMID:27386550

The presence of clathrates in comet 67P/Churyumov-Gerasimenko

PubMed Central

Luspay-Kuti, Adrienn; Mousis, Olivier; Hässig, Myrtha; Fuselier, Stephen A.; Lunine, Jonathan I.; Marty, Bernard; Mandt, Kathleen E.; Wurz, Peter; Rubin, Martin

2016-01-01

Cometary nuclei are considered to most closely reflect the composition of the building blocks of our solar system. As such, comets carry important information about the prevalent conditions in the solar nebula before and after planet formation. Recent measurements of the time variation of major and minor volatile species in the coma of the Jupiter family comet 67P/Churyumov-Gerasimenko (67P) by the ROSINA (Rosetta Orbiter Spectrometer for Ion and Neutral Analysis) instrument onboard Rosetta provide insight into the possible origin of this comet. The observed outgassing pattern indicates that the nucleus of 67P contains crystalline ice, clathrates, and other ices. The observed outgassing is not consistent with gas release from an amorphous ice phase with trapped volatile gases. If the building blocks of 67P were formed from crystalline ices and clathrates, then 67P would have agglomerated from ices that were condensed and altered in the protosolar nebula closer to the Sun instead of more pristine ices originating from the interstellar medium or the outskirts of the disc, where amorphous ice may dominate. PMID:27152351

The presence of clathrates in comet 67P/Churyumov-Gerasimenko.

PubMed

Luspay-Kuti, Adrienn; Mousis, Olivier; Hässig, Myrtha; Fuselier, Stephen A; Lunine, Jonathan I; Marty, Bernard; Mandt, Kathleen E; Wurz, Peter; Rubin, Martin

2016-04-01

Cometary nuclei are considered to most closely reflect the composition of the building blocks of our solar system. As such, comets carry important information about the prevalent conditions in the solar nebula before and after planet formation. Recent measurements of the time variation of major and minor volatile species in the coma of the Jupiter family comet 67P/Churyumov-Gerasimenko (67P) by the ROSINA (Rosetta Orbiter Spectrometer for Ion and Neutral Analysis) instrument onboard Rosetta provide insight into the possible origin of this comet. The observed outgassing pattern indicates that the nucleus of 67P contains crystalline ice, clathrates, and other ices. The observed outgassing is not consistent with gas release from an amorphous ice phase with trapped volatile gases. If the building blocks of 67P were formed from crystalline ices and clathrates, then 67P would have agglomerated from ices that were condensed and altered in the protosolar nebula closer to the Sun instead of more pristine ices originating from the interstellar medium or the outskirts of the disc, where amorphous ice may dominate.

Irradiation influence on the detection of genetic-modified soybeans

NASA Astrophysics Data System (ADS)

Villavicencio, A. L. C. H.; Araújo, M. M.; Baldasso, J. G.; Aquino, S.; Konietzny, U.; Greiner, R.

2004-09-01

Three soybean varieties were analyzed to evaluate the irradiation influence on the detection of genetic modification. Samples were treated in a 60Co facility at dose levels of 0, 500, 800, and 1000Gy. The seeds were at first analyzed by Comet Assay as a rapid screening irradiation detection method. Secondly, germination test was performed to detect the viability of irradiated soybeans. Finally, because of its high sensitivity, its specificity and rapidity the polimerase chain reaction was the method applied for genetic modified organism detection. The analysis of DNA by the single technique of microgel electrophoresis of single cells (DNA Comet Assay) showed that DNA damage increased with increasing radiation doses. No negative influence of irradiation on the genetic modification detection was found.

The Gas Production Rate and Coma Structure of Comet C/1995 01 (Hale-Bopp)

NASA Technical Reports Server (NTRS)

Morgenthaler, Jeffrey P.; Harris, Walter M.; Roesler, Frederick L.; Scherb, Frank; Anderson, Christopher M.; Doane, Nathaniel E.; Oliversen, Ronald J.

2002-01-01

The University of Wisconsin-Madison and NASA-Goddard conducted a comprehensive multi-wavelength observing campaign of coma emissions from comet Hale-Bopp, including OH 3080 A, [O I] 6300 A, H2O(+) 6158 A, H Balmer-alpha 6563 A, NH2 6330 A, [C I] 9850 A CN 3879 A, C2 5141 A, C3 4062 A, C I 1657 A, and the UV and optical continua. In this work, we concentrate on the results of the H2O daughter studies. Our wide-field OH 3080 A measured flux agrees with other, similar observations and the expected value calculated from published water production rates using standard H2O and OH photochemistry. However, the total [O I] 6300 A flux determined spectroscopically over a similar field-of-view was a factor of 3 - 4 higher than expected. Narrow-band [O I] images show this excess came from beyond the H2O scale length, suggesting either a previously unknown source of [O I] or an error in the standard OH + upsilon to O((sup I)D) + H branching ratio. The Hale-Bopp OH and [O I] distributions, both of which were imaged to cometocentric distances greater than 1 x 10(exp 6) km, were more spatially extended than those of comet Halley (after correcting for brightness differences), suggesting a higher bulk outflow velocity. Evidence of the driving mechanism for this outflow is found in the H(alpha) line profile, which was narrower than in comet Halley (though likely because of opacity effects, not as narrow as predicted by Monte-Carlo models). This is consistent with greater collisional coupling between the suprathermal H photodissociation products and Hale-Bopp's dense coma. Presumably because of mass loading of the solar wind by ions and ions by the neutrals, the measured acceleration of H2O(+) down the ion tail was much smaller than in comet Halley. Tailward extensions in the azimuthal distributions of OH 3080 A, [O I], and [C I], as well as a Doppler asymmetry in the [O I] line profile, suggest ion-neutral coupling. While the tailward extension in the OH can be explained by increased neutral acceleration, the [O I] 6300 A and [C I] 9850 A emissions show 13% and less than 200% excesses in this direction (respectively), suggesting a non-negligible contribution from dissociative recombination of CO(+) and/or electron collisional excitation. Thus, models including the effects of photo-and collisional chemistry are necessary for the full interpretation of these data.

Tungsten carbide-cobalt as a nanoparticulate reference positive control in in vitro genotoxicity assays.

PubMed

Moche, Hélène; Chevalier, Dany; Barois, Nicolas; Lorge, Elisabeth; Claude, Nancy; Nesslany, Fabrice

2014-01-01

With the increasing human exposure to nanoparticles (NP), the evaluation of their genotoxic potential is of significant importance. However, relevance for NP of the routinely used in vitro genotoxicity assays is often questioned, and a nanoparticulate reference positive control would therefore constitute an important step to a better testing of NP, ensuring that test systems are really appropriate. In this study, we investigated the possibility of using tungsten carbide-cobalt (WC-Co) NP as reference positive control in in vitro genotoxicity assays, including 2 regulatory assays, the mouse lymphoma assay and the micronucleus assay, and in the Comet assay, recommended for the toxicological evaluation of nanomedicines by the French Agency of Human Health Products (Afssaps). Through these assays, we were able to study different genetic endpoints in 2 cell types commonly used in regulatory genotoxicity assays: the L5178Y mouse lymphoma cell line and primary cultures of human lymphocytes. Our results showed that the use of WC-Co NP as positive control in in vitro genotoxicity assays was conceivable, but that different parameters have to be considered, such as cell type and treatment schedule. L5178Y mouse lymphoma cells did not provide satisfactory results in the 3 performed tests. However, human lymphocytes were more sensitive to genotoxic effects induced by WC-Co NP, particularly after a 24-h treatment in the in vitro micronucleus assay and after a 4-h treatment in the in vitro Comet assay. Under such conditions, WC-Co could be used as a nanoparticulate reference positive control in these assays.

Sensitivity and fragmentation calibration of the time-of-flight mass spectrometer RTOF on board ESA's Rosetta mission

NASA Astrophysics Data System (ADS)

Gasc, Sébastien; Altwegg, Kathrin; Jäckel, Annette; Le Roy, Léna; Rubin, Martin; Fiethe, Björn; Mall, Urs; Rème, Henri

2014-05-01

The European Space Agency's Rosetta mission will rendez-vous comet 67P/Churyumov-Gerasimenko (67P) in September 2014. The Rosetta spacecraft with the Rosetta Orbiter Spectrometer for Ion and Neutral Analysis (ROSINA) onboard will follow and survey 67P for more than a year until the comet reaches its perihelion and beyond. ROSINA will provide new information on the global molecular, elemental, and isotopic composition of the coma [1]. ROSINA consists of a pressure sensor (COPS) and two mass spectrometers, the Double Focusing Mass Spectrometer (DFMS) and the Reflectron Time Of Flight mass spectrometer (RTOF). RTOF has a wide mass range, from 1 amu/e to >300 amu/e, and contains two ion sources, a reflectron and two detectors. The two ion sources, the orthogonal and the storage source, are capable to measure cometary ions while the latter also allows measuring cometary neutral gas. In neutral gas mode the ionization is performed through electron impact. A built-in Gas Calibration Unit (GCU) contains a known gas mixture composed of He, CO2, and Kr that can be used for in-flight calibration of the instrument. Among other ROSINA specific scientific goals, RTOF's task will be to determine molecular composition of volatiles via measuring and separating heavy hydrocarbons; it has been designed to study the development of the cometary activity as well as the coma chemistry between 3.5 AU and perihelion. From the spectroscopic studies and in-situ observations of other comets, we expect to find molecules such as H2O, CO, CO2, hydrocarbons, alcohols, formaldehyde, and other organic compounds in the coma of 67P/Churyumov-Gerasimenko [2]. To demonstrate and quantify the sensitivity and functionality of RTOF, calibration measurements have been realized with more than 20 species among the most abundant molecules quoted above, as well as other species such as PAHs. We will describe the applied methods used to realize this calibration and will discuss our preliminary results, i.e. RTOF capabilities in terms of sensitivity, isotopic ratios, and fragmentation patterns. We will demonstrate that RTOF is well capable to meet the requirements to address the scientific questions discussed above. [1] Balsiger, H. et al.: ROSINA-Rosetta Orbiter Spectrometer for Ion and Neutral Analysis, Space Science Reviews, Vol. 128, 745-801, 2007. [2] Bockelée-Morvan, D., Crovisier, J., Mumma, M. J., and Weaver, H. A.: The Composition of Cometary Volatiles, in Comets II (M. C. Festou et al., eds), Univ. Arizona Press, Tucson, 2004

Charge exchange in solar wind-cometary interactions

NASA Technical Reports Server (NTRS)

Gombosi, T. I.; Horanyi, M.; Kecskemety, K.; Cravens, T. E.; Nagy, A. F.

1983-01-01

A simple model of a cometary spherically symmetrical atmosphere and ionosphere is considered. An analytic solution of the governing equations describing the radial distribution of the neutral and ion densities is found. The new solution is compared to the well-known solution of the equations containing only ionization terms. Neglecting recombination causes a significant overestimate of the ion density in the vicinity of the comet. An axisymmetric model of the solar wind-cometary interaction is considered, taking into account the loss of solar wind ions due to charge exchange. The calculations predict that for active comets, solar wind absorption due to charge exchange becomes important at a few thousand kilometers from the nucleus, and a surface separating the shocked solar wind from the cometary ionosphere develops in this region. These calculations are in reasonable agreement with the few observations available for the ionopause location at comets.

Centered reduced moments and associate density functions applied to alkaline comet assay.

PubMed

Castaneda, Roman; Pelaez, Alejandro; Marquez, Maria-Elena; Abad, Pablo

2005-01-01

The single cell gel electrophoresis assay is a sensitive, rapid, and visual technique for deoxyribonucleic acid (DNA) strand-break detection in individual mammalian cells, whose application has significantly increased in the past few years. The cells are embedded in agarose on glass slides followed by lyses of the cell membrane. Thereafter, damaged DNA strands are electrophoresed away from the nucleus towards the anode giving the appearance of a comet tail. Nowadays, charge coupled device cameras are attached at optical microscopes for recording the images of the cells, and digital image processing is applied for obtaining quantitative descriptors. However, the conventional software is usually expensive, inflexible and, in many cases, can only provide low-order descriptors based in image segmentation, determination of centers of mass, and Euclidean distances. Associated density functions and centered reduced moments offer an effective and flexible alternative for quantitative analysis of the comet cells. We will show how the position of the center of mass, the lengths and orientation of the main semiaxes, and the eccentricity of such images can be accurately determined by this method.

Evaluating the potential genotoxicity of phthalates esters (PAEs) in perfumes using in vitro assays.

PubMed

Al-Saleh, Iman; Al-Rajudi, Tahreer; Al-Qudaihi, Ghofran; Manogaran, Pulicat

2017-10-01

We previously reported high levels of phthalate esters (PAEs) added as solvents or fixatives in 47 brands of perfumes. Diethyl phthalate was the most abundant compound (0.232-23,649 ppm), and 83.3% of the perfumes had levels >1 ppm, the threshold limit cited by a Greenpeace investigation. All samples had dimethyl phthalate levels higher than its threshold limit of 0.1 ppm, and 88, 38, and 7% of the perfumes had benzyl butyl phthalate, di(2-ethylhexyl) phthalate, and dibutyl phthalate levels, respectively, above their threshold limits. The role of PAEs as endocrine disruptors has been well documented, but their effect on genotoxic behavior has received little attention. We used in vitro single-cell gel electrophoresis (comet) and micronucleus (MN) assays with human lymphoblastoid TK6 cells to evaluate the genotoxic potency of 42 of the same perfumes and to determine its association with PAEs. All perfumes induced more DNA damage than a negative control (NEG), ≥ 90% of the samples caused more damage than cells treated with the vehicles possibly used in perfume's preparations such as methanol (ME) and ethanol (ET), and 11.6% of the perfumes caused more DNA damage than a positive control (hydrogen peroxide). Chromosome breakage expressed as MN frequency was higher in cells treated with 71.4, 64.3, 57.1, and 4.8% of the perfumes than in NEG, cells treated with ME or ET, and another positive control (x-rays), respectively. The genotoxic responses in the comet and MN assays were not correlated. The comet assay indicated that the damage in TK6 cells treated with five PAEs at concentrations of 0.05 and 0.2 ppm either individually or as a mixture did not differ significantly from the damage in cells treated with the perfumes. Unlike the comet assay, the sensitivity of the MN assay to PAEs was weak at both low and high concentrations, and MN frequencies were generally low. This study demonstrates for the first time the possible contribution of PAEs in perfumes to DNA damage and suggests that their use as solvents or fixatives should be regulated. Other ingredients with mutagenic/genotoxic properties, however, may also have contributed to the DNA damage. Future studies should focus on applying a series of assays that use different cellular models with various endpoints to identify the spectrum of genotoxic mechanisms involved.

A Cell Line-Based Neutralization Assay for Primary Human Immunodeficiency Virus Type 1 Isolates That Use either the CCR5 or the CXCR4 Coreceptor

PubMed Central

Trkola, Alexandra; Matthews, Jamie; Gordon, Cynthia; Ketas, Tom; Moore, John P.

1999-01-01

We describe here a cell line-based assay for the evaluation of human immunodeficiency virus type 1 (HIV-1) neutralization. The assay is based on CEM.NKR cells, transfected to express the HIV-1 coreceptor CCR5 to supplement the endogenous expression of CD4 and the CXCR4 coreceptor. The resulting CEM.NKR-CCR5 cells efficiently replicate primary HIV-1 isolates of both R5 and X4 phenotypes. A comparison of the CEM.NKR-CCR5 cells with mitogen-activated peripheral blood mononuclear cells (PBMC) in neutralization assays with sera from HIV-1-infected individuals or specific anti-HIV-1 monoclonal antibodies shows that the sensitivity of HIV-1 neutralization is similar in the two cell types. The CEM.NKR-CCR5 cell assay, however, is more convenient to perform and eliminates the donor-to-donor variation in HIV-1 replication efficiency, which is one of the principal drawbacks of the PBMC-based neutralization assay. We suggest that this new assay is suitable for the general measurement of HIV-1 neutralization by antibodies. PMID:10516002

Summary of major conclusions from the 6th International Workshop on Genotoxicity Testing (IWGT), Foz do Iguacu, Brazil

EPA Science Inventory

The paper describes major conclusions of working groups convened in the following areas: comet assay; micronucleus test in the liver and organs other than bone marrow; pig-A assay; quantitative approaches to genotoxicity risk assessment; and approaches for identifying germ cell m...

Comparative analysis of three sperm DNA damage assays and sperm nuclear protein content in couples undergoing assisted reproduction treatment.

PubMed

Simon, L; Liu, L; Murphy, K; Ge, S; Hotaling, J; Aston, K I; Emery, B; Carrell, D T

2014-05-01

Is there an association between sperm DNA damage, measured by three different assays, sperm nuclear protein content and clinical outcomes in assisted reproduction treatment (ART)? Sperm DNA damage measured by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling (TUNEL) and the Comet assay were significantly associated with ART outcomes in our single institution study. Abnormal protamine expression is known to be associated with sperm DNA damage and male infertility. A number of studies have shown a significant relationship between sperm DNA damage and ART outcomes. To date, there are no large studies providing direct comparisons of DNA damage tests within the same study population. Thus, the prognostic value for each method remains unknown. Cross-sectional study of 238 men from infertile couples undergoing ART at the University Center for Reproductive Medicine, Utah, USA, between April 2011 and March 2013. Sperm from men undergoing ART were tested for DNA damage using the alkaline Comet assay, TUNEL and flow cytometric chromatin evaluation (FCCE) assays. Histone retention was analysed using the aniline blue staining method, whereas protamine content (proteins P1 and P2) and ratio were analysed using acid urea gel electrophoresis. The prognostic value of each sperm DNA test to predict clinical pregnancy was calculated. Histone retention was associated with sperm DNA damage (P < 0.001), reduced embryo quality (P = 0.005) and clinical pregnancies (P < 0.001). The mean percentage of sperm with DNA damage was significantly higher in sperm from non-pregnant couples compared with that from pregnant couples, as measured by TUNEL assay (15.04 ± 1.16% versus 8.79 ± 0.56%; P < 0.001) and alkaline Comet assay (72.79 ± 2.49% versus 55.86 ± 2.29%; P < 0.001). There was no association between clinical pregnancies and DNA fragmentation index measured by FCCE (12.97 ± 1.46 versus 14.93 ± 1.65; P = 0.379). Of the protamine parameters analysed, only the P1/P2 ratio was associated with sperm count (P = 0.013), men's age (P = 0.037), maturity (P = 0.049) and blastocyst quality (P = 0.012). Histone retention and sperm DNA damage measured by Comet and TUNEL assays were associated with fertilization rate (P < 0.05), embryo quality (P < 0.05) and implantation rate (P < 0.05). A potential drawback of this study is that it is cross-sectional. Generally in such studies there is more than one variable that could cause the effect. Analysing sperm is one part of the equation; there are also a number of female factors that have the potential to influence ART outcomes. Therefore, given the large and well-established role of female factors in infertility, normal sperm DNA integrity and protamination do not necessarily ensure clinical pregnancy in ART. Thus, female factors can reduce the prognostic value of sperm DNA tests. Further, our use of native semen instead of prepared sperm may have iatrogenically increased the DNA damage. Alteration in sperm nuclear protein affects sperm DNA integrity. Further, with the current dataset, TUNEL and Comet assays appeared more predictive of ART success than FCCE. No personal or direct financial support has been received for any of this work. The authors declare no competing interests. N/A.

Automated image-based assay for evaluation of HIV neutralization and cell-to-cell fusion inhibition.

PubMed

Sheik-Khalil, Enas; Bray, Mark-Anthony; Özkaya Şahin, Gülsen; Scarlatti, Gabriella; Jansson, Marianne; Carpenter, Anne E; Fenyö, Eva Maria

2014-08-30

Standardized techniques to detect HIV-neutralizing antibody responses are of great importance in the search for an HIV vaccine. Here, we present a high-throughput, high-content automated plaque reduction (APR) assay based on automated microscopy and image analysis that allows evaluation of neutralization and inhibition of cell-cell fusion within the same assay. Neutralization of virus particles is measured as a reduction in the number of fluorescent plaques, and inhibition of cell-cell fusion as a reduction in plaque area. We found neutralization strength to be a significant factor in the ability of virus to form syncytia. Further, we introduce the inhibitory concentration of plaque area reduction (ICpar) as an additional measure of antiviral activity, i.e. fusion inhibition. We present an automated image based high-throughput, high-content HIV plaque reduction assay. This allows, for the first time, simultaneous evaluation of neutralization and inhibition of cell-cell fusion within the same assay, by quantifying the reduction in number of plaques and mean plaque area, respectively. Inhibition of cell-to-cell fusion requires higher quantities of inhibitory reagent than inhibition of virus neutralization.

The effect of mass loading outside cometary bow shock for the plasma and wave measurements in the coming cometary missions

NASA Astrophysics Data System (ADS)

Sagdeev, R. Z.; Shapiro, V. D.; Shevchenko, V. I.; Szego, K.

1987-02-01

The neutral gas emitted by comets is partly photoionized along its path. The interaction of the ions with the solar wind leads to observable particle and wave effects in the ambient plasma. These are described in the present paper.

Expected scientific results on ballistic spacecraft missions to comet Encke during the 1980 apparition

NASA Technical Reports Server (NTRS)

Mumma, M. J.

1976-01-01

Summarized are three proposed ballistic spacecraft missions to intercept P/Encke during the 1980 apparition. A baseline physical activity model for P/Encke is established and the performances of the neutral mass spectrometer and of the imaging experiment on each intercept mission are assessed.

[Essential oil from Artemisia lavandulaefolia induces apoptosis and necrosis of HeLa cells].

PubMed

Zhang, Lu-min; Lv, Xue-wei; Shao, Lin-xiang; Ma, Yan-fang; Cheng, Wen-zhao; Gao, Hai-tao

2013-12-01

To investigate the effects of Artemisia lavandulaefolia essential oil on apoptosis and necrosis of HeLa cells. Cell viability was assayed using MTT method. The morphological and structure alterations in HeLa cells were observed by microscopy. Furthermore, cell apoptosis was measured by DNA Ladder and flow cytometry. DNA damage was measured by comet assay, and the protein expression was examined by Western blot analysis. MTT assay displayed essential oil from Artemisia lavandulaefolia could inhibit the proliferation of HeLa cells in a dose-dependent manner. After treated with essential oil of Artemisia lavadulaefolia for 24 h, HeLa cells in 100 and 200 microg/mL experiment groups exhibited the typical morphology changes of undergoing apoptosis, such as cell shrinkage and nucleus chromatin condensed. However, the cells in the 400 microg/mL group showed the necrotic morphology changes including cytomembrane rupture and cytoplasm spillover. In addition, DNA Ladder could be demonstrated by DNA electrophoresis in each experiment group. Apoptosis peak was also evident in flow cytometry in each experiment group. After treating the HeLa cells with essential oil of Artemisia lavadulaefolia for 6 h, comet tail was detected by comet assay. Moreover, western blotting analysis showed that caspase-3 was activated and the cleavage of PARP was inactivated. Essential oil from Artemisia lavadulaefolia can inhibit the proliferation of HeLa cells in vitro. Low concentration of essential oil from Artemisia lavadulaefolia can induce apoptosis, whereas high concentration of the compounds result in necrosis of HeLa cells. And,the mechanism may be related to the caspase-3-mediated-PARP apoptotic signal pathway.

Comet assay with gill cells of Mytilus galloprovincialis end point tools for biomonitoring of water antibiotic contamination: Biological treatment is a reliable process for detoxification.

PubMed

Mustapha, Nadia; Zouiten, Amina; Dridi, Dorra; Tahrani, Leyla; Zouiten, Dorra; Mosrati, Ridha; Cherif, Ameur; Chekir-Ghedira, Leila; Mansour, Hedi Ben

2016-04-01

This article investigates the ability of Pseudomonas peli to treat industrial pharmaceuticals wastewater (PW). Liquid chromatography-mass spectrometry (MS)/MS analysis revealed the presence, in this PW, of a variety of antibiotics such as sulfathiazole, sulfamoxole, norfloxacine, cloxacilline, doxycycline, and cefquinome.P. peli was very effective to be grown in PW and inducts a remarkable increase in chemical oxygen demand and biochemical oxygen demand (140.31 and 148.51%, respectively). On the other hand, genotoxicity of the studied effluent, before and after 24 h of shaking incubation with P. peli, was evaluated in vivo in the Mediterranean wild mussels Mytilus galloprovincialis using comet assay for quantification of DNA fragmentation. Results show that PW exhibited a statistically significant (p< 0.001) genotoxic effect in a dose-dependent manner; indeed, the percentage of genotoxicity was 122.6 and 49.5% after exposure to 0.66 ml/kg body weight (b.w.); 0.33 ml/kg b.w. of PW, respectively. However, genotoxicity decreased strongly when tested with the PW obtained after incubation with P. peli We can conclude that using comet assay genotoxicity end points are useful tools to biomonitor the physicochemical and biological quality of water. Also, it could be concluded that P. peli can treat and detoxify the studied PW. © The Author(s) 2013.

Use of sensitive methods for detection of DNA damage on human lymphocytes exposed to p,p'-DDT: Comet assay and new criteria for scoring micronucleus test.

PubMed

Gajski, Goran; Ravlic, Sanda; Capuder, Zeljka; Garaj-Vrhovac, Vera

2007-08-01

Wide distribution, stability and long persistence in the environment of dichlorodiphenyltrichloroethane (DDT), probably the best-known and most useful insecticide in the world, imposes the need for further examination of the effect of this chemical on human health and especially on the human genome. In this study, peripheral blood human lymphocytes from a healthy donor were exposed to 0.025 mg/L concentration of p,p'-DDT at different time periods (1, 2, 24 and 48 h). For the assessment of genotoxic effect, the new criteria for scoring micronucleus test and alkaline comet assay were used. Both methods showed that p,p'-DDT induces DNA damage in low concentration used in this research. Results of micronucleus test showed a statistically significant (p < 0.05) genotoxic effect of p,p'-DDT on human lymphocytes compared with corresponding control and a different exposure time. A comet assay also showed increased DNA damage caused in p,p'-DDT-exposed human lymphocytes than in corresponding control cells for the tail length. Results obtained by measuring the level of DNA migration and incidence of micronuclei (MN), nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) indicate the sensitivity of these tests and their application in detection of primary genome damage after long-term exposure to establish the effect of p,p'-DDT on human genome.

DNA damage induced by coal dust, fly and bottom ash from coal combustion evaluated using the micronucleus test and comet assay in vitro.

PubMed

Matzenbacher, Cristina Araujo; Garcia, Ana Letícia Hilario; Dos Santos, Marcela Silva; Nicolau, Caroline Cardoso; Premoli, Suziane; Corrêa, Dione Silva; de Souza, Claudia Telles; Niekraszewicz, Liana; Dias, Johnny Ferraz; Delgado, Tânia Valéria; Kalkreuth, Wolfgang; Grivicich, Ivana; da Silva, Juliana

2017-02-15

Coal mining and combustion generating huge amounts of bottom and fly ash are major causes of environmental pollution and health hazards due to the release of polycyclic aromatic hydrocarbons (PAH) and heavy metals. The Candiota coalfield in Rio Grande do Sul, is one of the largest open-cast coal mines in Brazil. The aim of this study was to evaluate genotoxic and mutagenic effects of coal, bottom ash and fly ash samples from Candiota with the comet assay (alkaline and modified version) and micronucleus test using the lung fibroblast cell line (V79). Qualitative and quantitative analysis of PAH and inorganic elements was carried out by High Performance Liquid Chromatography (HPLC) and by Particle-Induced X-ray Emission (PIXE) techniques respectively. The samples demonstrated genotoxic and mutagenic effects. The comet assay modified using DNA-glicosilase formamidopirimidina (FPG) endonuclease showed damage related to oxidative stress mechanisms. The amount of PAHs was higher in fly ash followed by pulverized coal. The amount of inorganic elements was highest in fly ash, followed by bottom ash. It is concluded that the samples induce DNA damage by mechanisms that include oxidative stress, due to their complex composition, and that protective measures have to be taken regarding occupational and environmental hazards. Copyright © 2016 Elsevier B.V. All rights reserved.

Genetic damage induced by organic extract of coke oven emissions on human bronchial epithelial cells.

PubMed

Zhai, Qingfeng; Duan, Huawei; Wang, Yadong; Huang, Chuanfeng; Niu, Yong; Dai, Yufei; Bin, Ping; Liu, Qingjun; Chen, Wen; Ma, Junxiang; Zheng, Yuxin

2012-08-01

Coke oven emissions are known as human carcinogen, which is a complex mixture of polycyclic aromatic hydrocarbon. In this study, we aimed to clarify the mechanism of action of coke oven emissions induced carcinogenesis and to identify biomarkers of early biological effects in a human bronchial epithelial cell line with CYP1A1 activity (HBE-CYP1A1). Particulate matter was collected in the oven area on glass filter, extracted and analyzed by GC/MS. DNA breaks and oxidative damage were evaluated by alkaline and endonucleases (FPG, hOGG1 and ENDO III)-modified comet assays. Cytotoxicity and chromosomal damage were assessed by the cytokinesis-block micronucleus cytome (CBMN-Cyt) assay. The cells were treated with organic extract of coke oven emissions (OE-COE) representing 5, 10, 20, 40μg/mL extract for 24h. We found that there was a dose-effect relationship between the OE-COE and the direct DNA damage presented by tail length, tail intensity and Olive tail moment in the comet assay. The presence of lesion-specific endonucleases in the assays increased DNA migration after OE-COE treatment when compared to those without enzymes, which indicated that OE-COE produced oxidative damage at the level of pyrimidine and purine bases. The dose-dependent increase of micronuclei, nucleoplasmic bridges and nuclear buds in exposed cells was significant, indicating chromosomal and genomic damage induced by OE-COE. Based on the cytotoxic biomarkers in CBMN-Cyt assay, OE-COE may inhibit nuclear division, interfere with apoptosis, or induce cell necrosis. This study indicates that OE-COE exposure can induce DNA breaks/oxidative damage and genomic instability in HBE-CYP1A1 cells. The FPG-comet assay appears more specific for detecting oxidative DNA damage induced by complex mixtures of genotoxic substances. Copyright © 2012 Elsevier Ltd. All rights reserved.

The use of ex vivo human skin tissue for genotoxicity testing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Reus, Astrid A.; Usta, Mustafa; Krul, Cyrille A.M., E-mail: cyrille.krul@tno.nl

2012-06-01

As a result of the chemical legislation concerning the registration, evaluation, authorization and restriction of chemicals (REACH), and the Seventh Amendment to the Cosmetics Directive, which prohibits animal testing in Europe for cosmetics, alternative methods for safety evaluation of chemicals are urgently needed. Current in vitro genotoxicity assays are not sufficiently predictive for the in vivo situation, resulting in an unacceptably high number of misleading positives. For many chemicals and ingredients of personal care products the skin is the first site of contact, but there are no in vitro genotoxicity assays available in the skin for additional evaluation of positivemore » or equivocal responses observed in regulatory in vitro genotoxicity assays. In the present study ex vivo human skin tissue obtained from surgery was used for genotoxicity evaluation of chemicals by using the comet assay. Fresh ex vivo human skin tissue was cultured in an air–liquid interface and topically exposed to 20 chemicals, including true positive, misleading positive and true negative genotoxins. Based on the results obtained in the present study, the sensitivity, specificity and accuracy of the ex vivo skin comet assay to predict in vivo genotoxicity were 89%, 90% and 89%, respectively. Donor and experimental variability were mainly reflected in the magnitude of the response and not the difference between the presence and absence of a genotoxic response. The present study indicates that human skin obtained from surgery is a promising and robust model for safety evaluation of chemicals that are in direct contact with the skin. -- Highlights: ► We use human skin obtained from surgery for genotoxicity evaluation of chemicals. ► We use the comet assay as parameter for genotoxicity in ex vivo human skin. ► Sensitivity, specificity and accuracy to predict in vivo genotoxins are determined. ► Sensitivity, specificity and accuracy are 89%, 90% and 90%, respectively. ► The method is suitable for evaluation of chemicals that are in contact with skin.« less

Genoprotective effects of green tea (Camellia sinensis) in human subjects: results of a controlled supplementation trial.

PubMed

Han, K C; Wong, W C; Benzie, Iris F F

2011-01-01

Green tea is rich in polyphenolic antioxidants and has widely reported but largely unsubstantiated health benefits. In the present study, genoprotective effects of two types of green tea were studied both in an in vitro and in a human supplementation trial. For the in vitro study, human lymphocytes were pre-incubated in tea (0·005-0·1 %, w/v), washed and subjected to oxidant challenge induced by H2O2. In a placebo-controlled, cross-over supplementation study, eighteen healthy volunteers took 2 x 150 ml/d of 1% (w/v) green tea ('Longjing' green tea or 'screw-shaped' green tea) or water (control) for 4 weeks (n 6). Subjects took all the three treatments in a random order, with 6 weeks' washout between each treatment. Fasting blood and urine were collected before and after each treatment. The comet assay was used to measure the resistance of lymphocytic DNA to H2O2-induced challenge. Basal oxidation-induced DNA damage was measured using the formamidopyrimidine glycosylase (Fpg) enzyme-assisted comet assay. Urine 7,8-dihydro-2-deoxyguanosine (8-oxodG, mol/mmol creatinine), a biomarker of whole-body oxidative stress, was measured by liquid chromatography with tandem MS. In vitro testing results of tea-treated cells showed increased (P < 0·05) resistance of DNA to the challenge. In the supplementation trial, a significant (P < 0·05) increase in resistance was also observed. Furthermore, the FPg comet data showed .20% decrease in DNA damage with tea supplementation: mean and standard deviation changes in %DNA in comet tail in the Fpg-assisted comet assay were: -5·96 (SD 3·83) % after Longjing tea; -6·22 (SD 3·34) % after screw-shaped tea; +0·91 (SD 5·79) % after water (P < 0·05). No significant changes in urine 8-oxodG were seen. The results indicate that green tea has significant genoprotective effects and provide evidence for green tea as a 'functional food'.

Monte Carlo simulation of nonadiabatic expansion in cometary atmospheres - Halley

NASA Astrophysics Data System (ADS)

Hodges, R. R.

1990-02-01

Monte Carlo methods developed for the characterization of velocity-dependent collision processes and ballistic transports in planetary exospheres form the basis of the present computer simulation of icy comet atmospheres, which iteratively undertakes the simultaneous determination of velocity distribution for five neutral species (water, together with suprathermal OH, H2, O, and H) in a flow regime varying from the hydrodynamic to the ballistic. Experimental data from the neutral mass spectrometer carried by Giotto for its March, 1986 encounter with Halley are compared with a model atmosphere.

DNA Strand Breaks in Mitotic Germ Cells of Caenorhabditis elegans Evaluated by Comet Assay

PubMed Central

Park, Sojin; Choi, Seoyun; Ahn, Byungchan

2016-01-01

DNA damage responses are important for the maintenance of genome stability and the survival of organisms. Such responses are activated in the presence of DNA damage and lead to cell cycle arrest, apoptosis, and DNA repair. In Caenorhabditis elegans, double-strand breaks induced by DNA damaging agents have been detected indirectly by antibodies against DSB recognizing proteins. In this study we used a comet assay to detect DNA strand breaks and to measure the elimination of DNA strand breaks in mitotic germline nuclei of C. elegans. We found that C. elegans brc-1 mutants were more sensitive to ionizing radiation and camptothecin than the N2 wild-type strain and repaired DNA strand breaks less efficiently than N2. This study is the first demonstration of direct measurement of DNA strand breaks in mitotic germline nuclei of C. elegans. This newly developed assay can be applied to detect DNA strand breaks in different C. elegans mutants that are sensitive to DNA damaging agents. PMID:26903030

Genotoxicity of Water Contaminants from the Basin of Lake Sevan, Armenia Evaluated by the Comet Assay in Gibel Carp (Carassius auratus gibelio) and Tradescantia Bioassays.

PubMed

Simonyan, Anna; Gabrielyan, Barduch; Minasyan, Seyran; Hovhannisyan, Galina; Aroutiounian, Rouben

2016-03-01

Combination of bioassays and chemical analysis was applied to determine the genotoxic/mutagenic contamination in four different sites of the basin of Lake Sevan in Armenia. Water genotoxicity was evaluated using the single cell gel electrophoresis technique (comet assay) in erythrocytes of gibel carp (Carassius auratus gibelio), Tradescantia micronucleus (Trad-MCN) and Tradescantia stamen hair mutation (Trad-SHM) assays. Significant inter-site differences in the levels of water genotoxicity according to fish and Trad-MCN bioassays have been revealed. Two groups of locations with lower (south-southwest of the village Shorzha and Peninsula of Lake Sevan) and higher (estuaries of Gavaraget and Dzknaget rivers) levels of water genotoxicity were distinguished. Correlation analysis support the hypothesis that the observed genetic alterations in fish and plant may be a manifestation of the effects of water contamination by nitrate ions, Si, Al, Fe, Mn and Cu. Increase of DNA damage in fish also correlated with content of total phosphorus.

Role of Macronutrients and Micronutrients in DNA Damage: Results From a Food Frequency Questionnaire

PubMed Central

Ladeira, Carina; Carolino, Elisabete; Gomes, Manuel C; Brito, Miguel

2017-01-01

The links between diet and genomic instability have been under investigation for several decades, and evidence suggests a significant causal or preventive role for various dietary factors. This study investigates the influence of macronutrients (calories, protein, and glucides) and micronutrients, such as vitamins and minerals, as assessed by a food frequency questionnaire, on genotoxicity biomarkers measured by cytokinesis-blocked micronucleus assay and comet assay. The results found significant positive and negative correlations. Micronucleus frequency tends to increase with higher intake of caffeine, calcium, magnesium, zinc, and protein (P < .05, Spearman correlation). Calorie and omega-6 intakes are negatively correlated with DNA damage measured by the comet assay. These results are somewhat controversial because some of the correlations found are contrary to dominant views in the literature; however, we suggest that unraveling the association between diet and genetic instability requires a much better understanding of the modulating role of macronutrients and micronutrients. PMID:28469462

Toxicity of tributyltin in the marine mollusc Mytilus edulis.

PubMed

Hagger, Josephine A; Depledge, Michael H; Galloway, Tamara S

2005-01-01

Our previous studies have demonstrated that tributyltin (TBT) is genotoxic to the early life stages of marine mussels and worms. Here, the toxicity of TBT to adult organisms was determined using a suite of biomarkers designed to detect cytotoxic, immunotoxic and genotoxic effects. Exposure of adult mussels, Mytilus edulis, to environmentally realistic concentrations of TBTO for 7 days resulted in a statistically significant decrease in cell viability at concentrations of 0.5 microg/l and above. TBT had no effect on phagocytic activity or antioxidant capacity (FRAP assay). There was a statistically significant increase in DNA damage detected using the comet and micronucleus assays between the controls and 0.5, 1 and 5 microg/l of TBTO (P > 0.0005). Furthermore there was a strong correlation between DNA strand breaks (comet assay) and formation of micronuclei (P = 0.0005; R2 = 61.5%). Possible mechanisms by which TBT could damage DNA either directly or indirectly are discussed including the possibility that TBT is genotoxic due to its ability to disrupt calcium homeostasis.

Radioprotective effects of honeybee venom (Apis mellifera) against 915-MHz microwave radiation-induced DNA damage in wistar rat lymphocytes: in vitro study.

PubMed

Gajski, Goran; Garaj-Vrhovac, Vera

2009-01-01

The aim of this study is to investigate the radioprotective effect of bee venom against DNA damage induced by 915-MHz microwave radiation (specific absorption rate of 0.6 W/kg) in Wistar rats. Whole blood lymphocytes of Wistar rats are treated with 1 microg/mL bee venom 4 hours prior to and immediately before irradiation. Standard and formamidopyrimidine-DNA glycosylase (Fpg)-modified comet assays are used to assess basal and oxidative DNA damage produced by reactive oxygen species. Bee venom shows a decrease in DNA damage compared with irradiated samples. Parameters of Fpg-modified comet assay are statistically different from controls, making this assay more sensitive and suggesting that oxidative stress is a possible mechanism of DNA damage induction. Bee venom is demonstrated to have a radioprotective effect against basal and oxidative DNA damage. Furthermore, bee venom is not genotoxic and does not produce oxidative damage in the low concentrations used in this study.

Recent advances in the characterization of HIV-1 neutralization assays for standardized evaluation of the antibody response to infection and vaccination.

PubMed

Polonis, Victoria R; Brown, Bruce K; Rosa Borges, Andrew; Zolla-Pazner, Susan; Dimitrov, Dimiter S; Zhang, Mei-Yun; Barnett, Susan W; Ruprecht, Ruth M; Scarlatti, Gabriella; Fenyö, Eva-Maria; Montefiori, David C; McCutchan, Francine E; Michael, Nelson L

2008-06-05

In AIDS vaccine development the pendulum has swung towards a renewed emphasis on the potential role for neutralizing antibodies in a successful global vaccine. It is recognized that vaccine-induced antibody performance, as assessed in the available neutralization assays, may well serve as a "gatekeeper" for HIV-1 subunit vaccine prioritization and advancement. As a result, development of a standardized platform for reproducible measurement of neutralizing antibodies has received considerable attention. Here we review current advancements in our knowledge of the performance of different types of antibodies in a traditional primary cell neutralization assay and the newer, more standardized TZM-bl reporter cell line assay. In light of recently revealed differences (see accompanying article) in the results obtained in these two neutralization formats, parallel evaluation with both platforms should be contemplated as an interim solution until a better understanding of immune correlates of protection is achieved.

The Noble Gas Abundances in the Coma of Comet 67P/Churyumov-Gerasimenko from Rosetta/ROSINA

NASA Astrophysics Data System (ADS)

Rubin, M.; Altwegg, K.; Balsiger, H. R.; Berthelier, J. J.; Briois, C.; Combi, M. R.; De Keyser, J.; Fiethe, B.; Fuselier, S. A.; Gasc, S.; Gombosi, T. I.; Hansen, K. C.; Jäckel, A.; Kopp, E.; Korth, A.; Mall, U.; Marty, B.; Mousis, O.; Owen, T.; Reme, H.; Schuhmann, M.; Schroeder, I. R. H. G.; Semon, T.; Tzou, C. Y.; Waite, J. H., Jr.; Wurz, P.

2017-12-01

The Rosetta Orbiter Spectrometer for Ion and Neutral Analysis (ROSINA), the mass spectrometer suite on board the European Space Agency's Rosetta mission, was dedicated to the measurement of the volatiles in the coma of comet 67P/Churyumov-Gerasimenko (67P) [1]. Among many other species, ROSINA DFMS, the Double Focusing Mass Spectrometer, detected and quantified the three noble gases argon, krypton, and xenon including their major isotopes [2,3]. Noble gases provide important clues to the physical and chemical conditions during and possibly even before and after the comet's formation in the early solar system. Furthermore, measurements of the isotope ratios provide constraints on the amount of cometary material brought to Earth and its early atmosphere. In this presentation, we will report on the measured coma densities and derived nucleus bulk abundances of these three noble gases and investigate correlations with other volatiles. Furthermore, we will discuss the measured isotope ratios and the implications of these results. AcknowledgementsUoB was funded by the State of Bern, the Swiss National Science Foundation and by the European Space Agency PRODEX Programme. Work at MPS was funded by the Max-Planck Gesellschaft and BMWI (contract 50QP1302), at Southwest Research institute by Jet Propulsion Laboratory (subcontract #1496541 and JPL subcontract to JWH NAS703001TONMO710889), at the University of Michigan by NASA (contract JPL-1266313). This work has been supported through the A*MIDEX project from the French National Research Agency (ANR) (n° ANR-11-IDEX- 0001-02) and by CNES grants at IRAP, LATMOS, LPC2E, LAM, CRPG, by the European Research Council (grant no. 267255 to B. Marty) and at BIRA-IASB by the Belgian Science Policy Office via PRODEX/ROSINA PEA C4000107705. References[1] Balsiger, H., et al., Rosina - Rosetta orbiter spectrometer for ion and neutral analysis. Space Science Reviews. 128, 745-801, 2007. [2] Balsiger, H., et al., Detection of argon in the coma of comet 67P/Churyumov-Gerasimenko. Sci Adv. 1, e1500377, 2015. [3] Marty B., et al., Xenon isotopes in 67P/Churyumov-Gerasimenko show that comets contributed to Earth's atmosphere, Science, 356, 1069 - 1072, 2017.

Chemical Composition of the Semi-Volatile Grains of Comet 67P/Churyumov-Gerasimenko

NASA Astrophysics Data System (ADS)

Wurz, P.; Altwegg, K.; Balsiger, H. R.; Berthelier, J. J.; De Keyser, J.; Fiethe, B.; Fuselier, S. A.; Gasc, S.; Gombosi, T. I.; Korth, A.; Mall, U.; Reme, H.; Rubin, M.; Tzou, C. Y.

2017-12-01

Rosetta was in orbit of comet 67P/Churyumov-Gerasimenko from August 2014 to September 2016. On board is the Rosetta Orbiter Spectrometer for Ion and Neutral Analysis (ROSINA) experiment that has been continuously collecting data on the chemical composition and activity of the coma from 3.5 AU to pericentre at 1.24 AU and out again to 3.5 AU. ROSINA consists of two mass spectrometers, the Double Focusing Mass Spectrometer (DFMS) and the Reflectron-type Time-Of-Flight (RTOF), as well as the COmet Pressure Sensor (COPS). ROSINA recorded the neutral gas and thermal plasma in the comet's coma. The two mass spectrometers have high dynamic ranges and complement each other with high mass resolution, and high time resolution and large mass range. COPS measures total gas densities, bulk velocities, and gas temperatures. Occasionally, a dust grain of cometary origin enters the ion source of a ROSINA instrument where the volatile part evaporates since these ion sources are hot. The release of volatiles from cometary dust grains was observed with all three ROSINA instruments on several occasions. Because the volatile content of such a dust grain is completely evaporated after a few seconds, the RTOF instrument is best suited for the investigation of its chemical composition since complete mass spectra are recorded during this time. During the mission 9 dust grains were observed with RTOF during the October 2014 to July 2016 time period. It is estimated that these grains contain about 10-15 g of volatiles. The mass spectra were interpreted with a set of 75 molecules, with the major groups of chemical species being hydrocarbons, oxygenated hydrocarbons, nitrogen-bearing molecules, sulphur-bearing molecules, halogenated molecules and others. About 70% of these grains are depleted in water compared to the comet coma, thus, can be considered as semi-volatile dust grains, and the other about 30% are water grains. The chemical composition varies considerably from grain to grain, indicating large chemical heterogeneity at these scales. In contrast, the elemental abundances vary much less.

Serum virus neutralization assay for detection and quantitation of serum neutralizing antibodies to influenza A virus in swine

USDA-ARS?s Scientific Manuscript database

The serum virus neutralization (SVN) assay is a serological test to detect the presence and magnitude of functional systemic antibodies that prevent infectivity of a virus. The SVN assay is a highly sensitive and specific test that may be applied to influenza A viruses (IAV) in swine to measure the ...

Expeditious neutralization assay for human metapneumovirus based on a recombinant virus expressing Renilla luciferase.

PubMed

Zhou, Min; Kitagawa, Yoshinori; Yamaguchi, Mayu; Uchiyama, Chika; Itoh, Masae; Gotoh, Bin

2013-01-01

Human metapneumovirus (HMPV) is a common cause of respiratory diseases in persons of all ages. Because of its slow replication and weak cytopathic effect in cultured cells, conventional neutralization assays for HMPV require around one week for completion. The purpose of this study is to establish a rapid neutralization assay based on a recombinant virus expressing Renilla luciferase (Rluc). A recombinant HMPV expressing both Rluc and green fluorescent protein (GFP) was created by reverse genetics method. Two-fold serial dilutions of human 23 sera were made in a 96-well plate and incubated with 50 pfu/well of the recombinant virus at 4°C for 1 h. The mixtures were then transferred to LLC-MK2 cells in a 96-well plate, incubated for 2 h, and replaced with trypsin-free fresh media. After incubation at 32°C for 24 h, the cells were lysed and measured for Rluc activity. The neutralization titer was defined as the reciprocal of the highest serum dilution that resulted in 50% reduction of Rluc activity. The novel assay could be completed within 24 h and eliminated the requirement of trypsin supporting multistep replication in cultured cells, as well as laborious processes including the plaque assay with immunostaining. Neutralization titers correlated well with those determined by a GFP-based assay previously developed. The neutralization assay based on Rluc activity is the fastest and the most straightforward of all previous assays, and may be available for high throughput screening of neutralizing antibodies. Copyright © 2012 Elsevier B.V. All rights reserved.

Clover-Tagged Porcine Reproductive and Respiratory Syndrome Virus Infectious Clones for Rapid Detection of Virus Neutralizing Antibodies.

PubMed

Huang, Baicheng; Xiao, Xia; Xue, Biyun; Zhou, En-Min

2018-06-24

Porcine reproductive and respiratory syndrome (PRRS), caused by the PRRS virus (PRRSV), is a widespread disease that affects domestic pigs of all ages. Accurate and rapid detection of PRRSV specific neutralizing antibodies levels in a pig herd is beneficial for the evaluation of the herd's immunity to combat the specific viral infection. However, the current methods for viral detection, including fluorescent focus neutralization (FFN) and cytopathic effect (CPE) reduction neutralizing assays, are subjective and time-consuming. Therefore, a Clover-tagged PRRSV virus neutralization assay were developed that instrumentally measures the fluorescence signal of Clover stably expressing by a PRRSV infectious clone for at least 10 passages. Herein, the results showed that the proposed Clover-tagged PRRSV neutralization assay is reliable using instrumental measurements of the fluorescence signal of Clover and allows for rapid detection of neutralizing antibodies against PRRSV. The assay was evaluated by testing swine sera from experimental and field samples, and comparisons were made with the traditional FFN and CPE reduction assays. These results suggest that the Clover-tagged PRRSV infectious clone offers a fast and reliable testing method for neutralizing antibodies and could permit high-throughput screening of new antiviral agents. Copyright © 2018. Published by Elsevier B.V.

A Bayesian, generalized frailty model for comet assays.

PubMed

Ghebretinsae, Aklilu Habteab; Faes, Christel; Molenberghs, Geert; De Boeck, Marlies; Geys, Helena

2013-05-01

This paper proposes a flexible modeling approach for so-called comet assay data regularly encountered in preclinical research. While such data consist of non-Gaussian outcomes in a multilevel hierarchical structure, traditional analyses typically completely or partly ignore this hierarchical nature by summarizing measurements within a cluster. Non-Gaussian outcomes are often modeled using exponential family models. This is true not only for binary and count data, but also for, example, time-to-event outcomes. Two important reasons for extending this family are for (1) the possible occurrence of overdispersion, meaning that the variability in the data may not be adequately described by the models, which often exhibit a prescribed mean-variance link, and (2) the accommodation of a hierarchical structure in the data, owing to clustering in the data. The first issue is dealt with through so-called overdispersion models. Clustering is often accommodated through the inclusion of random subject-specific effects. Though not always, one conventionally assumes such random effects to be normally distributed. In the case of time-to-event data, one encounters, for example, the gamma frailty model (Duchateau and Janssen, 2007 ). While both of these issues may occur simultaneously, models combining both are uncommon. Molenberghs et al. ( 2010 ) proposed a broad class of generalized linear models accommodating overdispersion and clustering through two separate sets of random effects. Here, we use this method to model data from a comet assay with a three-level hierarchical structure. Although a conjugate gamma random effect is used for the overdispersion random effect, both gamma and normal random effects are considered for the hierarchical random effect. Apart from model formulation, we place emphasis on Bayesian estimation. Our proposed method has an upper hand over the traditional analysis in that it (1) uses the appropriate distribution stipulated in the literature; (2) deals with the complete hierarchical nature; and (3) uses all information instead of summary measures. The fit of the model to the comet assay is compared against the background of more conventional model fits. Results indicate the toxicity of 1,2-dimethylhydrazine dihydrochloride at different dose levels (low, medium, and high).

Protective effect of boric acid on lead- and cadmium-induced genotoxicity in V79 cells.

PubMed

Ustündağ, Aylin; Behm, Claudia; Föllmann, Wolfram; Duydu, Yalçin; Degen, Gisela H

2014-06-01

The toxic heavy metals cadmium (Cd) and lead (Pb) are important environmental pollutants which can cause serious damage to human health. As the metal ions (Cd(2+) and Pb(2+)) accumulate in the organism, there is special concern regarding chronic toxicity and damage to the genetic material. Metal-induced genotoxicity has been attributed to indirect mechanisms, such as induction of oxidative stress and interference with DNA repair. Boron is a naturally occurring element and considered to be an essential micronutrient, although the cellular activities of boron compounds remain largely unexplored. The present study has been conducted to evaluate potential protective effects of boric acid (BA) against genotoxicity induced by cadmium chloride (CdCl2) and lead chloride (PbCl2) in V79 cell cultures. Cytotoxicity assays (neutral red uptake and cell titer blue assay) served to determine suitable concentrations for subsequent genotoxicity assays. Chromosomal damage and DNA strand breaks were assessed by micronucleus tests and comet assays. Both PbCl2 and CdCl2 (at 3, 5 and 10 µM) were shown to induce concentration-dependent increases in micronucleus frequencies and DNA strand breaks in V79 cells. BA itself was not cytotoxic (up to 300 µM) and showed no genotoxic effects. Pretreatment of cells with low levels of BA (2.5 and 10 µM) was found to strongly reduce the genotoxic effects of the tested metals. Based on the findings of this in vitro study, it can be suggested that boron provides an efficient protection against the induction of DNA strand breaks and micronuclei by lead and cadmium. Further studies on the underlying mechanisms for the protective effect of boron are needed.

Measuring Sperm DNA Fragmentation and Clinical Outcomes of Medically Assisted Reproduction: A Systematic Review and Meta-Analysis.

PubMed

Cissen, Maartje; Wely, Madelon van; Scholten, Irma; Mansell, Steven; Bruin, Jan Peter de; Mol, Ben Willem; Braat, Didi; Repping, Sjoerd; Hamer, Geert

2016-01-01

Sperm DNA fragmentation has been associated with reduced fertilization rates, embryo quality, pregnancy rates and increased miscarriage rates. Various methods exist to test sperm DNA fragmentation such as the sperm chromatin structure assay (SCSA), the sperm chromatin dispersion (SCD) test, the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labelling (TUNEL) assay and the single cell gel electrophoresis (Comet) assay. We performed a systematic review and meta-analysis to assess the value of measuring sperm DNA fragmentation in predicting chance of ongoing pregnancy with IVF or ICSI. Out of 658 unique studies, 30 had extractable data and were thus included in the meta-analysis. Overall, the sperm DNA fragmentation tests had a reasonable to good sensitivity. A wide variety of other factors may also affect the IVF/ICSI outcome, reflected by limited to very low specificity. The constructed hierarchical summary receiver operating characteristic (HSROC) curve indicated a fair discriminatory capacity of the TUNEL assay (area under the curve (AUC) of 0.71; 95% CI 0.66 to 0.74) and Comet assay (AUC of 0.73; 95% CI 0.19 to 0.97). The SCSA and the SCD test had poor predictive capacity. Importantly, for the TUNEL assay, SCD test and Comet assay, meta-regression showed no differences in predictive value between IVF and ICSI. For the SCSA meta-regression indicated the predictive values for IVF and ICSI were different. The present review suggests that current sperm DNA fragmentation tests have limited capacity to predict the chance of pregnancy in the context of MAR. Furthermore, sperm DNA fragmentation tests have little or no difference in predictive value between IVF and ICSI. At this moment, there is insufficient evidence to recommend the routine use of sperm DNA fragmentation tests in couples undergoing MAR both for the prediction of pregnancy and for the choice of treatment. Given the significant limitations of the evidence and the methodological weakness and design of the included studies, we do urge for further research on the predictive value of sperm DNA fragmentation for the chance of pregnancy after MAR, also in comparison with other predictors of pregnancy after MAR.

Abundant molecular oxygen in the coma of comet 67P/Churyumov-Gerasimenko.

PubMed

Bieler, A; Altwegg, K; Balsiger, H; Bar-Nun, A; Berthelier, J-J; Bochsler, P; Briois, C; Calmonte, U; Combi, M; De Keyser, J; van Dishoeck, E F; Fiethe, B; Fuselier, S A; Gasc, S; Gombosi, T I; Hansen, K C; Hässig, M; Jäckel, A; Kopp, E; Korth, A; Le Roy, L; Mall, U; Maggiolo, R; Marty, B; Mousis, O; Owen, T; Rème, H; Rubin, M; Sémon, T; Tzou, C-Y; Waite, J H; Walsh, C; Wurz, P

2015-10-29

The composition of the neutral gas comas of most comets is dominated by H2O, CO and CO2, typically comprising as much as 95 per cent of the total gas density. In addition, cometary comas have been found to contain a rich array of other molecules, including sulfuric compounds and complex hydrocarbons. Molecular oxygen (O2), however, despite its detection on other icy bodies such as the moons of Jupiter and Saturn, has remained undetected in cometary comas. Here we report in situ measurement of O2 in the coma of comet 67P/Churyumov-Gerasimenko, with local abundances ranging from one per cent to ten per cent relative to H2O and with a mean value of 3.80 ± 0.85 per cent. Our observations indicate that the O2/H2O ratio is isotropic in the coma and does not change systematically with heliocentric distance. This suggests that primordial O2 was incorporated into the nucleus during the comet's formation, which is unexpected given the low upper limits from remote sensing observations. Current Solar System formation models do not predict conditions that would allow this to occur.

Formation of ions and radicals from icy grains in comets

NASA Technical Reports Server (NTRS)

Jackson, William M.; Gerth, Christopher; Hendricks, Charles

1991-01-01

Ion and radical formation in comets are thought to occur primarily by photodissociation of gas phase molecules. Experimental evidence and theoretical calculations are presented that show that some of the radical and ions can come directly from ice grains. The experimental evidence suggest that if the frozen molecules on the surface of grains undergo direct dissociation then they may be able to release radicals directly in the gas phase. If the molecules undergo predissociation it is unlikely that they will release radicals in the gas phase since they should be quenched. Calculations of this direct photodissociation mechanism further indicate that even if the parent molecule undergoes direct dissociation, the yield will not be high enough to explain the rays structure in comets unless the radicals are stored in the grains and then released when the grain evaporates. Calculations were also performed to determine the maximum number of ions that can be stored in an icy grain's radius. This number is compared with the ratio of the ion to neutral molecular density. The comparison suggests that some of the ions observed near the nucleus of the comet could have originally been present in the cometary nucleus. It is also pointed out that the presence of these ions in icy grains could lead to radical formation via electron recombination. Finally, an avalanche process was evaluated as another means of producing ions in comets.

Comet C/2011 W3 (Lovejoy) between 2 and 10 Solar Radii: Physical Parameters of the Comet and the Corona

NASA Astrophysics Data System (ADS)

Raymond, J. C.; Downs, Cooper; Knight, Matthew M.; Battams, Karl; Giordano, Silvio; Rosati, Richard

2018-05-01

Comet C/2011 W3 (Lovejoy) is the first sungrazing comet in many years to survive perihelion passage. We report ultraviolet observations with the Ultraviolet Coronagraph Spectrometer (UVCS) spectrometer aboard the Solar and Heliospheric Observatory satellite at five heights as the comet approached the Sun. The brightest line, Lyα, shows dramatic variations in intensity, velocity centroid, and width during the observation at each height. We derive the outgassing rates and the abundances of N, O, and Si relative to H, and we estimate the effective diameter of the nucleus to be several hundred meters. We consider the effects of the large outgassing rate on the interaction between the cometary gas and the solar corona and find good qualitative agreement with the picture of a bow shock resulting from mass loading by cometary neutrals. We obtain estimates of the solar wind density, temperature, and speed, and compare them with predictions of a global magnetohydrodynamic simulation, finding qualitative agreement within our uncertainties. We also determine the sublimation rate of silicate dust in the comet’s tail by comparing the visible brightness from the Large Angle Spectroscopic Coronagraphs with the Si III intensity from UVCS. The sublimation rates lie between the predicted rates for olivines and pyroxenes, suggesting that the grains are composed of a mixture of those minerals.

Anti-proliferative effect of biogenic gold nanoparticles against breast cancer cell lines (MDA-MB-231 & MCF-7)

NASA Astrophysics Data System (ADS)

K. S., Uma Suganya; Govindaraju, K.; Ganesh Kumar, V.; Prabhu, D.; Arulvasu, C.; Stalin Dhas, T.; Karthick, V.; Changmai, Niranjan

2016-05-01

Breast cancer is a major complication in women and numerous approaches are being developed to overcome this problem. In conventional treatments such as chemotherapy and radiotherapy the post side effects cause an unsuitable effect in treatment of cancer. Hence, it is essential to develop a novel strategy for the treatment of this disease. In the present investigation, a possible route for green synthesis of gold nanoparticles (AuNPs) using leaf extract of Mimosa pudica and its anticancer efficacy in the treatment of breast cancer cell lines is studied. The synthesized nanoparticles were found to be effective in killing cancer cells (MDA-MB-231 & MCF-7) which were studied using various anticancer assays (MTT assay, cell morphology determination, cell cycle analysis, comet assay, Annexin V-FITC/PI staining and DAPI staining). Cell morphological analysis showed the changes occurred in cancer cells during the treatment with AuNPs. Cell cycle analysis revealed apoptosis in G0/G1 to S phase. Similarly in Comet assay, there was an increase in tail length in treated cells in comparison with the control. Annexin V-FITC/PI staining assay showed prompt fluorescence in treated cells indicating the translocation of phosphatidylserine from the inner membrane. PI and DAPI staining showed the DNA damage in treated cells.

Compounds used to produce cloned animals are genotoxic and mutagenic in mammalian assays in vitro and in vivo

PubMed Central

Oliveira, R.J.; Mantovani, M.S.; da Silva, A.F.; Pesarini, J.R.; Mauro, M.O.; Ribeiro, L.R.

2014-01-01

The compounds 6-dimethylaminopurine and cycloheximide promote the successful production of cloned mammals and have been used in the development of embryos produced by somatic cell nuclear transfer. This study investigated the effects of 6-dimethylaminopurine and cycloheximide in vitro, using the thiazolyl blue tetrazolium bromide colorimetric assay to assess cytotoxicity, the trypan blue exclusion assay to assess cell viability, the comet assay to assess genotoxicity, and the micronucleus test with cytokinesis block to test mutagenicity. In addition, the comet assay and the micronucleus test were also performed on peripheral blood cells of 54 male Swiss mice, 35 g each, to assess the effects of the compounds in vivo. The results indicated that both 6-dimethylaminopurine and cycloheximide, at the concentrations and doses tested, were cytotoxic in vitro and genotoxic and mutagenic in vitro and in vivo, altered the nuclear division index in vitro, but did not diminish cell viability in vitro. Considering that alterations in DNA play important roles in mutagenesis, carcinogenesis, and morphofunctional teratogenesis and reduce embryonic viability, this study indicated that 6-dimethylaminopurine and cycloheximide utilized in the process of mammalian cloning may be responsible for the low embryo viability commonly seen in nuclear transfer after implantation in utero. PMID:24714812

Compounds used to produce cloned animals are genotoxic and mutagenic in mammalian assays in vitro and in vivo.

PubMed

Oliveira, R J; Mantovani, M S; Silva, A F da; Pesarini, J R; Mauro, M O; Ribeiro, L R

2014-04-01

The compounds 6-dimethylaminopurine and cycloheximide promote the successful production of cloned mammals and have been used in the development of embryos produced by somatic cell nuclear transfer. This study investigated the effects of 6-dimethylaminopurine and cycloheximide in vitro, using the thiazolyl blue tetrazolium bromide colorimetric assay to assess cytotoxicity, the trypan blue exclusion assay to assess cell viability, the comet assay to assess genotoxicity, and the micronucleus test with cytokinesis block to test mutagenicity. In addition, the comet assay and the micronucleus test were also performed on peripheral blood cells of 54 male Swiss mice, 35 g each, to assess the effects of the compounds in vivo. The results indicated that both 6-dimethylaminopurine and cycloheximide, at the concentrations and doses tested, were cytotoxic in vitro and genotoxic and mutagenic in vitro and in vivo, altered the nuclear division index in vitro, but did not diminish cell viability in vitro. Considering that alterations in DNA play important roles in mutagenesis, carcinogenesis, and morphofunctional teratogenesis and reduce embryonic viability, this study indicated that 6-dimethylaminopurine and cycloheximide utilized in the process of mammalian cloning may be responsible for the low embryo viability commonly seen in nuclear transfer after implantation in utero.

The JaCVAM international validation study on the in vivo comet assay: Selection of test chemicals.

PubMed

Morita, Takeshi; Uno, Yoshifumi; Honma, Masamitsu; Kojima, Hajime; Hayashi, Makoto; Tice, Raymond R; Corvi, Raffaella; Schechtman, Leonard

2015-07-01

The Japanese Center for the Validation of Alternative Methods (JaCVAM) sponsored an international prevalidation and validation study of the in vivo rat alkaline pH comet assay. The main objective of the study was to assess the sensitivity and specificity of the assay for correctly identifying genotoxic carcinogens, as compared with the traditional rat liver unscheduled DNA synthesis assay. Based on existing carcinogenicity and genotoxicity data and chemical class information, 90 chemicals were identified as primary candidates for use in the validation study. From these 90 chemicals, 46 secondary candidates and then 40 final chemicals were selected based on a sufficiency of carcinogenic and genotoxic data, differences in chemical class or genotoxic or carcinogenic mode of action (MOA), availability, price, and ease of handling. These 40 chemicals included 19 genotoxic carcinogens, 6 genotoxic non-carcinogens, 7 non-genotoxic carcinogens and 8 non-genotoxic non-carcinogens. "Genotoxicity" was defined as positive in the Ames mutagenicity test or in one of the standard in vivo genotoxicity tests (primarily the erythrocyte micronucleus assay). These chemicals covered various chemicals classes, MOAs, and genotoxicity profiles and were considered to be suitable for the purpose of the validation study. General principles of chemical selection for validation studies are discussed. Copyright © 2015 Elsevier B.V. All rights reserved.

Evaluation of genetic damage induced by glyphosate isopropylamine salt using Tradescantia bioassays.

PubMed

Alvarez-Moya, Carlos; Silva, Mónica Reynoso; Arámbula, Alma Rosa Villalobos; Sandoval, Alfonso Islas; Vasquez, Hugo Castañeda; González Montes, Rosa María

2011-01-01

Glyphosate is noted for being non-toxic in fishes, birds and mammals (including humans). Nevertheless, the degree of genotoxicity is seriously controversial. In this work, various concentrations of a glyphosate isopropylamine salt were tested using two methods of genotoxicity assaying, viz., the pink mutation assay with Tradescantia (4430) and the comet assay with nuclei from staminal cells of the same plant. Staminal nuclei were studied in two different forms, namely nuclei from exposed plants, and nuclei exposed directly. Using the pink mutation assay, isopropylamine induced a total or partial loss of color in staminal cells, a fundamental criterion utilized in this test. Consequently, its use is not recommended when studying genotoxicity with agents that produce pallid staminal cells. The comet assay system detected statistically significant (p < 0.01) genotoxic activity by isopropylamine, when compared to the negative control in both the nuclei of treated plants and directly treated nuclei, but only the treated nuclei showed a dose-dependent increase. Average migration in the nuclei of treated plants increased, when compared to that in treated nuclei. This was probably due, either to the permanence of isopropylamine in inflorescences, or to the presence of secondary metabolites. In conclusion, isopropylamine possesses strong genotoxic activity, but its detection can vary depending on the test systems used.

Evaluation of genetic damage induced by glyphosate isopropylamine salt using Tradescantia bioassays

PubMed Central

Alvarez-Moya, Carlos; Silva, Mónica Reynoso; Arámbula, Alma Rosa Villalobos; Sandoval, Alfonso Islas; Vasquez, Hugo Castañeda; González Montes, Rosa María

2011-01-01

Glyphosate is noted for being non-toxic in fishes, birds and mammals (including humans). Nevertheless, the degree of genotoxicity is seriously controversial. In this work, various concentrations of a glyphosate isopropylamine salt were tested using two methods of genotoxicity assaying, viz., the pink mutation assay with Tradescantia (4430) and the comet assay with nuclei from staminal cells of the same plant. Staminal nuclei were studied in two different forms, namely nuclei from exposed plants, and nuclei exposed directly. Using the pink mutation assay, isopropylamine induced a total or partial loss of color in staminal cells, a fundamental criterion utilized in this test. Consequently, its use is not recommended when studying genotoxicity with agents that produce pallid staminal cells. The comet assay system detected statistically significant (p < 0.01) genotoxic activity by isopropylamine, when compared to the negative control in both the nuclei of treated plants and directly treated nuclei, but only the treated nuclei showed a dose-dependent increase. Average migration in the nuclei of treated plants increased, when compared to that in treated nuclei. This was probably due, either to the permanence of isopropylamine in inflorescences, or to the presence of secondary metabolites. In conclusion, isopropylamine possesses strong genotoxic activity, but its detection can vary depending on the test systems used. PMID:21637555

Indoor and outdoor genotoxic load detected by the Comet assay in leaves of Nicotiana tabacum cultivars Bel B and Bel W3.

PubMed

Restivo, Francesco Maria; Laccone, Maria Concetta; Buschini, Annamaria; Rossi, Carlo; Poli, Paola

2002-03-01

Environmental pollution assessment and control are priority issues for both developed and developing countries of the world. The use of plant material for a more complete picture of environmental health appears to be particularly appealing. Here we validate a previous plant-adapted Comet assay on leaf tissues of Nicotiana tabacum cultivars Bel B and Bel W3. The effects of H(2)O(2) on DNA damage in Bel B and Bel W3 agree with the hypothesis that some component of the machinery that protects DNA integrity from oxidative stress may be impaired in cv. Bel W3. Exposure in the field on sunny summer days (peak ozone concentration >80 p.p.b.) showed significantly higher DNA damage in cv. Bel W3 if plants were collected and subjected to the Comet assay when the air ozone concentration was reaching its peak value, but not when plants were sampled early in the morning and hence after a period of low ozone concentration. The different results suggest that Bel W3 possesses a less efficient recovery apparatus that requires a longer period of activity to be effective and/or is less protected against reactive oxygen species production during exposure to ozone. However, it cannot be excluded that the increase in mean DNA damage is the result of the presence of a genotoxic agent(s) other than ozone. Interestingly, Bel W3 also appears to be more responsive, compared with Bel B, when exposed to ambient indoor pollutants. The use of cv. Bel W3 increases the sensitivity of the assay under both indoor and field conditions. However, different classes of mutagens should be tested to define the range of profitable utilization of this tobacco cultivar for environmental genotoxicity detection.

Comparison in vivo Study of Genotoxic Action of High- Versus Very Low Dose-Rate γ-Irradiation

PubMed Central

Osipov, A. N.; Klokov, D. Y.; Elakov, A. L.; Rozanova, O. M.; Zaichkina, S. I.; Aptikaeva, G. F.; Akhmadieva, A. Kh.

2004-01-01

The aim of the present study was to compare genotoxicity induced by high- versus very low dose-rate exposure of mice to γ-radiation within a dose range of 5 to 61 cGy using the single-cell gel electrophoresis (comet) assay and the micronucleus test. CBA/lac male mice were irradiated at a dose rate of 28.2 Gy/h (high dose rate) or 0.07 mGy/h (very low dose rate). The comet assay study on spleen lymphocytes showed that very low dose-rate irradiation resulted in a statistically significant increase in nucleoid relaxation (DNA breaks), starting from a dose of 20 cGy. Further prolongation of exposure time and, hence, increase of a total dose did not, however, lead to further increase in the extent of nucleoid relaxation. Doses of 20 and 61 cGy were equal in inducing DNA breaks in mouse spleen lymphocytes as assayed by the comet assay. Of note, the level of DNA damage by 20–61 cGy doses of chronic irradiation (0.07 mGy/h) was similar to that an induced by an acute (28.2 Gy/h) dose of 14 cGy. The bone marrow micronucleus test revealed that an increase in polychromatic erythrocytes with micronuclei over a background level was induced by very low-level γ-irradiation with a dose of 61 cGy only, with the extent of the cytogenetic effect being similar to that of 10 cGy high-dose-rate exposure. In summary, presented results support the hypothesis of the nonlinear threshold nature of mutagenic action of chronic low dose-rate irradiation. PMID:19330145

Protective effects of melatonin-loaded lipid-core nanocapsules on paraquat-induced cytotoxicity and genotoxicity in a pulmonary cell line.

PubMed

Charão, Mariele F; Baierle, Marília; Gauer, Bruna; Goethel, Gabriela; Fracasso, Rafael; Paese, Karina; Brucker, Natália; Moro, Angela M; Bubols, Guilherme B; Dias, Bruna B; Matte, Ursula S; Guterres, Silvia S; Pohlmann, Adriana R; Garcia, Solange C

2015-06-01

Many acute poisonings lack effective and specific antidotes. Due to both intentional and accidental exposures, paraquat (PQ) causes thousands of deaths annually, especially by pulmonary fibrosis. Melatonin (Mel), when incorporated into lipid-core nanocapsules (Mel-LNC), has enhanced antioxidant properties. The effects of such a formulation have not yet been studied with respect to mitigation of PQ- induced cytotoxicity and DNA damage. Here, we have tested whether Mel-LNC can ameliorate PQ-induced toxicity in the A549 alveolar epithelial cell line. Physicochemical characterization of the formulations was performed. Cellular uptake was measured using nanocapsules marked with rhodamine B. Cell viability was determined by the MTT assay and DNA damage was assessed by the comet assay. The enzyme-modified comet assay with endonuclease III (Endo III) and formamidopyrimidine glycosylase (FPG) were used to investigate oxidative DNA damage. Incubation with culture medium for 24h did not alter the granulometric profile of Mel-LNC formulations. Following treatment (3 and 24h), red fluorescence was detected around the cell nucleus, indicating internalization of the formulation. Melatonin solution (Mel), Mel-LNC, and LNC did not have significant effects on cell viability or DNA damage. Pre-treatment with Mel-LNC enhanced cell viability and showed a remarkable reduction in % DNA in tail compared to the PQ group; this was not observed in cells pre-treated with Mel. PQ induces oxidative DNA damage detected with the enzyme-modified comet assay. Mel-LNC reduced this damage more effectively than did Mel. In summary, Mel-LNC is better than Mel at protecting A549 cells from the cytotoxic and genotoxic effects of PQ. Copyright © 2015 Elsevier B.V. All rights reserved.

DNA damage and repair capacity in workers exposed to low concentrations of benzene.

PubMed

Lovreglio, Piero; Doria, Denise; Fracasso, Maria Enrica; Barbieri, Anna; Sabatini, Laura; Drago, Ignazio; Violante, Francesco S; Soleo, Leonardo

2016-03-01

DNA damage and cellular repair capacity were studied in 18 male fuel tanker drivers and 13 male filling-station attendants exposed to low and very low concentrations of benzene, respectively, and compared to 20 males with no occupational exposure (controls). Exposure to airborne benzene was measured using passive personal samplers, and internal doses were assayed through the biomarkers t,t-muconic acid, S-phenylmercapturic acid and urinary benzene. DNA damage was evaluated using tail intensity (TI) determined by the comet assay in peripheral lymphocytes. Urinary 7-hydro-8-oxo-2'-deoxyguanosine (8-oxodG) was measured as a biomarker of oxidative damage. DNA repair kinetics were assessed using the comet assay in lymphocytes sampled 20 and 60 min post H2O2 exposure. Benzene exposure differed significantly between the drivers (median 246.3 µg/m(3)), attendants (median 13.8 µg/m(3)), and controls (median 4.1 µg/m(3)). There were no differences in TI and 8-oxodG among the three groups, or between smokers and non-smokers. DNA repair kinetics were similar among the drivers, attendants and controls, although the comet assay on H2 O2 -damaged lymphocytes after 60 min revealed significantly lower levels of TI only in drivers. The DNA repair process in smokers was similar to that observed in drivers. In conclusion, this study found no relationship between low levels of benzene exposure and DNA damage, although there was evidence that exposure interferes with DNA repair kinetics. The biological impact of this finding on the onset of genotoxic effects in exposed workers has still to be ascertained. © 2015 Wiley Periodicals, Inc.

Modelling the solar wind interaction with Mercury by a quasi-neutral hybrid model

NASA Astrophysics Data System (ADS)

Kallio, E.; Janhunen, P.

2003-11-01

Quasi-neutral hybrid model is a self-consistent modelling approach that includes positively charged particles and an electron fluid. The approach has received an increasing interest in space plasma physics research because it makes it possible to study several plasma physical processes that are difficult or impossible to model by self-consistent fluid models, such as the effects associated with the ions’ finite gyroradius, the velocity difference between different ion species, or the non-Maxwellian velocity distribution function. By now quasi-neutral hybrid models have been used to study the solar wind interaction with the non-magnetised Solar System bodies of Mars, Venus, Titan and comets. Localized, two-dimensional hybrid model runs have also been made to study terrestrial dayside magnetosheath. However, the Hermean plasma environment has not yet been analysed by a global quasi-neutral hybrid model.

Plasma waves at comet 67P/Churyumov-Gerasimenko: in the diamagnetic cavity and outside it

NASA Astrophysics Data System (ADS)

Gunell, Herbert; Altwegg, Kathrin; Cessateur, Gaël; De Keyser, Johan; Dhooghe, Frederik; Eriksson, Anders; Gibbons, Andrew; Glassmeier, Karl-Heinz; Goetz, Charlotte; Karlsson, Tomas; Hamrin, Maria; Henri, Pierre; Maggiolo, Romain; Nilsson, Hans; Odelstad, Elias; Rubin, Martin; Wedlund, Cyril Simon; Stenberg Wieser, Gabriella; Tzou, Chia-Yu; Vallieres, Xavier

2017-04-01

We present observations of waves at Comet 67P/Churyumov-Gerasimenko performed on 20 January 2015, when the activity of the comet was low, and in July and August 2015 when the activity had increased and the Rosetta spacecraft passed through the diamagnetic cavity several times. We use distribution functions obtained by the Ion Composition Analyser of the Rosetta Plasma Consortium (RPC-ICA) and electron temperature estimates from the Langmuir Probes (RPC-LAP) to compute dispersion relations for waves on the ion timescale, and we compare the results to spectra obtained by RPC-LAP. On 20 January 2015, at low activity, peaks of the wave spectra appeared at frequencies near 500 Hz, and we identify these waves as ion acoustic. We performed cross-calibrations between RPC-ICA, RPC-LAP, and the Mutual Impedance Probe (RPC-MIP) in order to determine the plasma density. Matching the dispersion relations to the wave observations also helps us estimating the density. We explore the relationship between the waves, the ion distribution functions, and the neutral density, which was measured by the ROSINA-COPS instrument. It is found that when the waves are seen, the ion temperature is low (approximately 0.01 eV). At times the ion temperature is higher (approximately 1 eV), approaching the electron temperature, which leads to strong damping of the ion acoustic waves. This happens when the neutral density is high, suggesting that the ions are heated by being accelerated by the solar wind electric field and scattered in collisions with the neutrals. These results are compared to measurements of wave spectra when Rosetta was inside the diamagnetic cavity in July and August 2015. In the cavity, the plasma is effectively unmagnetised. We identify cavity passages using the magnetometer RPC-MAG. The waves are analysed in the same way as in the earlier measurements outside the cavity, and the two cases are compared.

The singing comet 67P: utilizing fully kinetic simulations to study its interaction with the solar wind plasma

NASA Astrophysics Data System (ADS)

Deca, J.; Divin, A. V.; Horanyi, M.; Henri, P.

2016-12-01

We present preliminary results of the first 3-D fully kinetic and electromagnetic simulations of the solar wind interaction with 67P/Churyumov-Gerasimenko at 3 AU, before the comet transitions into its high-activity phase. We focus on the global cometary environment and the electron-kinetic activity of the interaction. In addition to the background solar wind plasma flow, our model includes also plasma-driven ionization of cometary neutrals and collisional effects. We approximate mass loading of cold cometary oxygen and hydrogen using a hyperbolic relation with distance to the comet. We consider two primary cases: a weak outgassing comet (with the peak ion density 10x the solar wind density) and a moderately outgassing comet (with the peak ion density 50x the solar wind density). The weak comet is characterized by the formation of a narrow region containing a compressed solar wind (the density of the solar wind ion population is 3x the value far upstream of the comet) and a magnetic barrier ( 2x to 4x the interplanetary magnetic field). Blobs of plasma are detached continuously from this sheath region. Standing electromagnetic waves are excited in the cometary wake due to a strong anisotropy in the plasma pressure, as the density and the magnetic field magnitude are anti-correlated.The moderate mass-loading case shows more dynamics at the dayside region. The stagnation of the solar wind flow is accompanied by the formation of elongated density stripes, indicating the presence of a Rayleigh-Taylor instability. These density cavities are elongated in the direction of the magnetic field and encompass the dayside ionopause. To conclude, we believe that our results provide vital information to disentangle the observations made by the Rosetta spacecraft and compose a global solar wind - comet interaction model.

A Novel Assay for Antibody-Dependent Cell-Mediated Cytotoxicity against HIV-1- or SIV-Infected Cells Reveals Incomplete Overlap with Antibodies Measured by Neutralization and Binding Assays

PubMed Central

Alpert, Michael D.; Heyer, Lisa N.; Williams, David E. J.; Harvey, Jackson D.; Greenough, Thomas; Allhorn, Maria

2012-01-01

The resistance of human immunodeficiency virus type 1 (HIV-1) to antibody-mediated immunity often prevents the detection of antibodies that neutralize primary isolates of HIV-1. However, conventional assays for antibody functions other than neutralization are suboptimal. Current methods for measuring the killing of virus-infected cells by antibody-dependent cell-mediated cytotoxicity (ADCC) are limited by the number of natural killer (NK) cells obtainable from individual donors, donor-to-donor variation, and the use of nonphysiological targets. We therefore developed an ADCC assay based on NK cell lines that express human or macaque CD16 and a CD4+ T-cell line that expresses luciferase from a Tat-inducible promoter upon HIV-1 or simian immunodeficiency virus (SIV) infection. NK cells and virus-infected targets are mixed in the presence of serial plasma dilutions, and ADCC is measured as the dose-dependent loss of luciferase activity. Using this approach, ADCC titers were measured in plasma samples from HIV-infected human donors and SIV-infected macaques. For the same plasma samples paired with the same test viruses, this assay was approximately 2 orders of magnitude more sensitive than optimized assays for neutralizing antibodies—frequently allowing the measurement of ADCC in the absence of detectable neutralization. Although ADCC correlated with other measures of Env-specific antibodies, neutralizing and gp120 binding titers did not consistently predict ADCC activity. Hence, this assay affords a sensitive method for measuring antibodies capable of directing ADCC against HIV- or SIV-infected cells expressing native conformations of the viral envelope glycoprotein and reveals incomplete overlap of the antibodies that direct ADCC and those measured in neutralization and binding assays. PMID:22933282

A Protosolar Nebula Origin for the Ices Agglomerated by Comet 67P/Churyumov-Gerasimenko

NASA Astrophysics Data System (ADS)

Mousis, O.; Lunine, J. I.; Luspay-Kuti, A.; Guillot, T.; Marty, B.; Ali-Dib, M.; Wurz, P.; Altwegg, K.; Bieler, A.; Hässig, M.; Rubin, M.; Vernazza, P.; Waite, J. H.

2016-03-01

The nature of the icy material accreted by comets during their formation in the outer regions of the protosolar nebula (PSN) is a major open question in planetary science. Some scenarios of comet formation predict that these bodies agglomerated from crystalline ices condensed in the PSN. Concurrently, alternative scenarios suggest that comets accreted amorphous ice originating from the interstellar cloud or from the very distant regions of the PSN. On the basis of existing laboratory and modeling data, we find that the N2/CO and Ar/CO ratios measured in the coma of the Jupiter-family comet 67P/Churyumov-Gerasimenko by the Rosetta Orbiter Spectrometer for Ion and Neutral Analysis instrument on board the European Space Agency’s Rosetta spacecraft match those predicted for gases trapped in clathrates. If these measurements are representative of the bulk N2/CO and Ar/CO ratios in 67P/Churyumov-Gerasimenko, it implies that the ices accreted by the comet formed in the nebula and do not originate from the interstellar medium, supporting the idea that the building blocks of outer solar system bodies have been formed from clathrates and possibly from pure crystalline ices. Moreover, because 67P/Churyumov-Gerasimenko is impoverished in Ar and N2, the volatile enrichments observed in Jupiter’s atmosphere cannot be explained solely via the accretion of building blocks with similar compositions and require an additional delivery source. A potential source may be the accretion of gas from the nebula that has been progressively enriched in heavy elements due to photoevaporation.

Physical properties of asteroids in comet-like orbits in the infrared asteroidal survey catalogs

NASA Astrophysics Data System (ADS)

Kim, Y.; Ishiguro, M.; Usui, F.

2014-07-01

Dormant comet and Infrared Asteroidal Survey Catalogs. Comet nucleus is a solid body consisting of dark refractory material and ice. Cometary volatiles sublimate from subsurface layer by solar heating, leaving behind large dust grains on the surface. Eventually, the appearance could turn into asteroidal rather than cometary. It is, therefore, expected that there would be ''dormant comets'' in the list of known asteroids. Over past decade, several ground-based studies have been performed to dig out such dormant comets. One common approach is applying a combination of optical and dynamical properties learned from active comet nucleus to the list of known asteroids. Typical comet nucleus has (i) Tisserand parameter with respect to Jupiter, T_{J}<3, (ii) low geometric albedo, p_{v}<0.1 and (iii) reddish or neutral spectra, similar to P, D, C-type asteroids. Following past ground-based surveys, infrared space missions gave us an opportunity to work on further study of dormant comets. To the present, three infrared asteroidal catalogs taken with IRAS[1], AKARI[2] and WISE[3] are available, providing information of sizes and albedos which are useful to study the physical properties of dormant comets as well as asteroids. Usui et al. (2014) merged three infrared asteroidal catalogs with valid sizes and albedos into single catalog, what they called I-A-W[4]. We applied a huge dataset of asteroids in I-A-W to investigate the physical properties of asteroids in comet-like orbits (ACOs, whose orbits satisfy Q>4.5 au and T_{J}<3). Here we present a study of ACOs in infrared asteroidal catalogs taken with AKARI, IRAS and WISE. In this presentation, we aim to introduce albedo and size properties of ACOs in infrared asteroidal survey catalogs, in combination with orbital and spectral properties from literature. Results and Implications. We summarize our finding and implication as followings: - are 123 ACOs (Q>4.5 au and T_J<3) in I-A-W catalog after rejection of objects with large orbital uncertainties. - Majority (˜80 %) of ACOs have low albedo (p_{v}<0.1), showing similar albedo distribution to active comet nuclei. - Low-albedo ACOs have the cumulative size distribution shallower than that of active comet nuclei. - High-albedo (p_{v}≥0.1) ACOs consist of small (D<3 km) bodies are concentrated in near-Earth space. - We suggest that such high-albedo, small near-Earth asteroids are susceptible to Yarkovsky effect and injected into comet-like orbits.

Antioxidants and the Comet assay.

PubMed

Cemeli, Eduardo; Baumgartner, Adolf; Anderson, Diana

2009-01-01

It is widely accepted that antioxidants, either endogenous or from the diet, play a key role in preserving health. They are able to quench radical species generated in situations of oxidative stress, either triggered by pathologies or xenobiotics, and they protect the integrity of DNA from genotoxicants. Nevertheless, there are still many compounds with unclear or unidentified prooxidant/antioxidant activities. This is of concern since there is an increase in the number of compounds synthesized or extracted from vegetables to which humans might be exposed. Despite the well-established protective effects of fruit and vegetables, the antioxidant(s) responsible have not all been clearly identified. There might also be alternative mechanisms contributing to the protective effects for which a comprehensive description is lacking. In the last two decades, the Comet assay has been extensively used for the investigation of the effects of antioxidants and many reports can be found in the literature. The Comet assay, a relatively fast, simple, and sensitive technique for the analysis of DNA damage in all cell types, has been applied for the screening of chemicals, biomonitoring and intervention studies. In the present review, several of the most well-known antioxidants are considered. These include: catalase, superoxide dismutase, glutathione peroxidase, selenium, iron chelators, melatonin, melanin, vitamins (A, B, C and E), carotenes, flavonoids, isoflavones, tea polyphenols, wine polyphenols and synthetic antioxidants. Investigations showing beneficial as well as non-beneficial properties of the antioxidants selected, either at the in vitro, ex vivo or in vivo level are discussed.

Evaluation of DNA Damage in Common Carp (Cyprinus carpio L.) by Comet Assay for Determination of Possible Pollution in Lake Mogan (Ankara)

PubMed Central

Çok, İsmet; Ulutaş, Onur Kenan; Okuşluk, Öncü; Durmaz, Emre; Demir, Nilsun

2011-01-01

Contamination of the aquatic environment with various concentrations of pollutants results in unexpected threats to humans and wildlife. The consequences of exposure and metabolism of pollutants/xenobiotics, especially carcinogens and mutagens, can be suitably assessed by investigating severe events, such as DNA damage; for example, DNA adducts and DNA strand breaks. One of the commonly used techniques to detect DNA damage in aquatic organisms is single-cell gel electrophoresis (comet assay). This study was carried out using Cyprinus carpio in order to identify the possible pollution in Lake Mogan, near Ankara, Turkey, where the city's sewer system and pesticides used in agriculture are believed to be the common causes of pollution. From the comet assay, the tail length (μm), tail intensity (%), and tail moment values of fish caught from Lake Mogan were found to be 31.10 ± 10.39, 7.77 ± 4.51, 1.50 ± 1.48, respectively, whereas for clean reference sites they were found to be 22.80 ± 1.08, 3.47 ± 1.59, 0.40 ± 0.51, respectively. The values are statistically different from each other (p < 0.0001, p < 0.0001, and p < 0.0013, respectively). These results indicate that Lake Mogan may be polluted with substances that have genotoxic effects and constitute an early warning for the lake system. Further detailed research is needed to establish the source of the pollution and the chemicals responsible. PMID:21805014

Detection of in vivo DNA damage induced by ethanol in multiple organs of pregnant mice using the alkaline single cell gel electrophoresis (Comet) assay.

PubMed

Kido, Ryoko; Sato, Itaru; Tsuda, Shuji

2006-01-01

Ethanol is principal ingredient of alcohol beverage, but considered as human carcinogen, and has neurotoxicity. Alcohol consumption during pregnancy often causes fetal alcohol syndrome. The DNA damage is one of the important factors in carcinogenicity or teratogenicity. To detect the DNA damage induced by ethanol, we used an in vivo alkaline single cell gel electrophoresis (Comet) assay in pregnant mice organs and embryos. Pregnant ICR mice on Day 7 of gestation were treated with 2, 4 or 8 g/kg ethanol, and maternal organs/tissues and embryos were subjected to the Comet assay at 4, 8, 12 and 24 hr after ethanol treatment. Four and 8 g/kg ethanol induced DNA damage in brain, lung and embryos at 4 or 8 hr after the treatment. Two g/kg ethanol did not cause any DNA damage, and 8 g/kg ethanol only increased the duration of DNA damage without distinct increase in the degree of the damage. No significant DNA damage was observed in the liver. To detect the effect of acetaldehyde, disulfiram, acetaldehyde dehydrogenase inhibitor, was administered before 4 g/kg ethanol treatment. No significant increase of DNA damage was observed in the disulfiram pre-treated group. These data indicate that ethanol induces DNA damage, which might be related to ethanol toxicity. Since pre-treatment of disulfiram did not increase DNA damage, DNA damage observed in this study might not be the effect of acetaldehyde.

Evaluation of DNA damage induced by gamma radiation in gill and muscle tissues of Cyprinus carpio and their relative sensitivity.

PubMed

M K, Praveen Kumar; Shyama, Soorambail K; D'Costa, Avelyno; Kadam, Samit B; Sonaye, Bhagatsingh Harisingh; Chaubey, Ramesh Chandra

2017-10-01

The effect of radiation on the aquatic environment is of major concern in recent years. Limited data is available on the genotoxicity of gamma radiation on different tissues of aquatic organisms. Hence, the present investigation was carried out to study the DNA damage induced by gamma radiation in the gill and muscle tissues and their relative sensitivity using the comet assay in the freshwater teleost fish, common carp (Cyprinus carpio). The comet assay was optimized and validated in common carp using cyclophosphamide (CP), a reference genotoxic agent. The fish were exposed (acute) to various doses of gamma radiation (2, 4, 6, 8 and 10Gy) and samplings (gill and muscle tissue) were done at regular intervals (24, 48 and 72h) to assess the DNA damage. A significant increase in DNA damage was observed as indicated by an increase in % tail DNA for all doses of gamma radiation in both tissues. We also observed a dose-related increase and a time-dependent decrease of DNA damage. In comparison, DNA damage showed different sensitivity among the tissues at different doses. This shows that a particular dose may have different effects on different tissues which could be due to physiological factors of the particular tissue. Our study also suggests that the gills and muscle of fish are sensitive and reliable tissues for evaluating the genotoxic effects of reference and environmental agents, using the comet assay. Copyright © 2017. Published by Elsevier Inc.

EL4 cell-based colorimetric toxin neutralization activity assays for determination of neutralizing anti-ricin antibodies.

PubMed

Lindsey, Changhong Y; Brown, J Edward; Torabazar, Nahid R; Smith, Leonard A

2013-01-01

A recombinant ricin toxin A-chain 1-33/44-198 vaccine (RVEc), developed at the United States Army Medical Research Institute of Infectious Diseases as a vaccine candidate, is under investigation in a phase 1 clinical study. To effectively evaluate the immunogenicity of this ricin vaccine and to eliminate the use of radioactive material, an EL4 cell-based colorimetric toxin neutralization activity (TNA) assay using a CellTiter 96 AQueous One Solution Cell Proliferation Assay Reagent has been developed, optimized, and applied in the vaccine efficacy studies. The TNA assay measures the protective neutralizing anti-ricin antibodies in animal sera by determining the cell viability after ricin exposure in the assay system and comparing it to a purified mouse polyclonal antiricin IgG standard curve. The standard curve of the anti-ricin TNA assay closely fits a four-parameter logistic regression model. The unknown test sample concentration was expressed as microg/mL, but not the 50% effective concentration (EC50), which was determined by most TNA assays. The neutralizing endpoint titers, not the 50% effective dilution (ED50), of human specimens were measured with the TNA assay in support of the clinical study of the RVEc vaccine. The optimal amount of ricin toxin, EL4 cells, and concentration of standards used in the assay system was established to minimize false-negative and false-positive results of serum specimens from the nonclinical and clinical studies of RVEc. The testing conditions were adjusted to optimize assay performance. The colorimetric TNA assay replaced a radioactive TNA assay previously used in the ricin vaccine studies.

Molecular nitrogen in comet 67P/Churyumov-Gerasimenko indicates a low formation temperature.

PubMed

Rubin, M; Altwegg, K; Balsiger, H; Bar-Nun, A; Berthelier, J-J; Bieler, A; Bochsler, P; Briois, C; Calmonte, U; Combi, M; De Keyser, J; Dhooghe, F; Eberhardt, P; Fiethe, B; Fuselier, S A; Gasc, S; Gombosi, T I; Hansen, K C; Hässig, M; Jäckel, A; Kopp, E; Korth, A; Le Roy, L; Mall, U; Marty, B; Mousis, O; Owen, T; Rème, H; Sémon, T; Tzou, C-Y; Waite, J H; Wurz, P

2015-04-10

Molecular nitrogen (N2) is thought to have been the most abundant form of nitrogen in the protosolar nebula. It is the main N-bearing molecule in the atmospheres of Pluto and Triton and probably the main nitrogen reservoir from which the giant planets formed. Yet in comets, often considered the most primitive bodies in the solar system, N2 has not been detected. Here we report the direct in situ measurement of N2 in the Jupiter family comet 67P/Churyumov-Gerasimenko, made by the Rosetta Orbiter Spectrometer for Ion and Neutral Analysis mass spectrometer aboard the Rosetta spacecraft. A N2/CO ratio of (5.70 ± 0.66) × 10(-3) (2σ standard deviation of the sampled mean) corresponds to depletion by a factor of ~25.4 ± 8.9 as compared to the protosolar value. This depletion suggests that cometary grains formed at low-temperature conditions below ~30 kelvin. Copyright © 2015, American Association for the Advancement of Science.

Chicken fetal liver DNA damage and adduct formation by activation-dependent DNA-reactive carcinogens and related compounds of several structural classes.

PubMed

Williams, Gary M; Duan, Jian-Dong; Brunnemann, Klaus D; Iatropoulos, Michael J; Vock, Esther; Deschl, Ulrich

2014-09-01

The chicken egg genotoxicity assay (CEGA), which utilizes the liver of an intact and aseptic embryo-fetal test organism, was evaluated using four activation-dependent DNA-reactive carcinogens and four structurally related less potent carcinogens or non-carcinogens. In the assay, three daily doses of test substances were administered to eggs containing 9-11-day-old fetuses and the fetal livers were assessed for two endpoints, DNA breaks using the alkaline single cell gel electrophoresis (comet) assay and DNA adducts using the (32)P-nucleotide postlabeling (NPL) assay. The effects of four carcinogens of different structures requiring distinct pathways of bioactivation, i.e., 2-acetylaminofluorene (AAF), aflatoxin B1 (AFB1), benzo[a]pyrene (B[a]P), and diethylnitrosamine (DEN), were compared with structurally related non-carcinogens fluorene (FLU) and benzo[e]pyrene (B[e]P) or weak carcinogens, aflatoxin B2 (AFB2) and N-nitrosodiethanolamine (NDELA). The four carcinogens all produced DNA breaks at microgram or low milligram total doses, whereas less potent carcinogens and non-carcinogens yielded borderline or negative results, respectively, at higher doses. AAF and B[a]P produced DNA adducts, whereas none was found with the related comparators FLU or B[e]P, consistent with comet results. DEN and NDELA were also negative for adducts, as expected in the case of DEN for an alkylating agent in the standard NPL assay. Also, AFB1 and AFB2 were negative in NPL, as expected, due to the nature of ring opened aflatoxin adducts, which are resistant to enzymatic digestion. Thus, the CEGA, using comet and NPL, is capable of detection of the genotoxicity of diverse DNA-reactive carcinogens, while not yielding false positives for non-carcinogens. © The Author 2014. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Gas Production at Comet 67P/Churyumov-Gerasimenko as Measured by the ROSINA Instrument: Long Term Trends and Correlations with H2O and CO2

NASA Astrophysics Data System (ADS)

Hansen, K. C.; Altwegg, K.; Berthelier, J. J.; Combi, M. R.; De Keyser, J.; Fiethe, B.; Fougere, N.; Fuselier, S. A.; Gombosi, T. I.; Huang, Z.; Rubin, M.; Tenishev, V.; Toth, G.; Tzou, C. Y.

2017-12-01

The Rosetta Orbiter Spectrometer for Ion and Neutral Analysis (ROSINA) instrument onboard the Rosetta spacecraft measured the in situ gas density of comet 67P/Churyumov-Gerasimenko during the full perihelion passage of the comet within 3.5au. During this time, ROSINA sampled the neutral coma, measuring the broad range of cometary species including both the major constituents such as H2O, CO2, CO as well as many other species that are interesting to the general astrophysical community, such as O2, Xe, Si and even amino acids. Many of these species are hard to detect and therefore measurements are limited to when the spacecraft was close to the comet or the production rate was high. In contrast, in this work we will consider species that are most easily measured due to either their higher production rates or the ease with which their mass peaks are located (H2O, CO2, CO, O2, 18OH, HDO, OCS, SO2, H2S, CN, HCN, NH3, CH4, C2H2, C2H3, CH3OH and F). The advantage of examining these species is that we are able to present measurements over the entire perihelion passage at reasonably high time resolution. In this work we will present two important results. First, we will examine the long-term trend and heliocentric distance dependence of the production of these species over the entire perihelion passage of 67P. Second we will consider the correlation of the production of each species with the production of H2O and CO2. The study will consider both the long term correspondence between production of different species as well as the shorter term correlation.

Optimization and Validation of the TZM-bl Assay for Standardized Assessments of Neutralizing Antibodies Against HIV-1

PubMed Central

Sarzotti-Kelsoe, Marcella; Bailer, Robert T; Turk, Ellen; Lin, Chen-li; Bilska, Miroslawa; Greene, Kelli M.; Gao, Hongmei; Todd, Christopher A.; Ozaki, Daniel A.; Seaman, Michael S.; Mascola, John R.; Montefiori, David C.

2014-01-01

The TZM-bl assay measures antibody-mediated neutralization of HIV-1 as a function of reductions in HIV-1 Tat-regulated firefly luciferase (Luc) reporter gene expression after a single round of infection with Env-pseudotyped viruses. This assay has become the main endpoint neutralization assay used for the assessment of preclinical and clinical trial samples by a growing number of laboratories worldwide. Here we present the results of the formal optimization and validation of the TZM-bl assay, performed in compliance with Good Clinical Laboratory Practice (GCLP) guidelines. The assay was evaluated for specificity, accuracy, precision, limits of detection and quantitation, linearity, range and robustness. The validated manual TZM-bl assay was also adapted, optimized and qualified to an automated 384-well format. PMID:24291345

Inhibitory Activities of Butanol Fraction from Butea monosperma (Lam.) Taub. Bark Against Free Radicals, Genotoxins and Cancer Cells.

PubMed

Kaur, Varinder; Kumar, Manish; Kaur, Paramjeet; Kaur, Sandeep; Kaur, Satwinderjeet

2017-06-01

The present study was undertaken to investigate antioxidant, antigenotoxic, and antiproliferative activity of butanol fraction (Bmbu) from bark of medicinal plant Butea monosperma. Antioxidant potency of Bmbu was examined by various in vitro assays. It was also investigated for antigenotoxic activity using Escherichia coli. PQ37 employing SOS chromotest. Further, cytotoxic and apoptosis inducing activity of Bmbu was evaluated in MCF-7 breast cancer cells. Bmbu showed potent free radical scavenging ability in ABTS assay (IC 50 56.70 μg/ml) and anti-lipid peroxidation ability (IC 50 40.39 μg/ml). 4NQO and H 2 O 2 induced genotoxicity was suppressed by Bmbu in SOS chromotest by 74.26% and 82.02% respectively. It also inhibited the growth of MCF-7 cells with GI 50 value of 158.71 μg/ml. Induction of apoptosis in MCF-7 cells by Bmbu treatment was deciphered using confocal microscopy, flow cytometry, and neutral comet assay. Bmbu treatment increased cell population in sub-G 1 phase (69.6%) indicating apoptotic cells. Further, Bmbu treatment resulted in increased reactive oxygen species generation and decreased mitochondrial membrane potential indicating involvement of mitochondrial dependent pathway of apoptosis. HPLC profiling showed the presence of polyphenols such as ellagic acid, catechin, quercetin, and gallic acid as its major constituents. Consequently, it is suggested that the phytoconstituents from this plant may be further exploited for development of novel drug formulation with possible therapeutic implication. © 2017 Wiley-VHCA AG, Zurich, Switzerland.

Arsenic is Cytotoxic and Genotoxic to Primary Human Lung Cells

PubMed Central

Xie, Hong; Huang, ShouPing; Martin, Sarah; Wise, John P.

2014-01-01

Arsenic originates from both geochemical and numerous anthropogenic activities. Exposure of the general public to significant levels of arsenic is widespread. Arsenic is a well-documented human carcinogen. Long-term exposure to high levels of arsenic in drinking water have been linked to bladder, lung, kidney, liver, prostate, and skin cancer. Among them, lung cancer is of great public concern. However, little is known about how arsenic causes lung cancer and few studies have considered effects in normal human lung cells. The purpose of this study was to determine the cytotoxicity and genotoxicity of arsenic in human primary bronchial fibroblast and epithelial cells. Our data show that arsenic induces a concentration-dependent decrease in cell survival after short (24 h) or long (120 h) exposures. Arsenic induces concentration-dependent but not time-dependent increases in chromosome damage in fibroblasts. No chromosome damage is induced after either 24 h or 120 h arsenic exposure in epithelial cells. Using neutral comet assay and gamma-H2A.X foci forming assay, we found that 24 h or 120 h exposure to arsenic induces increases in DNA double strand breaks in both cell lines. These data indicate that arsenic is cytotoxic and genotoxic to human lung primary cells but lung fibroblasts are more sensitive to arsenic than epithelial cells. Further research is needed to understand the specific mechanisms involved in arsenic-induced genotoxicity in human lung cells. PMID:24291234

Extended atmospheres of outer planet satellites and comets

NASA Technical Reports Server (NTRS)

Smyth, W. H.; Combi, M. R.

1985-01-01

Collisions between neutral hydrogen atoms in the interstellar medium and those in the so-called Titan hydrogen torus may provide an additional lifetime sink for atoms in the Saturn environment. Progress toward re-sorting the Voyager UVS scans of neutral hydrogen in the Saturn system to enable both a factor of two increase in the amount of data to be analyzed as well as to help identify near-Titan hydrogen is discussed. Progress toward development of the cometary carbon and oxygen models is also discussed and a preliminary model run for the H2O source of cometary oxygen is presented.

Modeling of Pickup Ion Distributions in the Halley Cometo-Sheath: Empirical Rates of Ionization, Diffusion, Loss and Creation of Fast Neutral Atoms

NASA Technical Reports Server (NTRS)

Huddleston, D.; Neugebauer, M.; Goldstein, B.

1994-01-01

The shape of the velocity distribution of water-group ions observed by the Giotto ion mass spectrometer on its approach to comet Halley is modeled to derive empirical values for the rates on ionization, energy diffusion, and loss in the mid-cometosheath.

End-of-mission ROSINA/COPS measurements as a probe of the innermost coma of comet 67P/Churyumov-Gerasimenko

NASA Astrophysics Data System (ADS)

Tenishev, V.; Fougere, N.; Rubin, M.; Tzou, C. Y.; Combi, M. R.; Altwegg, K.; Gombosi, T. I.; Shou, Y.; Huang, Z.; Hansen, K. C.; Toth, G.

2017-12-01

A cometary coma is a unique phenomenon in the Solar system that represents an example of a planetary atmosphere influenced by little or no gravity. Due to the negligible gravity of a comet's nucleus, a coma has a characteristic size that exceeds that of the nucleus itself by many orders of magnitude. An extended dusty gas cloud that forms a coma is affected mainly by molecular collisions, radiative cooling, and photolytic, charge-exchange, and impact-ionization reactions. Such an environment has been extensively observed during the recent Rosetta mission, which was the first mission that escorts a comet along its way through the Solar system for an extended amount of time with the main scientific objectives of characterizing comet's nucleus, determining the surface composition, and studying the comet's activity development. The ROSINA (Rosetta Orbiter Spectrometer for Ion and Neutral Analysis) Comet Pressure Sensor (COPS) onboard the Rosetta spacecraft has performed one of the most exciting observations of the innermost coma during the spacecraft descend maneuver during the last ten hours of the mission when the random and outflow directed pressures in the coma have been measured all the way down to the comet's surface. Performed at such close proximity to the nucleus, these observations can help to characterize effects due to topological features and/or the gas local conditions at the surface of the nucleus. The major focus of the presented study is analyzing of the end-of-mission pressure measurements by the ROSINA/COPS instrument. Because the coma at a heliocentric distance of 3.8 AU was in a collisionless regime, it can be described by solving the Liouville equation, as we have done in our analysis. We have used the SHAP5 nucleus model to account for the topology of the volatile source. Spacecraft trajectory and the instrument pointing with respect to the comet's nucleus have been obtained with the SPICE library. Here, we present results of our analysis and discuss the effects of the surface topology and that of the local surface volatile injection on the distribution of gas in the innermost coma of comet 67P/Churyumov-Gerasimenko.

Human papillomavirus status and the relative biological effectiveness of proton radiotherapy in head and neck cancer cells.

PubMed

Wang, Li; Wang, Xiaochun; Li, Yuting; Han, Shichao; Zhu, Jinming; Wang, Xiaofang; Molkentine, David P; Blanchard, Pierre; Yang, Yining; Zhang, Ruiping; Sahoo, Narayan; Gillin, Michael; Zhu, Xiaorong Ronald; Zhang, Xiaodong; Myers, Jeffrey N; Frank, Steven J

2017-04-01

Human papillomavirus (HPV)-positive oropharyngeal carcinomas response better to X-ray therapy (XRT) than HPV-negative disease. Whether HPV status influences the sensitivity of head and neck cancer cells to proton therapy or the relative biological effectiveness (RBE) of protons versus XRT is unknown. Clonogenic survival was used to calculate the RBE; immunocytochemical analysis and neutral comet assay were used to evaluate unrepaired DNA double-strand breaks. HPV-positive cells were more sensitive to protons and the unrepaired double-strand breaks were more numerous in HPV-positive cells than in HPV-negative cells (p < .001). Protons killed more cells than did XRT at all fraction sizes (all RBEs > 1.06). Cell line type and radiation fraction size influenced the RBE. HPV-positive cells were more sensitive to protons than HPV-negative cells maybe through the effects of HPV on DNA damage and repair. The RBE for protons depends more on cell type and fraction size than on HPV status. © 2016 Wiley Periodicals, Inc. Head Neck 39: 708-715, 2017. © 2016 Wiley Periodicals, Inc.

The Science of Sungrazers, Sunskirters, and Other Near-Sun Comets

NASA Astrophysics Data System (ADS)

Jones, Geraint H.; Knight, Matthew M.; Battams, Karl; Boice, Daniel C.; Brown, John; Giordano, Silvio; Raymond, John; Snodgrass, Colin; Steckloff, Jordan K.; Weissman, Paul; Fitzsimmons, Alan; Lisse, Carey; Opitom, Cyrielle; Birkett, Kimberley S.; Bzowski, Maciej; Decock, Alice; Mann, Ingrid; Ramanjooloo, Yudish; McCauley, Patrick

2018-02-01

This review addresses our current understanding of comets that venture close to the Sun, and are hence exposed to much more extreme conditions than comets that are typically studied from Earth. The extreme solar heating and plasma environments that these objects encounter change many aspects of their behaviour, thus yielding valuable information on both the comets themselves that complements other data we have on primitive solar system bodies, as well as on the near-solar environment which they traverse. We propose clear definitions for these comets: We use the term near-Sun comets to encompass all objects that pass sunward of the perihelion distance of planet Mercury (0.307 AU). Sunskirters are defined as objects that pass within 33 solar radii of the Sun's centre, equal to half of Mercury's perihelion distance, and the commonly-used phrase sungrazers to be objects that reach perihelion within 3.45 solar radii, i.e. the fluid Roche limit. Finally, comets with orbits that intersect the solar photosphere are termed sundivers. We summarize past studies of these objects, as well as the instruments and facilities used to study them, including space-based platforms that have led to a recent revolution in the quantity and quality of relevant observations. Relevant comet populations are described, including the Kreutz, Marsden, Kracht, and Meyer groups, near-Sun asteroids, and a brief discussion of their origins. The importance of light curves and the clues they provide on cometary composition are emphasized, together with what information has been gleaned about nucleus parameters, including the sizes and masses of objects and their families, and their tensile strengths. The physical processes occurring at these objects are considered in some detail, including the disruption of nuclei, sublimation, and ionisation, and we consider the mass, momentum, and energy loss of comets in the corona and those that venture to lower altitudes. The different components of comae and tails are described, including dust, neutral and ionised gases, their chemical reactions, and their contributions to the near-Sun environment. Comet-solar wind interactions are discussed, including the use of comets as probes of solar wind and coronal conditions in their vicinities. We address the relevance of work on comets near the Sun to similar objects orbiting other stars, and conclude with a discussion of future directions for the field and the planned ground- and space-based facilities that will allow us to address those science topics.

Evaluation of genetic damage in tobacco and arsenic exposed population of Southern Assam, India using buccal cytome assay and comet assay.

PubMed

Roy, Prasenjit; Mukherjee, Anita; Giri, Sarbani

2016-02-01

Ground water is the principal source of drinking water in Assam. Ground water contamination of arsenic in drinking water is a great concern for human health and considered as a human carcinogen. The present cytogenetic biomonitoring study was undertaken to investigate the genotoxic effects associated with people of southern Assam consuming arsenic contaminated water and chewing tobacco. Employing the buccal cytome assay, exfoliated cells were analyzed in 138 individuals of age range 22-42 years and divided into four groups. Group I (n=54) are participants residing in localities where ground water contains arsenic concentration below the permissible limit (<10μg/l) and without any tobacco chewing history. Group II (n=32) participants from the same area but they are tobacco chewers. Group III (n=24) participants from localities where significantly high arsenic contamination in ground water were observed. Whereas the Group IV (n=28) consists of participants from the arsenic contaminated area and also tobacco chewers. Body mass index (BMI) in all the groups are found to be nearly same and in normal range. Statistically significant (P<0.001) increase in genotoxic, cell death parameters and cell proliferation biomarkers were observed in the Group IV compared to other groups. In the comet assay, percent of tail DNA gradually increases among the groups and has statistical significance. Spearman correlation revealed strong positive correlation between the arsenic exposed peoples and the binucleated cells (r=0.4763; P<0.001). Amount of chewing tobacco had significant positive correlation with micronucleus frequency (r=0.268; P<0.05) and karyolitic cells (r=0.217; P<0.05) and also in the percentage of tail DNA (r=0.5532, P<0.001). A statistically significant increase in glucose content and decrease in hemoglobin content as well as acetylcholine esterase in the blood of exposed individuals was observed. Our preliminary study indicate that population exposed to arsenic through drinking water may become more susceptible towards chewing tobacco induced nuclear damage as evaluated by buccal cytome assay and comet assay. Copyright © 2015 Elsevier Inc. All rights reserved.

Validation of freezing tissues and cells for analysis of DNA strand break levels by comet assay

PubMed Central

Jackson, Petra

2013-01-01

The comet analysis of DNA strand break levels in tissues and cells has become a common method of screening for genotoxicity. The large majority of published studies have used fresh tissues and cells processed immediately after collection. However, we have used frozen tissues and cells for more than 10 years, and we believe that freezing samples improve efficiency of the method. We compared DNA strand break levels measured in fresh and frozen bronchoalveolar cells, and lung and liver tissues from mice exposed to the known mutagen methyl methanesulphonate (0, 25, 75, 112.5mg/kg). We used a high-throughput comet protocol with fully automated scoring of DNA strand break levels. The overall results from fresh and frozen samples were in agreement [R 2 = 0.93 for %DNA in tail (%TDNA) and R 2 = 0.78 for tail length (TL)]. A slightly increased %TDNA was observed in lung and liver tissue from vehicle controls; and TL was slightly reduced in bronchoalveolar lavage cells from the high-dose group. In our comet protocol, a small block of tissue designated for comet analysis is frozen immediately at tissue collection and kept deep frozen until rapidly homogenised and embedded in agarose. To demonstrate the feasibility of long-term freezing of samples, we analysed the day-to-day variation of our internal historical negative and positive comet assay controls collected over a 10-year period (1128 observations, 11 batches of frozen untreated and H2O2-treated A549 lung epithelial cells). The H2O2 treatment explained most of the variation 57–77% and the day-to-day variation was only 2–12%. The presented protocol allows analysis of samples collected over longer time span, at different locations, with reduced variation by reducing number of electrophoreses and is suitable for both toxicological and epidemiological studies. The use of frozen tissues; however, requires great care during preparation before analysis, with handling as a major risk factor. PMID:24136994

Mixture genotoxicity of 2,4-dichlorophenoxyacetic acid, acrylamide, and maleic hydrazide on human Caco-2 cells assessed with comet assay.

PubMed

Syberg, Kristian; Binderup, Mona-Lise; Cedergreen, Nina; Rank, Jette

2015-01-01

Assessment of genotoxic properties of chemicals is mainly conducted only for single chemicals, without taking mixture genotoxic effects into consideration. The current study assessed mixture effects of the three known genotoxic chemicals, 2,4-dichlorophenoxyacetic acid (2,4-D), acrylamide (AA), and maleic hydrazide (MH), in an experiment with a fixed ratio design setup. The genotoxic effects were assessed with the single-cell gel electrophoresis assay (comet assay) for both single chemicals and the ternary mixture. The concentration ranges used were 0-1.4, 0-20, and 0-37.7 mM for 2,4-D, AA, and MH, respectively. Mixture toxicity was tested with a fixed ratio design at a 10:23:77% ratio for 2.4-D:AA:MH. Results indicated that the three chemicals yielded a synergistic mixture effect. It is not clear which mechanisms are responsible for this interaction. A few possible interactions are discussed, but further investigations including in vivo studies are needed to clarify how important these more-than-additive effects are for risk assessment.

In situ gas and ion measurements at comet Halley

NASA Astrophysics Data System (ADS)

Krankowsky, D.; Lammerzahl, P.; Herrwerth, I.; Woweries, J.; Eberhardt, P.; Dolder, U.; Herrmann, U.; Schulte, W.; Berthelier, J. J.; Illiano, J. M.; Hodges, R. R.; Hoffman, J. H.

1986-05-01

The neutral mass spectrometer experiment carried by the Giotto spacecraft was designed to determine the abundances and the chemical, elemental and isotopic composition of the gases and low-energy ions in the coma of comet Halley. Its first results show the predominance of water vapour with an H2O density of 4.7x107molecules cm-3 at 1,000 km. Limits on the abundances of CO2, NH3 and CH4 relative to H2O are given. The water-group ions H3O+, H2O+ and OH+ have been unambiguously identified, along with the ions 12C+, 12CH+, 16O+, Na+, 12C2+, 32S+, 34S+ and 56Fe+.

Multifluid MHD Simulations of the Plasma Environment of Comet Churyumov-Gerasimenko at Different Heliocentric Distances

NASA Astrophysics Data System (ADS)

Huang, Z.; Jia, X.; Rubin, M.; Fougere, N.; Gombosi, T. I.; Tenishev, V.; Combi, M. R.; Bieler, A. M.; Toth, G.; Hansen, K. C.; Shou, Y.

2014-12-01

We study the plasma environment of the comet Churyumov-Gerasimenko, which is the target of the Rosetta mission, by performing large scale numerical simulations. Our model is based on BATS-R-US within the Space Weather Modeling Framework that solves the governing multifluid MHD equations, which describe the behavior of the cometary heavy ions, the solar wind protons, and electrons. The model includes various mass loading processes, including ionization, charge exchange, dissociative ion-electron recombination, as well as collisional interactions between different fluids. The neutral background used in our MHD simulations is provided by a kinetic Direct Simulation Monte Carlo (DSMC) model. We will simulate how the cometary plasma environment changes at different heliocentric distances.

High-Throughput Assay Optimization and Statistical Interpolation of Rubella-Specific Neutralizing Antibody Titers

PubMed Central

Lambert, Nathaniel D.; Pankratz, V. Shane; Larrabee, Beth R.; Ogee-Nwankwo, Adaeze; Chen, Min-hsin; Icenogle, Joseph P.

2014-01-01

Rubella remains a social and economic burden due to the high incidence of congenital rubella syndrome (CRS) in some countries. For this reason, an accurate and efficient high-throughput measure of antibody response to vaccination is an important tool. In order to measure rubella-specific neutralizing antibodies in a large cohort of vaccinated individuals, a high-throughput immunocolorimetric system was developed. Statistical interpolation models were applied to the resulting titers to refine quantitative estimates of neutralizing antibody titers relative to the assayed neutralizing antibody dilutions. This assay, including the statistical methods developed, can be used to assess the neutralizing humoral immune response to rubella virus and may be adaptable for assessing the response to other viral vaccines and infectious agents. PMID:24391140

Genotoxicity assessment of nanomaterials: recommendations on best practices, assays and methods.

PubMed

Elespuru, Rosalie; Pfuhler, Stefan; Aardema, Marilyn; Chen, Tao; Doak, Shareen H; Doherty, Ann; Farabaugh, Christopher S; Kenny, Julia; Manjanatha, Mugimane; Mahadevan, Brinda; Moore, Martha M; Ouédraogo, Gladys; Stankowski, Leon F; Tanir, Jennifer Y

2018-04-26

Nanomaterials (NMs) present unique challenges in safety evaluation. An international working group, the Genetic Toxicology Technical Committee of the International Life Sciences Institute's Health and Environmental Sciences Institute, has addressed issues related to the genotoxicity assessment of NMs. A critical review of published data has been followed by recommendations on methods alterations and best practices for the standard genotoxicity assays: bacterial reverse mutation (Ames); in vitro mammalian assays for mutations, chromosomal aberrations, micronucleus induction, or DNA strand breaks (comet); and in vivo assays for genetic damage (micronucleus, comet and transgenic mutation assays). The analysis found a great diversity of tests and systems used for in vitro assays; many did not meet criteria for a valid test, and/or did not use validated cells and methods in the Organization for Economic Co-operation and Development Test Guidelines, and so these results could not be interpreted. In vivo assays were less common but better performed. It was not possible to develop conclusions on test system agreement, NM activity, or mechanism of action. However, the limited responses observed for most NMs were consistent with indirect genotoxic effects, rather than direct interaction of NMs with DNA. We propose a revised genotoxicity test battery for NMs that includes in vitro mammalian cell mutagenicity and clastogenicity assessments; in vivo assessments would be added only if warranted by information on specific organ exposure or sequestration of NMs. The bacterial assays are generally uninformative for NMs due to limited particle uptake and possible lack of mechanistic relevance, and are thus omitted in our recommended test battery for NM assessment. Recommendations include NM characterization in the test medium, verification of uptake into target cells, and limited assay-specific methods alterations to avoid interference with uptake or endpoint analysis. These recommendations are summarized in a Roadmap guideline for testing.

[Evaluation of cyto- and genotoxic action of ferronanomagnetic and constant magnetic field in in vivo system].

PubMed

Chekhun, V F; Lozovs'ka, Iu V; Luk'ianova, N Iu; Demash, D V; Todor, I M; Nalieskina, L A

2013-01-01

Cyto- and genotoxic effects of nanoparticles on the basis of FM, CMF or their combination have been studied in AKE cells, BM cells of erythroid line, and peripheral blood lymphocytes with the use of MN test and "DNA-comet" assay. It has been shown that expression of mentioned effects is related to FM concentration and duration of tested agent action. It has been also demonstrated that action of CMF alone in the studied cells did not cause any changes in cell architectonics or affect MN counts which are associated with DNA damage. When FM and CMF were used in combination there has been observed the phenomenon of induction of CMF action with FM nanoparticles. The obtained results allow recommend MN test and "DNA-comet" assay as the markers of genome stability in the tests of genotoxic effects of nanomaterials for development of vector nanosystems.

The comet assay for the evaluation of genotoxic potential of landfill leachate.

PubMed

Widziewicz, Kamila; Kalka, Joanna; Skonieczna, Magdalena; Madej, Paweł

2012-01-01

Genotoxic assessment of landfill leachate before and after biological treatment was conducted with two human cell lines (Me45 and NHDF) and Daphnia magna somatic cells. The alkali version of comet assay was used to examine genotoxicity of leachate by DNA strand breaks analysis and its repair dynamics. The leachate samples were collected from Zabrze landfill, situated in the Upper Silesian Industrial District, Poland. Statistically significant differences (Kruskal-Wallice ANOVA rank model) were observed between DNA strand breaks in cells incubated with leachate before and after treatment (P < 0.001). Nonparametric Friedman ANOVA confirmed time-reliable and concentration-reliable cells response to leachate concentration. Examinations of chemical properties showed a marked decrease in leachate parameters after treatment which correlate to reduced genotoxicity towards tested cells. Obtained results demonstrate that biological cotreatment of leachate together with municipal wastewater is an efficient method for its genotoxic potential reduction; however, treated leachate still possessed genotoxic character.

The Comet Assay for the Evaluation of Genotoxic Potential of Landfill Leachate

PubMed Central

Widziewicz, Kamila; Kalka, Joanna; Skonieczna, Magdalena; Madej, Paweł

2012-01-01

Genotoxic assessment of landfill leachate before and after biological treatment was conducted with two human cell lines (Me45 and NHDF) and Daphnia magna somatic cells. The alkali version of comet assay was used to examine genotoxicity of leachate by DNA strand breaks analysis and its repair dynamics. The leachate samples were collected from Zabrze landfill, situated in the Upper Silesian Industrial District, Poland. Statistically significant differences (Kruskal-Wallice ANOVA rank model) were observed between DNA strand breaks in cells incubated with leachate before and after treatment (P < 0.001). Nonparametric Friedman ANOVA confirmed time-reliable and concentration-reliable cells response to leachate concentration. Examinations of chemical properties showed a marked decrease in leachate parameters after treatment which correlate to reduced genotoxicity towards tested cells. Obtained results demonstrate that biological cotreatment of leachate together with municipal wastewater is an efficient method for its genotoxic potential reduction; however, treated leachate still possessed genotoxic character. PMID:22666120

Cytogenetic investigation of subjects professionally exposed to radiofrequency radiation.

PubMed

Maes, Annemarie; Van Gorp, Urbain; Verschaeve, Luc

2006-03-01

Nowadays, virtually everybody is exposed to radiofrequency radiation (RFR) from mobile phone base station antennas or other sources. At least according to some scientists, this exposure can have detrimental health effects. We investigated cytogenetic effects in peripheral blood lymphocytes from subjects who were professionally exposed to mobile phone electromagnetic fields in an attempt to demonstrate possible RFR-induced genetic effects. These subjects can be considered well suited for this purpose as their RFR exposure is 'normal' though rather high, and definitely higher than that of the 'general population'. The alkaline comet assay, sister chromatid exchange (SCE) and chromosome aberration tests revealed no evidence of RFR-induced genetic effects. Blood cells were also exposed to the well known chemical mutagen mitomycin C in order to investigate possible combined effects of RFR and the chemical. No cooperative action was found between the electromagnetic field exposure and the mutagen using either the comet assay or SCE test.

A PROTOSOLAR NEBULA ORIGIN FOR THE ICES AGGLOMERATED BY COMET 67P/CHURYUMOV–GERASIMENKO

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mousis, O.; Vernazza, P.; Lunine, J. I.

The nature of the icy material accreted by comets during their formation in the outer regions of the protosolar nebula (PSN) is a major open question in planetary science. Some scenarios of comet formation predict that these bodies agglomerated from crystalline ices condensed in the PSN. Concurrently, alternative scenarios suggest that comets accreted amorphous ice originating from the interstellar cloud or from the very distant regions of the PSN. On the basis of existing laboratory and modeling data, we find that the N{sub 2}/CO and Ar/CO ratios measured in the coma of the Jupiter-family comet 67P/Churyumov–Gerasimenko by the Rosetta Orbitermore » Spectrometer for Ion and Neutral Analysis instrument on board the European Space Agency’s Rosetta spacecraft match those predicted for gases trapped in clathrates. If these measurements are representative of the bulk N{sub 2}/CO and Ar/CO ratios in 67P/Churyumov–Gerasimenko, it implies that the ices accreted by the comet formed in the nebula and do not originate from the interstellar medium, supporting the idea that the building blocks of outer solar system bodies have been formed from clathrates and possibly from pure crystalline ices. Moreover, because 67P/Churyumov–Gerasimenko is impoverished in Ar and N{sub 2}, the volatile enrichments observed in Jupiter’s atmosphere cannot be explained solely via the accretion of building blocks with similar compositions and require an additional delivery source. A potential source may be the accretion of gas from the nebula that has been progressively enriched in heavy elements due to photoevaporation.« less

Solar wind interaction with comet 67P: Impacts of corotating interaction regions

NASA Astrophysics Data System (ADS)

Edberg, N. J. T.; Eriksson, A. I.; Odelstad, E.; Vigren, E.; Andrews, D. J.; Johansson, F.; Burch, J. L.; Carr, C. M.; Cupido, E.; Glassmeier, K.-H.; Goldstein, R.; Halekas, J. S.; Henri, P.; Koenders, C.; Mandt, K.; Mokashi, P.; Nemeth, Z.; Nilsson, H.; Ramstad, R.; Richter, I.; Wieser, G. Stenberg

2016-02-01

We present observations from the Rosetta Plasma Consortium of the effects of stormy solar wind on comet 67P/Churyumov-Gerasimenko. Four corotating interaction regions (CIRs), where the first event has possibly merged with a coronal mass ejection, are traced from Earth via Mars (using Mars Express and Mars Atmosphere and Volatile EvolutioN mission) to comet 67P from October to December 2014. When the comet is 3.1-2.7 AU from the Sun and the neutral outgassing rate ˜1025-1026 s-1, the CIRs significantly influence the cometary plasma environment at altitudes down to 10-30 km. The ionospheric low-energy (˜5 eV) plasma density increases significantly in all events, by a factor of >2 in events 1 and 2 but less in events 3 and 4. The spacecraft potential drops below -20 V upon impact when the flux of electrons increases. The increased density is likely caused by compression of the plasma environment, increased particle impact ionization, and possibly charge exchange processes and acceleration of mass-loaded plasma back to the comet ionosphere. During all events, the fluxes of suprathermal (˜10-100 eV) electrons increase significantly, suggesting that the heating mechanism of these electrons is coupled to the solar wind energy input. At impact the magnetic field strength in the coma increases by a factor of 2-5 as more interplanetary magnetic field piles up around the comet. During two CIR impact events, we observe possible plasma boundaries forming, or moving past Rosetta, as the strong solar wind compresses the cometary plasma environment. We also discuss the possibility of seeing some signatures of the ionospheric response to tail disconnection events.

Genotoxicity assessment of propyl thiosulfinate oxide, an organosulfur compound from Allium extract, intended to food active packaging.

PubMed

Mellado-García, P; Maisanaba, S; Puerto, M; Llana-Ruiz-Cabello, M; Prieto, A I; Marcos, R; Pichardo, S; Cameán, A M

2015-12-01

Essential oils from onion (Allium cepa L.), garlic (Allium sativum L.), and their main components, such as propyl thiosulfinate oxide (PTSO) are being intended for active packaging with the purpose of maintaining and extending food product quality and shelf life. The present work aims to assess for the first time the potential mutagenicity/genotoxicity of PTSO (0-50 µM) using the following battery of genotoxicity tests: (1) the bacterial reverse-mutation assay in Salmonella typhimurium (Ames test, OECD 471); (2) the micronucleus test (OECD 487) (MN) and (3) the mouse lymphoma thymidine-kinase assay (OECD 476) (MLA) on L5178YTk(+/-), cells; and (4) the comet assay (with and without Endo III and FPG enzymes) on Caco-2 cells. The results revealed that PTSO was not mutagenic in the Ames test, however it was mutagenic in the MLA assay after 24 h of treatment (2.5-20 µM). The parent compound did not induce MN on mammalian cells; however, its metabolites (in the presence S9) produced positive results (from 15 µM). Data from the comet assay indicated that PTSO did not induce DNA breaks or oxidative DNA damage. Further in vivo genotoxicity tests are needed to confirm its safety before it is used as active additive in food packaging. Copyright © 2015 Elsevier Ltd. All rights reserved.

Genotoxicity evaluation of the naturally-derived food colorant, gardenia blue, and its precursor, genipin.

PubMed

Hobbs, Cheryl A; Koyanagi, Mihoko; Swartz, Carol; Davis, Jeffrey; Maronpot, Robert; Recio, Leslie; Hayashi, Shim-Mo

2018-06-04

Gardenia blue is widely used in Eastern Asia as a natural food colorant. To evaluate the genotoxic potential of gardenia blue, as well as genipin, the natural starting material from which it is produced, a GLP-compliant test battery was conducted according to OECD guidelines. No evidence of mutagenicity of gardenia blue was detected in a 5-strain bacterial reverse mutation assay, with or without metabolic activation; an equivocal response for genipin occurred in S. typhimurium TA97a without metabolic activation. In in vitro micronucleus and chromosome aberration assays, genipin tested positive under some test conditions; however, gardenia blue tested negative in both assays. In combined micronucleus/comet assays conducted in male and female B6C3F1 mice, exposure to genipin at doses reaching maximal toxicity (74 and 222 mg/kg bw/day for males and females, respectively) or gardenia blue tested up to the limit dose (2000 mg/kg bw/day) did not induce micronuclei in peripheral blood or DNA damage in several examined tissues. Modified ("reverse") comet assays showed no evidence of DNA crosslinking potential of either genipin, known to form crosslinks with other macromolecules, or gardenia blue. Our results indicate that consumption of gardenia blue in food products does not pose a significant genotoxic concern for humans. Copyright © 2018. Published by Elsevier Ltd.

Aspects of nitrogen dioxide toxicity in environmental urban concentrations in human nasal epithelium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Koehler, C.; Ginzkey, C.; Friehs, G.

Cytotoxicity and genotoxicity of nitrogen dioxide (NO{sub 2}) as part of urban exhaust pollution are widely discussed as potential hazards to human health. This study focuses on toxic effects of NO{sub 2} in realistic environmental concentrations with respect to the current limit values in a human target tissue of volatile xenobiotics, the epithelium of the upper aerodigestive tract. Nasal epithelial cells of 10 patients were cultured as an air-liquid interface and exposed to 0.01 ppm NO{sub 2}, 0.1 ppm NO{sub 2}, 1 ppm NO{sub 2}, 10 ppm NO{sub 2} and synthetic air for half an hour. After exposure, genotoxicity wasmore » evaluated by the alkaline single-cell microgel electophoresis (Comet) assay and by induction of micronuclei in the micronucleus test. Depression of proliferation and cytotoxic effects were determined using the micronucleus assay and trypan blue exclusion assay, respectively. The experiments revealed genotoxic effects by DNA fragmentation starting at 0.01 ppm NO{sub 2} in the Comet assay, but no micronucleus inductions, no changes in proliferation, no signs of necrosis or apoptosis in the micronucleus assay, nor did the trypan blue exclusion assay show any changes in viability. The present data reveal a possible genotoxicity of NO{sub 2} in urban concentrations in a screening test. However, permanent DNA damage as indicated by the induction of micronuclei was not observed. Further research should elucidate the effects of prolonged exposure.« less

Modeling the size of the very dynamic diamagnetic cavity of comet 67P/Churyumov-Gerasimenko

NASA Astrophysics Data System (ADS)

Timar, Aniko; Nemeth, Zoltan; Madanian, Hadi; Glassmeier, Karl-Heinz; Goetz, Charlotte; Richter, Ingo; Szego, Karoly

2017-04-01

After the first detection of the diamagnetic cavity of comet 67P/Churyumov-Gerasimenko (Goetz et al. 2015) it became apparent that the boundary of this plasma region is very dynamic. To date hundreds of short cavity crossing events were detected (Nemeth et al. 2016, Goetz et al. in press), none lasting longer than an hour. This intermittent set of short crossing events is very different from the classical cavity observation near 1P/Halley, where Giotto remained for a long time continuously inside the cavity. The distance of the boundary is larger than that predicted by recent models, so it was not clear whether these short cavity-like regions are connected to a global diamagnetic cavity, or they are due to some local effects causing similar magnetic and plasma signatures. Here we revisit the neutral-drag model of Cravens 1986 to provide a very good phenomenological approximation for the highly variable size of this dynamic region. The model uses the cometary neutral production rate and the solar wind dynamic pressure as inputs. For the production rate we use averaged and detrended data derived from ROSINA neutral density measurements (Hansen et al. 2016). The solar wind pressure comes from space weather models and independently from the magnetic field measurements of MAG derived by using a method proposed by Madanian et al. 2016. The changes in the production rate and the dynamic pressure allows us to accurately predict the size of the cavity. In addition we show that instead of the local neutral pressure, the global production rate drives the size of the cavity. We can also explain the observed asymmetry between inbound and outbound crossings of the cavity boundary.

Evaluation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) adduct levels and DNA strand breaks in human peripheral blood lymphocytes exposed in vitro to polycyclic aromatic hydrocarbons with or without animal metabolic activation.

PubMed

Isabel, Rodríguez-Romero María; Sandra, Gómez-Arroyo; Rafael, Villalobos-Pietrini; Carmen, Martínez-Valenzuela; Josefina, Cortés-Eslava; del Carmen, Calderón-Ezquerro María; Rocío, García-Martínez; Francisco, Arenas-Huertero; Elena, Calderón-Segura María

2012-04-01

The polycyclic aromatic hydrocarbons (PAHs) dibenzo(a,h)anthracene, benzo(ghi)perylene, benzo(b)fluoranthene and benzo(a)pyrene have been identified in urban air from Mexico City and some of them are classified as human carcinogens. In the present study, human peripheral blood lymphocytes were exposed in vitro to different concentrations of PAHs with (+S9) or without (-S9) metabolic activation. The genotoxic and cytotoxic effects of each PAH were examined with an alkaline comet assay and trypan blue dye exclusion, and oxidative DNA damage was determined via the detection of 8-hydroxy-2'-deoxyguanosine (8-OhdG) adduct levels by enzyme-linked immunosorbent assay (ELISA). The DNA damage was evaluated with two genotoxicity parameters: the frequency of comets and the comet tail length. Concentrations of 20, 40, 80, 160 and 320 µM DB(a,h)A-S9; 20, 40, 80, 160 and 240 µM B(ghi)P-S9; 20, 30, 40, 60 and 80 µM B(b)F-S9; and 80 µM B(a)P-S9 for 24 h induced a small but significant increase in the means of comet frequency, in the tail length and in the 8-oHDg levels in relation to the control (0.5% DMSO-S9). However, all PAHs+S9 produced a more significant increase in DNA strand breaks and the level of 8-OHdG compared with the control (0.5% DMSO+S9), with a concentration-effect relationship. The viability of lymphocytes exposed to all PAHs-S9 and PAHs+S9 was not modified compared with the control. The results of this study demonstrate that the comet and ELISA are rapid, suitable and sensitive methods to detect in vitro PAH-induced DNA damage in human peripheral lymphocytes.

5-AED enhances survival of irradiated mice in a G-CSF-dependent manner, stimulates innate immune cell function, reduces radiation-induced DNA damage and induces genes that modulate cell cycle progression and apoptosis

PubMed Central

Grace, Marcy B.; Singh, Vijay K.; Rhee, Juong G.; Jackson, William E.; Kao, Tzu-Cheg; Whitnall, Mark H.

2012-01-01

The steroid androst-5-ene-3ß,17ß-diol (5-androstenediol, 5-AED) elevates circulating granulocytes and platelets in animals and humans, and enhances survival during the acute radiation syndrome (ARS) in mice and non-human primates. 5-AED promotes survival of irradiated human hematopoietic progenitors in vitro through induction of Nuclear Factor-κB (NFκB)-dependent Granulocyte Colony-Stimulating Factor (G-CSF) expression, and causes elevations of circulating G-CSF and interleukin-6 (IL-6). However, the in vivo cellular and molecular effects of 5-AED are not well understood. The aim of this study was to investigate the mechanisms of action of 5-AED administered subcutaneously (s.c.) to mice 24 h before total body γ- or X-irradiation (TBI). We used neutralizing antibodies, flow cytometric functional assays of circulating innate immune cells, analysis of expression of genes related to cell cycle progression, DNA repair and apoptosis, and assessment of DNA strand breaks with halo-comet assays. Neutralization experiments indicated endogenous G-CSF but not IL-6 was involved in survival enhancement by 5-AED. In keeping with known effects of G-CSF on the innate immune system, s.c. 5-AED stimulated phagocytosis in circulating granulocytes and oxidative burst in monocytes. 5-AED induced expression of both bax and bcl-2 in irradiated animals. Cdkn1a and ddb1, but not gadd45a expression, were upregulated by 5-AED in irradiated mice. S.c. 5-AED administration caused decreased DNA strand breaks in splenocytes from irradiated mice. Our results suggest 5-AED survival enhancement is G-CSF-dependent, and that it stimulates innate immune cell function and reduces radiation-induced DNA damage via induction of genes that modulate cell cycle progression and apoptosis. PMID:22843381

5-AED enhances survival of irradiated mice in a G-CSF-dependent manner, stimulates innate immune cell function, reduces radiation-induced DNA damage and induces genes that modulate cell cycle progression and apoptosis.

PubMed

Grace, Marcy B; Singh, Vijay K; Rhee, Juong G; Jackson, William E; Kao, Tzu-Cheg; Whitnall, Mark H

2012-11-01

The steroid androst-5-ene-3ß,17ß-diol (5-androstenediol, 5-AED) elevates circulating granulocytes and platelets in animals and humans, and enhances survival during the acute radiation syndrome (ARS) in mice and non-human primates. 5-AED promotes survival of irradiated human hematopoietic progenitors in vitro through induction of Nuclear Factor-κB (NFκB)-dependent Granulocyte Colony-Stimulating Factor (G-CSF) expression, and causes elevations of circulating G-CSF and interleukin-6 (IL-6). However, the in vivo cellular and molecular effects of 5-AED are not well understood. The aim of this study was to investigate the mechanisms of action of 5-AED administered subcutaneously (s.c.) to mice 24 h before total body γ- or X-irradiation (TBI). We used neutralizing antibodies, flow cytometric functional assays of circulating innate immune cells, analysis of expression of genes related to cell cycle progression, DNA repair and apoptosis, and assessment of DNA strand breaks with halo-comet assays. Neutralization experiments indicated endogenous G-CSF but not IL-6 was involved in survival enhancement by 5-AED. In keeping with known effects of G-CSF on the innate immune system, s.c. 5-AED stimulated phagocytosis in circulating granulocytes and oxidative burst in monocytes. 5-AED induced expression of both bax and bcl-2 in irradiated animals. Cdkn1a and ddb1, but not gadd45a expression, were upregulated by 5-AED in irradiated mice. S.c. 5-AED administration caused decreased DNA strand breaks in splenocytes from irradiated mice. Our results suggest 5-AED survival enhancement is G-CSF-dependent, and that it stimulates innate immune cell function and reduces radiation-induced DNA damage via induction of genes that modulate cell cycle progression and apoptosis.

The genotoxic effect of oxcarbazepine on mice blood lymphocytes.

PubMed

Akbar, Huma; Khan, Ajmal; Mohammadzai, Imdadullah; Khisroon, Muhammad; Begum, Ilham

2018-04-01

This study was conducted to assess the amount of DNA damage caused by Oxcarbazepine (OXC) through single cell gel electrophoresis (SCGE) technique/comet assay. OXC derived from dibenzazepine series is an effective second generation antiepileptic drug (AED) for both children and adults. Side effects like genotoxic effects of AEDs are of prime importance resulting from toxic metabolites, free radicals and reactive oxygen species (ROS). Forty Eight adult male Bagg's albino mice (BALB/c) were randomly classified into eight groups, each comprising of six animals. Two of these groups were control and six were tested groups. Control groups were injected with 1% tween 80 while tested groups were injected with 10, 20, and 40 mg/kg-day OXC for seven days (acute therapy) and 28 days (subchronic therapy) in peritoneal cavity. Blood samples were collected by cardiac puncture and subjected to comet assay for the analysis of DNA damage. Per sample 100 cells were scored and classified according to comet tail length. The results showed that OXC in acute and long term therapies had significantly higher (p < 0.05) genotoxicity in treated groups as compared to control groups. Our study suggests that OXC may cause significant DNA damage in both acute as well as in subchronic therapies.

Primary DNA damage in chrome-plating workers.

PubMed

Gambelunghe, A; Piccinini, R; Ambrogi, M; Villarini, M; Moretti, M; Marchetti, C; Abbritti, G; Muzi, G

2003-06-30

In order to evaluate the primary DNA damage due to occupational exposure to chromium (VI), DNA strand-breaks and apoptosis in peripheral lymphocytes were measured in a group of 19 chrome-plating workers. DNA strand-breaks was assessed by alkaline (pH>13) single-cell microgel electrophoresis ('comet') assay, while apoptosis was measured by flow-cytometry after propidium iodide staining of the cells. Concentrations of chromium in urine, erythrocytes and lymphocytes were investigated as biological indicators of exposure. A group of 18 hospital workers (control group I) and another 20 university personnel (control group II) without exposure to chromium were also studied as controls. The results of the study show that chrome-plating workers have higher levels of chromium in urine, erythrocytes and lymphocytes than unexposed workers. Comet tail moment values, assumed as index of DNA damage, are increased in chromium-exposed workers and results are significantly correlated to chromium lymphocyte concentrations. No difference emerged in the percentage of apoptotic nuclei in exposed and unexposed workers. The study confirms that measurements of chromium in erythrocytes and lymphocytes may provide useful information about recent and past exposure to hexavalent chromium at the workplace. The increase in DNA strand-breaks measured by comet assay suggests this test is valid for the biological monitoring of workers exposed to genotoxic compounds such as chromium (VI).

High doses of alcohol during pregnancy cause DNA damages in osteoblasts of newborns rats.

PubMed

Carvalho, Isabel Chaves Silva; Dutra, Tamires Pereira; Andrade, Dennia Perez De; Balducci, Ivan; Pacheco-Soares, Cristina; Rocha, Rosilene Fernandes da

2016-02-01

Alcohol exerts teratogenic effects and its consumption during pregnancy can cause deficit of bone development. The aim of the current study was to evaluate the genotoxic effects of prenatal exposure to ethanol on newborn rat osteoblasts. Wistar rats were initially divided into two groups: Ethanol group which received Ethanol 20% V/V in liquid diet and solid diet ad libitum, and Control group, which received solid diet and water ad libitum. Each group received a specific diet for 8 weeks before breeding and throughout three weeks of gestation and the treatment was finished on the day the pups were killed. On the fifth day of life, the pups from each group were killed for removal of the calvaria and isolation of osteogenic cells by sequential enzymatic digestion. The cells were cultured for a maximum period of 14 days. The detection of genotoxic effects of alcohol was investigated by the comet and the micronucleus assay. Micronucleus and comet assay showed significant increases in DNA damage at 7 days in Ethanol group (p = 0.0302, p = 0.0446, respectively). However, at 14 days both assay showed no significant difference between the groups (p = 0.6194, p = 0.8326, respectively). Our results showed that prenatal exposure to ethanol induced DNA damage in osteoblasts, as shown by micronucleus formation and higher percentage of DNA in the comet tail. It can be concluded that prenatal exposure to ethanol damages osteoblast DNA in newborns exposed to high doses of ethanol during pregnancy, suggesting that prenatal ethanol consumption has a direct effect on fetal osteoblasts. © 2015 Wiley Periodicals, Inc.

DNA damage and micronuclei in parthenogenetic and bisexual Darevskia rock lizards from the areas with different levels of soil pollution.

PubMed

Simonyan, Anna; Hovhannisyan, Galina; Sargsyan, Anzhela; Arakelyan, Marine; Minasyan, Seyran; Aroutiounian, Rouben

2018-06-15

Natural species are widely used as indicator organisms to estimate of the impact of environmental pollution. Here we present the results of first study of a reliability of parthenogenetic Darevskia аrmeniaca and bisexual Darevskia raddei rock lizards as sentinels for monitoring of environmental genotoxicity. The comet assay and micronucleus test were applied to the lizards sampled in six areas in Armenia and Artsakh with different levels of soil contamination. The results obtained showed a clear relationship between the pollution level of lizards' habitats and the frequency of DNA damage in the comet assay. Low baseline frequency of micronuclei in D. аrmeniaca and D. raddei, however, makes this parameter ineffective for environmental genotoxicity evaluation. The parthenogenetic lizards D. аrmeniaca showed higher sensitivity toward genotoxic pollutions compared with bisexual D. raddei living in the same environment. The correlations between soil content of heavy metals Cr, Cu, Zn, Mo, Pb and DNA damage in D. аrmeniaca and between Cu, As, Mo, Pb and DNA damage in D. raddei were revealed. Overall, the lizards D. raddei and D. аrmeniaca appeared to be sensitive species in detecting soil pollution in natural environment. The application of the comet assay in Darevskia lizard species can be considered as a more appropriate method than a micronucleus test. The use of parthenogenetic lizards D. аrmeniaca as bioindicator will permit to assess the environmental genotoxicity independent of the genetic polymorphism of bisexual species. Copyright © 2018. Published by Elsevier Inc.

Effect of pollution on DNA damage and essential fatty acid profile in Cirrhinus mrigala from River Chenab

NASA Astrophysics Data System (ADS)

Hussain, Bilal; Sultana, Tayyaba; Sultana, Salma; Al-Ghanim, K. A.; Mahboob, Shahid

2017-05-01

The objective of this study was to evaluate the effect of anthropogenic pollution on DNA damage and the fatty acid profile of the bottom dweller fish ( Cirrhinus mrigala), collected from the River Chenab, in order to assess the effect of the toxicants on the quality of the fish meat. The levels of Cd, Hg, Cu, Mn, Zn, Pb, Cr and Sn and of phenols from this river were significantly higher than the permissible limits set by the USEPA. Comet assays showed DNA damage in Cirrhinus mrigala collected from three different sampling sites in the polluted area of the river. Significant differences were observed for DNA damage through comet assay in fish collected from polluted compared to control sites. No significant differences were observed for DNA damage between farmed and fish collected from upstream. The micronucleus assay showed similar trends. Fish from the highly polluted sites showed less number of fatty acids and more saturated fatty acids in their meat compared to fish from less polluted areas. Several fatty acids were missing in fish with higher levels of DNA in comet tail and micronucleus induction. Long-chain polyunsaturated fatty acids, eicosapentaenoic acid (20:5n-3) was found missing in the fish from polluted environment while it was found in considerable amount in farmed fish 7.8±0.4%. Docosahexaenoic acid (22:6n-3) also showed significant differences as 0.1±0.0 and 7.0±0.1% respectively, in wild polluted and farmed fishes.

Evaluation of the genotoxic potential of 3-monochloropropane-1,2-diol (3-MCPD) and its metabolites, glycidol and beta-chlorolactic acid, using the single cell gel/comet assay.

PubMed

El Ramy, R; Ould Elhkim, M; Lezmi, S; Poul, J M

2007-01-01

3-monochloropropane-1,2-diol (3-MCPD) is a member of a group of chemicals known as chloropropanols. It is found in many foods and food ingredients as a result of food processing. 3-MCPD is regarded as a rat carcinogen known to induce Leydig-cell and mammary gland tumours in males and kidney tumours in both genders. The aim of our study was to clarify the possible involvement of genotoxic mechanisms in 3-MCPD induced carcinogenicity at the target organ level. For that purpose, we evaluated DNA damages in selected target (kidneys and testes) and non-target (blood leukocytes, liver and bone marrow) male rat organs by the in vivo alkaline single cell gel electrophoresis (comet) assay, 3 and 24 h after 3-MCPD oral administration to Sprague-Dawley and Fisher 344 adult rats. 3-MCPD may be metabolised to a genotoxic intermediate, glycidol, whereas the predominant urinary metabolite in rats following 3-MCPD administration is beta-chlorolactic acid. Therefore, we also studied the DNA damaging effects of 3-MCPD and its metabolites, glycidol and beta-chlorolactic acid, in the in vitro comet assay on CHO cells. Our results show the absence of genotoxic potential of 3-MCPD in vivo in the target as well as in the non-target organs. Glycidol, the epoxide metabolite, induced DNA damages in CHO cells. beta-Chlorolactic acid, the main metabolite of 3-MCPD in rats, was shown to be devoid of DNA-damaging effects in vitro in mammalian cells.

The Gas Production Rate and Coma Structure of Comet C/1995 O1 (Hale-Bopp)

NASA Astrophysics Data System (ADS)

Morgenthaler, Jeffrey P.; Harris, Walter M.; Roesler, Frederick L.; Scherb, Frank; Anderson, Christopher M.; Doane, Nathaniel E.; Oliversen, Ronald J.

2002-06-01

The University of Wisconsin-Madison and NASA-Goddard conducted acomprehensive multi-wavelength observing campaign of coma emissionsfrom comet Hale-Bopp, including OH 3080 Å, [O I] 6300 Å H2O+ 6158 Å, H Balmer-α 6563 Å, NH2 6330 Å, [C I] 9850 ÅCN 3879 Å, C2 5141 Å, C3 4062 Å,C I 1657 Å, and the UV and optical continua. In thiswork, we concentrate on the results of the H2O daughter studies.Our wide-field OH 3080 Å measured flux agrees with other, similarobservations and the expected value calculated from published waterproduction rates using standard H2O and OH photochemistry.However, the total [O I] 6300 Å flux determined spectroscopically overa similar field-of-view was a factor of 3-4 higher than expected.Narrow-band [O I] images show this excess came from beyond theH2O scale length, suggesting either a previously unknown source of[O I] or an error in the standard OH + ν→ O(1 D) + H branching ratio. The Hale-Bopp OH and[O I] distributions, both of which were imaged tocometocentric distances >1 × 106 km, were more spatiallyextended than those of comet Halley (after correcting for brightnessdifferences), suggesting a higher bulk outflow velocity. Evidence ofthe driving mechanism for this outflow is found in the Hα lineprofile, which was narrower than in comet Halley (though likelybecause of opacity effects, not as narrow as predicted by Monte-Carlomodels). This is consistent with greater collisional coupling betweenthe suprathermal H photodissociation products and Hale-Bopp's densecoma. Presumably because of mass loading of the solar wind by ionsand ions by the neutrals, the measured acceleration of H2O+ downthe ion tail was much smaller than in comet Halley. Tailwardextensions in the azimuthal distributions of OH 3080 Å,[O I], and [C I] , as well as a Doppler asymmetry in the[O I] line profile, suggest ion-neutral coupling. While thetailward extension in the OH can be explained by increased neutralacceleration, the [O I] 6300 Å and [C I] 9850 Å emissions show 13%and >200% excesses in this direction (respectively), suggesting anon-negligible contribution from dissociative recombination of CO+and/or electron collisional excitation. Thus, models including theeffects of photo- and collisional chemistry are necessary for the fullinterpretation of these data.

Modeling of pickup ion distributions in the Halley cometosheath: Empirical limits on rates of ionization, diffusion, loss and creation of fast neutral atoms

NASA Technical Reports Server (NTRS)

Huddleston, D. E.; Neugebauer, M.; Goldstein, B. E.

1994-01-01

The shape of the velocity distribution of water group ions observed by the Giotto ion mass spectrometer on its approach to comet Halley is modeled to derive empirical values for the rates of ionization, energy diffusion, and loss in the midcometosheath. The model includes the effect of rapid pitch angle scattering into a bispherical shell distribution as well as the effect of the magnetization of the plasma on the charge exchange loss rate. It is found that the average rate of ionization of cometary neutrals in this region of the cometosheath appears to be of the order of a factor 3 faster than the `standard' rates approx. 1 x 10(exp -6)/s that are generally assumed to model the observations in most regions of the comet environment. For the region of the coma studied in the present work (approx. 1 - 2 x 10(exp 5) km from the nucleus), the inferred energy diffusion coefficient is D(sub 0) approx. equals 0.0002 to 0.0005 sq km/cu s, which is generally lower than values used in other models. The empirically obtained loss rate appears to be about an order of magnitude greater than can be explained by charge exchange with the `standard' cross section of approx. 2 x 10(exp -15)sq cm. However such cross sections are not well known and for water group ion/water group neutral interactions, rates as high as 8 x 10(exp -15) sq cm have previously been suggested in the literature. Assuming the entire loss rate is due to charge exchange yields a rate of creation of fast neutral atoms of the order of approx. 10(exp -4)/s or higher, depending on the level of velocity diffusion. The fast neutrals may, in turn, be partly responsible for the higher-than-expected ionization rate.

Ion composition and upstream solar wind observations at comet Giacobini-Zinner

NASA Astrophysics Data System (ADS)

Coplan, M. A.; Ogilvie, K. W.; A'Hearn, M. F.; Bochsler, P.; Geiss, J.

1987-01-01

The observations by the ion composition instrument (ICI) on the ICE spacecraft made during the encounter with comet P/Giacobini-Zinner (Ogilvie et al., 1986) are discussed in detail. Solar wind He-4(2+) kinetic temperatures, densities, and velocities before, during, and after the encounter are presented. These data combined with He-4(2+) velocity distributions provide evidence for the existence of a thick diffuse shock. Relative abundances of water group ions and CO(+) are derived along with an estimate of the abundance of an ion with M/Q = 24 + or - 1 amu/e. The ICI data are compared with electron data from two other experiments (Bame et al., 1986; Meyer-Vernet et al., 1986) and found to be in reasonable agreement in the region outside the tail. Spectroscopic data for several neutral and ionic species are compared with the ICI results for the water group ions and CO(+). The spectroscopic data are also used to eliminate Mg(+) and CN(+) as candidates for the M/Q = 24 peak. The two most likely candidates are C2(+) and Na(+), but neither photoionization of parent neutrals nor sputtering from dust grains is sufficient to explain the observed abundance relative to H2O(+).

Evaluation of the genotoxicity of waters impacted by domestic and industrial effluents of a highly industrialized region of São Paulo State, Brazil, by the comet assay in HTC cells.

PubMed

Manzano, Bárbara Cassu; Roberto, Matheus Mantuanelli; Hoshina, Márcia Miyuki; Menegário, Amauri Antônio; Marin-Morales, Maria Aparecida

2015-01-01

The problems that most affect the quality of the waters of rivers and lakes are associated with the discharges performed in these environments, mainly industrial and domestic effluents inappropriately treated or untreated. The comet assay is a sensitive tool and is recommended for studies of environmental biomonitoring, which aim to determine the genotoxicity potential of water pollutants. This study aimed to assess the genotoxic potential of the Ribeirão Tatu waters, region of Limeira, São Paulo (SP), by the comet assay with mammalian cells (hepatoma tissue culture (HTC)). Water samples were collected along the Ribeirão Tatu at three distinct periods: November 2008, February 2009 and August 2009, and five collection sites were established: P1, source of the stream; P2, site located downstream the urban perimeter of the municipality of Cordeirópolis and after receiving the pollution load of this city; P3, collection site located upstream the urban perimeter of the city of Limeira; P4, urban area of Limeira; and P5, rural area of Limeira, downstream the discharges of the city sewage. The results showed that for the November 2008 collection, there was no water sample-induced genotoxicity; for the February 2009 collection, the sites P1 and P2 were statistically significant in relation to the negative control (NC), and for the August 2009 collection, the site P5 was statistically significant. These results could be explained by the content of different metals during the different seasons that are under the influence of domestic, industrial and agricultural effluents and also due to the seasonality, since the water samples collected in the period of heavy rain (February 2009) presented a higher genotoxicity possibly due to the entrainment of contaminants into the bed of the stream promoted by the outflow of rainwaters. The comet assay showed to be a useful and sensitive tool in the evaluation of hydric resources impacted by pollutants of diverse origins, and a constant monitoring should be done in order to verify the influence of different factors (season, amount of contaminants) in the water quality.

Assessment of the in vitro and in vivo genotoxicity of extracts and indole monoterpene alkaloid from the roots of Galianthe thalictroides (Rubiaceae).

PubMed

Fernandes, L M; Garcez, W S; Mantovani, M S; Figueiredo, P O; Fernandes, C A; Garcez, F R; Guterres, Z R

2013-09-01

Roots of Galianthe thalictroides K. Schum. (Rubiaceae) are used in folk medicine in the State of Mato Grosso do Sul, Brazil, for treating and preventing cancer. To gain information about the genotoxicity of extracts (aqueous and EtOH), the CHCl₃ phase resulting from partition of the EtOH extract and the indole monoterpene alkaloid 1 obtained from this plant. The genotoxicity of 1 and extracts was evaluated in vivo through the Drosophila melanogaster wing Somatic Mutation and Recombination Test - SMART, while in vitro cytotoxic (MTT) and Comet assays were performed only with alkaloid 1. The results obtained with the SMART test indicated that the aqueous extract had no genotoxic activity. The EtOH extract was not genotoxic to ST descendants but genotoxic to HB ones. The CHCl₃ phase was genotoxic and cytotoxic. Alkaloid 1 showed significant mutational events with SMART, in the cytotoxicity assay (MTT), it showed a high cytotoxicity for human hepatoma cells (HepG2), whereas for the Comet assay, not showing genotoxic activity. The ethanol extract was shown to be genotoxic to HB descendants in the SMART assay, while the results obtained in this test for the monoterpene indole alkaloid 1 isolated from this extract. Copyright © 2013 Elsevier Ltd. All rights reserved.

17β-Estradiol induces cyto-genotoxicity on blood cells of common carp (Cyprinus carpio).

PubMed

Orozco-Hernández, Luis; Gutiérrez-Gómez, Adriana Andrea; SanJuan-Reyes, Nely; Islas-Flores, Hariz; García-Medina, Sandra; Galar-Martínez, Marcela; Dublán-García, Octavio; Natividad, Reyna; Gómez-Oliván, Leobardo Manuel

2018-01-01

17β-Estradiol, a natural hormone present at high concentrations in aquatic ecosystems, affects and modifies endocrine function in animals. In recent years research workers have expressed concern over its potential effects on aquatic organisms; however, little is known about its capacity to induce genetic damage or the pro-apoptotic effects of such damage on fish. Therefore, this study aimed to evaluate 17β-estradiol-induced cyto-genotoxicity in blood cells of the common carp Cyprinus carpio exposed to different concentrations (1 ng, 1 μg and 1 mg L -1 ). Peripheral blood samples were collected and evaluated by comet assay, micronucleus test, determination of caspase-3 activity and TUNEL assay at 12, 24, 48, 72 and 96 h of exposure. Increases in frequency of micronuclei, TUNEL-positive cells and caspase-3 activity were observed, particularly at the highest concentration. In contrast, the comet assay detected significant increases at 24 and 96 h with the 1 μg and 1 ng L -1 concentrations respectively. The set of assays used in the present study constitutes a reliable early warning biomarker for evaluating the toxicity induced by this type of emerging contaminants on aquatic species. Copyright © 2017 Elsevier Ltd. All rights reserved.

Effect of different procedures of ejaculate collection, extenders and packages on DNA integrity of boar spermatozoa following freezing-thawing.

PubMed

Fraser, L; Strzezek, J

2007-06-01

Whole ejaculate or sperm-rich fraction, collected from four sexually mature boars, was frozen in an extender containing lactose-hen egg yolk with glycerol (lactose-HEY-G) or extender containing lactose, lyophilized lipoprotein fractions isolated from ostrich egg yolk and glycerol (lactose-LPFo-G), and Orvus Es Paste, respectively. The sperm samples were also frozen in a standard boar semen extender (Kortowo-3), without the addition of cryoprotective substances. Sperm DNA integrity was assessed using a modified neutral comet assay. Sperm characteristics such as motility, plasma membrane integrity (SYBR-14/PI), mitochondrial function (rhodamine 123) and acrosome integrity were monitored. Freezing-thawing caused a significant increase (P<0.05) in sperm DNA fragmentation, irrespective of the procedures of ejaculate collection and extender type. Sperm DNA fragmentation was significantly lower (P<0.05) in the whole ejaculate compared with the sperm-rich fraction, indicating that spermatozoa maintained in the whole seminal plasma prior to its removal for freezing-thawing procedure were less vulnerable to cryo-induced DNA fragmentation. Furthermore, spermatozoa frozen in lactose-HEY-G or lactose-LPFo-G extender exhibited lower (P<0.05) DNA fragmentation than those frozen in the absence of cryoprotective substances. The levels of sperm DNA damage, as expressed by comet tail length and tail moment values, were significantly higher (P<0.05) in sperm samples frozen in the absence of cryoprotective substances. The deterioration in post-thaw sperm DNA integrity was concurrent with reduced sperm characteristics. It can be suggested that evaluation of DNA integrity, coupled with different sperm characteristics such as motility, plasma membrane integrity and mitochondrial function, may aid in determining the quality of frozen-thawed boar semen.

Incomplete Neutralization and Deviation from Sigmoidal Neutralization Curves for HIV Broadly Neutralizing Monoclonal Antibodies

PubMed Central

McCoy, Laura E.; Falkowska, Emilia; Doores, Katie J.; Le, Khoa; Sok, Devin; van Gils, Marit J.; Euler, Zelda; Burger, Judith A.; Seaman, Michael S.; Sanders, Rogier W.; Schuitemaker, Hanneke; Poignard, Pascal; Wrin, Terri; Burton, Dennis R.

2015-01-01

The broadly neutralizing HIV monoclonal antibodies (bnMAbs) PG9, PG16, PGT151, and PGT152 have been shown earlier to occasionally display an unusual virus neutralization profile with a non-sigmoidal slope and a plateau at <100% neutralization. In the current study, we were interested in determining the extent of non-sigmoidal slopes and plateaus at <100% for HIV bnMAbs more generally. Using both a 278 panel of pseudoviruses in a CD4 T-cell (U87.CCR5.CXCR4) assay and a panel of 117 viruses in the TZM-bl assay, we found that bnMAbs targeting many neutralizing epitopes of the spike had neutralization profiles for at least one virus that plateaued at <90%. Across both panels the bnMAbs targeting the V2 apex of Env and gp41 were most likely to show neutralization curves that plateaued <100%. Conversely, bnMAbs targeting the high-mannose patch epitopes were less likely to show such behavior. Two CD4 binding site (CD4bs) Abs also showed this behavior relatively infrequently. The phenomenon of incomplete neutralization was also observed in a large peripheral blood mononuclear cells (PBMC)-grown molecular virus clone panel derived from patient viral swarms. In addition, five bnMAbs were compared against an 18-virus panel of molecular clones produced in 293T cells and PBMCs and assayed in TZM-bl cells. Examples of plateaus <90% were seen with both types of virus production with no consistent patterns observed. In conclusion, incomplete neutralization and non-sigmoidal neutralization curves are possible for all HIV bnMAbs against a wide range of viruses produced and assayed in both cell lines and primary cells with implications for the use of antibodies in therapy and as tools for vaccine design. PMID:26267277

Incomplete Neutralization and Deviation from Sigmoidal Neutralization Curves for HIV Broadly Neutralizing Monoclonal Antibodies.

PubMed

McCoy, Laura E; Falkowska, Emilia; Doores, Katie J; Le, Khoa; Sok, Devin; van Gils, Marit J; Euler, Zelda; Burger, Judith A; Seaman, Michael S; Sanders, Rogier W; Schuitemaker, Hanneke; Poignard, Pascal; Wrin, Terri; Burton, Dennis R

2015-08-01

The broadly neutralizing HIV monoclonal antibodies (bnMAbs) PG9, PG16, PGT151, and PGT152 have been shown earlier to occasionally display an unusual virus neutralization profile with a non-sigmoidal slope and a plateau at <100% neutralization. In the current study, we were interested in determining the extent of non-sigmoidal slopes and plateaus at <100% for HIV bnMAbs more generally. Using both a 278 panel of pseudoviruses in a CD4 T-cell (U87.CCR5.CXCR4) assay and a panel of 117 viruses in the TZM-bl assay, we found that bnMAbs targeting many neutralizing epitopes of the spike had neutralization profiles for at least one virus that plateaued at <90%. Across both panels the bnMAbs targeting the V2 apex of Env and gp41 were most likely to show neutralization curves that plateaued <100%. Conversely, bnMAbs targeting the high-mannose patch epitopes were less likely to show such behavior. Two CD4 binding site (CD4bs) Abs also showed this behavior relatively infrequently. The phenomenon of incomplete neutralization was also observed in a large peripheral blood mononuclear cells (PBMC)-grown molecular virus clone panel derived from patient viral swarms. In addition, five bnMAbs were compared against an 18-virus panel of molecular clones produced in 293T cells and PBMCs and assayed in TZM-bl cells. Examples of plateaus <90% were seen with both types of virus production with no consistent patterns observed. In conclusion, incomplete neutralization and non-sigmoidal neutralization curves are possible for all HIV bnMAbs against a wide range of viruses produced and assayed in both cell lines and primary cells with implications for the use of antibodies in therapy and as tools for vaccine design.

Optimization and validation of a high throughput method for detecting neutralizing antibodies against human papillomavirus (HPV) based on pseudovirons.

PubMed

Nie, Jianhui; Huang, Weijin; Wu, Xueling; Wang, Youchun

2014-09-01

The pseudoviron-based neutralization assay is accepted as the gold standard to evaluate the functional humoral immune response against HPV. The goal of this study was to develop and optimize a human papillomavirus (HPV) neutralization assay using HPV pseudovirons with Gaussia luciferase (Gluc) as the reporter gene. For this purpose, high-titers Gluc pseudovirons were generated by cotransfecting 293TT cells with HPV structural genes and Gluc expressing plasmids. Six types of neutralizing monoclonal antibodies, vaccines immunized serum samples and WHO international antibody standard were used to validate the new developed assay. The ideal circumstances of the assay were identified for cell counts (30,000/well for 96-well plate), pseudoviron inoculating size (100 times RLU above background) and incubation time (72 hr). The sensitivity of the Gluc assay was comparable to secreted alkaline phosphatase (SEAP) assay and higher than the green florescent protein (GFP) assay. The non-specific background for different types of sample was significantly different (rabbit sera > human sera > mouse sera, P < 0.01). The non-specific neutralization effects were not attributed to IgG antibody. The cutoff value for this assay was determined as 50% inhibition at a dilution of 1:40. Without requirements of sample dilution and different incubation times at different temperature before processing, the detection time was shortened from more than 90 min to less than 5 min for a 96-well plate compared with the SEAP-based assay. With the advantages of short detection time and easy-to-use procedure, the newly developed assay is more suitable for large sero-epidemiological studies or clinical trials and more amenable to automation. © 2014 Wiley Periodicals, Inc.

Hydrogen halides at Comet 67P/Churyumov-Gerasimenko as detected by ROSINA-DFMS

NASA Astrophysics Data System (ADS)

Dhooghe, Frederik

2017-04-01

The Rosetta spacecraft has been studying the coma of comet 67P/Churyumov-Gerasimenko (67P/C-G) in-situ from the comet encounter in August 2014 up to end of mission in September 2016. The Rosetta Orbiter Spectrometer for Ion and Neutral Analysis (ROSINA) contains a double focussing mass spectrometer (DFMS) with a mass range 13-140 u/e. It is optimized for high mass resolution and large dynamic range for the chemical and isotopic characterization of the volatiles in the coma. Since comets retained information about the physical and chemical conditions of the protoplanetary disk from which they formed, they may provide insights into the halogen chemistry in the early Solar System. We have studied the halogen-bearing compounds in the coma with DFMS on 67P/C-G's inbound journey during four periods, as the gas production increased towards perihelion and as the comet's subsolar latitude moved from the northern to the southern hemisphere: (A) when Rosetta was close to the comet, during 1-31/10/2014, at 3.0-3.3 AU, (B) during the close flybys on 14/2/2015 and on 28/3/2015 at 2.3 AU and 2.0 AU, (C) post-equinox between 10/5/2015 and 1/6/2015, at 1.5-1.7 AU, and (D) around perihelion between 9/7/2015 and 31/8/2015, at 1.24-1.31 AU. The main halogen-bearing compounds in the comet atmosphere were found to be the hydrogen halides HF (hydrogen fluoride), HCl (hydrogen chloride) and HBr (hydrogen bromide). HF and HCl could be observed during all four periods, while hydrogen bromide could, due to its low abundance, only be detected during period A, when Rosetta was close to the comet. An increase in the halogen-to-oxygen ratio as a function of distance was observed which suggests a distributed source for HF and HCl, probably through progressive release of these compounds from grains. This contribution will address the abundance and variability of the hydrogen halides in the coma as well as the cometary isotopic ratios for 37Cl/35Cl and 81Br/79Br.

Development of a cell-based qualitative assay for detection of neutralizing anti-human interleukin-1 receptor antagonist (hIL-1Ra) antibodies in rats.

PubMed

Gao, Jin; Li, Jingjing; Yang, Minmin; Wu, Mingyuan; Tu, Ping; Yu, Yan; Han, Wei

2015-01-01

To determine the incidence of the positive neutralizing anti-human interleukin receptor antagonist (anti-IL-1Ra), a novel assay based on the proliferation of human melanoma A375.S2 cells was developed and validated. In the presence of a growth-limiting concentration of IL-1β, A375.S2 cells were able to regain proliferation following the addition of IL-1Ra in a concentration-dependent manner. This dose-response effect enabled the validation of a standard curve for calculation of the concentration of IL-1Ra or, inversely, the concentration of neutralizing anti-IL-1Ra antibodies in cell culture medium or sera. The assay used CCK-8 as an indicator of proliferation. The dose-response relationship between rhIL-1Ra (dose range of 5-75 ng/ml rhIL-1Ra) and A375.S2 cell proliferation was sigmoidal and fitted a four-parameter logistic model. The percent coefficients of variation (%CVs) of quality control samples were 12.5 and 11.9% for intra-assay repeatability and 14.5 and 19.5% for inter-assay repeatability, while the total accuracy was in the range of 97.2-103.6%. For the neutralization assay, the optimal sample dilution factor was found to be 40-fold and the reasonable standard for positive and negative decision was calculated to be 59.4% neutralization rate. The %CVs of quality control samples were 12.7 and 24.0% for intra-assay repeatability and 11.6 and 30.0% for inter-assay repeatability. Analysis using the assay showed that rats could produce neutralizing anti-IL-1Ra antibodies after repeated intramuscular injection with rhIL-1Ra, and this response was not significantly dependent on the dose injected.

A novel method for detecting neutralizing antibodies against therapeutic proteins by measuring gene expression.

PubMed

Yu, Yanbin; Piddington, Christopher; Fitzpatrick, Dan; Twomey, Brian; Xu, Ren; Swanson, Steven J; Jing, Shuqian

2006-10-20

The presence of neutralizing antibodies against protein therapeutics is a concern in the biomedical field. Such antibodies not only reduce the efficacy of protein therapeutics, but also impose potential dangers to the patients receiving them. To date, a small number of in vitro cell-based bioassays for detecting neutralizing antibodies against therapeutic proteins have been developed. Most of the existing assays, however, either involve the use of radioactive materials or have limited sensitivities and/or poor specificities. With advances in mRNA profiling and detection techniques, we have established a novel and non-radioactive bioassay system using branched DNA (bDNA) technology for detecting protein-therapeutic neutralizing antibodies in patient serum. Our assay measures the variations of target gene expression that reflect the biologic effect of the therapeutic agent and the capability of the antibodies, if present, to neutralize the therapeutics. Compared with most existing assays, the new assay is more sensitive and specific, and completely eliminates the use of radioactive materials. Application of the new assay system can be widely expanded if new target genes and responding cell lines for other therapeutics are identified or engineered.

Cometary Plasma Probed by Rosetta

NASA Astrophysics Data System (ADS)

Galand, Marina; Vigren, Erik; Raghuram, Susarla; Schwartz, Steve; Eriksson, Anders; Edberg, Niklas; Lebreton, Jean-Pierre; Henri, Pierre; Burch, Jim; Fuselier, Stephen; Haessig, Myrtha; Mandt, Kathy; Altwegg, Kathrin; Tzou, Chia-You

2015-04-01

In Fall 2014, comet 67P/Churyumov-Gerasimenko, the main target of the Rosetta mission, was at 3 AU from the Sun. Its outgassing rate was only of the order of 5×1025 s-1 based on Rosetta Orbiter Spectrometer for Ion and Neutral Analysis (ROSINA) / Cometary Pressure Sensor (COPS). Despite such a thin coma, a plasma of cometary origin has been detected by Rosetta Plasma Consortium (RPC) sensors and ROSINA/ Double Focusing Mass Spectrometer (DFMS). Close to the comet they have revealed the presence of a cometary ionosphere, with a hot electron population, consistent with the deposition of Extreme UltraViolet (EUV) solar radiation. We will present a comparison between RPC sensors and an energy deposition model in terms of suprathermal electron intensities [RPC/ Ion and Electron Sensor (IES)] and electron temperature and density [RPC/ LAngmuir Probe (LAP) and RPC/ Mutual Impedance Probe (MIP)]. We will also compare ion composition among the main species, between our ionospheric model and ROSINA/DFMS. We will discuss effects of the space environment on the cometary plasma. Finally, we will highlight any evolution in the cometary plasma as the comet is getting closer to perihelion.

Signature of Metallic ion in the upper atmosphere of Mars following the passage of comet C/2013 A1 (Siding Spring)

NASA Astrophysics Data System (ADS)

Benna, M.; Grebowsky, J. M.; Mahaffy, P. R.; Plane, J. M. C.; Yelle, R. V.; Jakosky, B. M.

2017-09-01

The Mars Atmosphere and Volatile EvolutioN (MAVEN) mission made the first in situ detection of metal ions in the upper atmosphere of Mars. These ions result from the ablation of dust particles from comet Siding Spring. This detection was carried out by the Neutral Gas and Ion Mass Spectrometer (NGIMS) on board the MAVEN spacecraft. Metal ions of Na, Mg, Al, K, Ti, Cr, Mn, Fe, Co, Ni, Cu, and Zn, and possibly of Si, and Ca, were identified in the ion spectra collected at altitudes of 185 km. The measurements revealed that Na ion was the most abundant species, and that the remaining metals were depleted with respect to the CI (type 1 carbonaceous Chondrites) abundance of Na ion.

Detection of CN Emission from (2060) Chiron.

PubMed

Bus, S J; A'hearn, M F; Schleicher, D G; Bowell, E

1991-02-15

In the past decade there has been a gradual, but substantial change in our understanding of the physical nature of (2060) Chiron. Once thought to be the first known member of a population of asteroids orbiting between Saturn and Uranus, Chiron is now generally regarded as the largest known comet. The detection of CN emission in the spectrum of Chiron is reported. Not only do these observations underscore the cometary nature of Chiron, but, at a heliocentric distance exceeding 11 astronomical units, represent the most distant detection yet of a neutral gas species common in comets. These results are consistent with the outgassing from Chiron being primarily driven by isolated outbursts of CO(2) from a very small fraction of Chiron's surface. These may be indicative of primordial inhomogeneities.

Laboratory Research. [spectroscopic analysis, photochemical reactions, and proton irradiation of ice

NASA Technical Reports Server (NTRS)

Donn, B.

1981-01-01

To properly interpret the rapidly growing body of data from comet observations, many types of laboratory measurements are needed. These include: (1) molecular spectroscopy in the visible, ultraviolet, infrared and microwave region of the spectra; (2) laser fluorescent spectroscopy of photofragments; (3) laboratory cross-section or reaction rate measurements using flow tube techniques, fluorescent spectroscopy detection for neutrals and ion-molecule reaction techniques; (4) experiments to simulate solar-wind interactions with comets; (5) studies of the properties and behavior of ice mixtures; (6) experiments on the sublimation rate of ice, and the phase transition from amorphous to crystalline ice; (7) investigations of the irradiation of ice; and (8) the electron impact dissociation and excitation of molecules of cometary interest. A nearly completed experiment on the proton irradiation of ice is described.

The ESA mission to Comet Halley

NASA Technical Reports Server (NTRS)

Reinhard, R.

1981-01-01

The Europeon Space Agency's approximately Giotto mission plans for a launch in July 1985 with a Halley encounter in mid-March 1986 4 weeks after the comet's perihelion passage. Giotto carries 10 scientific experiments, a camera, neutral, ion and dust mass spectrometers, a dust impact detector system, various plasma analyzers, a magnetometer and an optical probe. The instruments are described, the principles on which they are based are described, and the experiment key performance data are summarized. The launch constraints the helicentric transfer trajectory, and the encounter scenario are analyzed. The Giotto spacecraft major design criteria, spacecraft subsystem and the ground system are described. The problem of hypervelocity dust particle impacts in the innermost part of the coma, the problem of spacecraft survival, and the adverse effects of impact-generated plasma aroung the spacecraft are considered.

Radiometric cytolysis inhibition assay, a new rapid test for neutralizing antibodies to intact and trypsin-cleaved poliovirus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hovi, T.; Roivainen, M.

1989-04-01

We have developed a new rapid test, the radiometric cytolysis inhibition assay (RACINA), for the determination of neutralizing poliovirus antibodies. HeLa cells prelabeled with /sup 51/Cr, (/sup 3/H)leucine, or, preferentially, with (/sup 3/H)uridine are used as sensitive quantitative indicators of residual infectious virus. Both suspensions and monolayer cultures of the indicator cells can be used. Neutralization of a fraction of a high-titer virus preparation can be scored after the first replication cycle at 8 to 10 h. By lowering the incubation temperature to 30/degree/C, the completion of the cytolysis due to the first replication cycle of poliovirus was delayed beyondmore » 21 h. This makes it possible to use the RACINA, unlike the standard microneutralization assay, for measuring antibodies to trypsin-cleaved polioviruses. The RACINA was found to be as sensitive as and more reproducible than the standard microneutralization assay in the measurement of neutralizing poliovirus antibodies. The RACINA is a rapid and reliable test for neutralizing antibodies and in principle it may be applicable for quantitation of neutralizing antibodies to other cytolytic agents as well.« less

Effects of ozone exposure on human epithelial adenocarcinoma and normal fibroblasts cells.

PubMed

Poma, Anna; Colafarina, Sabrina; Aruffo, Eleonora; Zarivi, Osvaldo; Bonfigli, Antonella; Di Bucchianico, Sebastiano; Di Carlo, Piero

2017-01-01

Previous studies show variable ozone cytotoxicity and genotoxicity in cell cultures, laboratory animals and humans directly exposed to tropospheric ozone. The aim of this study was therefore to investigate and compare the cyto and genotoxic effects of ozone using adenocarcinoma human alveolar basal epithelial cells A549 and normal human fibroblasts Hs27. A cell culture chamber with controlled atmosphere (a simulation reactor) was built to inject a flow of 120 ppb of ozone, which is two times the threshold value for the protection of human health, fixed by the EU legislation. Cell proliferation was evaluated by a luminescent cell viability assay while we assessed the genotoxic potential of ozone by the induction of micronuclei as well as evaluating DNA strand breaks by the induction of micronuclei evaluated by means of the cytokinesis-block micronucleus (CBMN) assay as well as evaluating DNA strand breaks by Alkaline Comet Assay (CA) or Comet Assay. A549 cells viability decreases significantly at 24 hours treatment with 120 ppb of O3 while at 48 hours and 72 hours O3 treated cells viability doesn't differ in respect to the control. However a significative decrease of A549 viability is shown at 72 hours vs. 48 hours in both treated and not-treated cells. The viability trend in the Hs27 cells did not show any significant changes in treated samples compared to the control in all conditions. The two genotoxicity biomarkers, the micronucleus and the comet tests, showed in both the cell types exposed to ozone, a significant increase in the number of micronuclei and in the tail DNA % in respect to the control even if at different times/cell type. Moreover, we found that O3 provokes genotoxic effects more evident in A549 cancer cells than in normal fibroblasts Hs27 ones. We applied a cell growth simulation model referred to ozone treated or not cell lines to confirm that the ozone exposure causes a slackening in the cells replication.

LabKey Server NAb: A tool for analyzing, visualizing and sharing results from neutralizing antibody assays

PubMed Central

2011-01-01

Background Multiple types of assays allow sensitive detection of virus-specific neutralizing antibodies. For example, the extent of antibody neutralization of HIV-1, SIV and SHIV can be measured in the TZM-bl cell line through the degree of luciferase reporter gene expression after infection. In the past, neutralization curves and titers for this standard assay have been calculated using an Excel macro. Updating all instances of such a macro with new techniques can be unwieldy and introduce non-uniformity across multi-lab teams. Using Excel also poses challenges in centrally storing, sharing and associating raw data files and results. Results We present LabKey Server's NAb tool for organizing, analyzing and securely sharing data, files and results for neutralizing antibody (NAb) assays, including the luciferase-based TZM-bl NAb assay. The customizable tool supports high-throughput experiments and includes a graphical plate template designer, allowing researchers to quickly adapt calculations to new plate layouts. The tool calculates the percent neutralization for each serum dilution based on luminescence measurements, fits a range of neutralization curves to titration results and uses these curves to estimate the neutralizing antibody titers for benchmark dilutions. Results, curve visualizations and raw data files are stored in a database and shared through a secure, web-based interface. NAb results can be integrated with other data sources based on sample identifiers. It is simple to make results public after publication by updating folder security settings. Conclusions Standardized tools for analyzing, archiving and sharing assay results can improve the reproducibility, comparability and reliability of results obtained across many labs. LabKey Server and its NAb tool are freely available as open source software at http://www.labkey.com under the Apache 2.0 license. Many members of the HIV research community can also access the LabKey Server NAb tool without installing the software by using the Atlas Science Portal (https://atlas.scharp.org). Atlas is an installation of LabKey Server. PMID:21619655

Early Activity of Churyumov-Gerasimenko: ROSINA/RTOF Results

NASA Astrophysics Data System (ADS)

Wurz, P.; Altwegg, K.; Balsiger, H. R.; Gasc, S.; Galli, A.; Rubin, M.; Jäckel, A.; Le Roy, L.; Calmonte, U.; Tzou, C. Y.; Mall, U. A.; Korth, A.; Fiethe, B.; De Keyser, J. M.; Berthelier, J. J.; Rème, H.; Gombosi, T. I.; Fuselier, S.

2014-12-01

The European Space Agency's Rosetta spacecraft is now close to the comet 67P/Churyumov-Gerasimenko (67P/C-G). On board is the Rosetta Orbiter Spectrometer for Ion and Neutral Analysis (ROSINA) instrument suite. ROSINA consists of two mass spectrometers, the Double Focusing Mass Spectrometer (DFMS) and the Reflectron-type Time-Of-Flight (RTOF), as well as the COmet Pressure Sensor (COPS). The first signal with ROSINA/RTOF of the gaseous environment of the comet was a significant increase in water density observed on DOY 218.1 of 2014 (at 3.5 AU) by RTOF above the gaseous envelope of the Rosetta spacecraft. A similar density increase is observed by COPS at the same time. A preliminary analysis shows that the water density is nH2O ≈ 1012 m-3 at 100 km distance from the comet (located at 3.5 AU from the Sun). This gives a density at the surface of nH2O ≈ 6.4·1015 m-3 and a vertical column density of water of NCH2O ≈ 6.5·1018 m-2. Assuming an active area of 4% we arrive at a production rate of QH2O ≈ 5.8·1024 mole s-1. These values are preliminary and will be refined by forthcoming observations. Other than water, no signal related to cometary activity could be observed above the molecular background from the spacecraft at present, e.g. cometary CO and CO2 are not observed in the RTOF data so far. This hints at a possible deficiency of carbon bearing compounds in the comet.

Coelomocyte biomarkers in the earthworm Eisenia fetida exposed to 2,4,6-trinitrotoluene (TNT).

PubMed

Fuchs, Julio; Piola, Lucas; González, Elio Prieto; Oneto, María Luisa; Basack, Silvana; Kesten, Eva; Casabé, Norma

2011-04-01

Contamination by 2,4,6-trinitrotoluene (TNT) is a global environmental problem at sites of former explosive production, handling, or storage, and could have deleterious consequences for human and ecological health. We investigated its sublethal effects to Eisenia fetida, using two nonspecific biomarkers. In coelomocytes of earthworms exposed 24, 48, or 72 h, we evaluated DNA damage (comet assay) and neutral red retention time (NRRT), using the filter paper contact test. Both percentage of damage (D%) and calculated damage index showed significant DNA damage at almost all concentrations, at all time points assayed. Along exposure time, two different patterns were observed. At the lower TNT concentrations (0.25-0.5 μg/cm2) an increased DNA migration at 48 h, with a decrease close to initial levels after 72 h exposure, was observed. This decrease could be attributed to activation of the DNA repair system. At higher concentrations (1.0-2.0 μg/cm2), the high DNA damage observed remained constant during the 72 h exposure, suggesting that the rate of DNA repair was not enough to compensate such damage. Analysis of NRRT results showed a significant interaction between time and treatment. After 48 h, a significant decrease was observed at 4.0 μg/cm2. After 72 h, NRRT presented a concentration-dependent decrease, significantly different with respect to control at 0.5, 1.0, 2.0, and 4.0 μg/cm2. The two assayed methods, performed on the same sample, showed clear responses to sublethal TNT exposure in E. fetida, providing sensitive unspecific biomarkers of cell injury and DNA damage.

Post-thaw sperm characteristics following long-term storage of boar semen in liquid nitrogen.

PubMed

Fraser, L; Strzeżek, J; Kordan, W

2014-06-30

This study investigated the effect of long-term liquid nitrogen storage of semen from individual boars on post-thaw sperm characteristics. Ejaculates, collected from five Polish large white (PLW) and five Polish landrace (PLR) boars, were frozen using a standard cryopreservation protocol. Post-thaw analysis was performed within a week (Period 1) and 42-48 months (Period 2) of semen storage in liquid nitrogen. Post-thaw sperm assessments included total motility, mitochondrial function (JC-1/PI assay), plasma membrane integrity (SYBR-14/PI assay), osmotic resistance test (ORT), lipid peroxidation (LPO) status and DNA fragmentation, analysed by the neutral Comet assay. Individual boar variability within breed and cryostorage periods had significant effects on the analysed parameters of frozen-thawed spermatozoa. Prolonged semen storage in liquid nitrogen (Period 2) induced a marked reduction in post-thaw sperm motility, mitochondrial function and plasma membrane integrity in most of the boars. Post-thaw semen of eight boars exhibited a marked decrease in osmotic resistance of the sperm acrosomal membrane, whereas a significant increase in the sperm cryo-susceptibility to induced LPO and DNA fragmentation was observed only in three boars after long-term semen storage. Additionally, frozen-thawed spermatozoa of PLR boars exhibited significantly lower osmotic resistance of the acrosomal membrane than PLW boars following prolonged semen storage in liquid nitrogen. The results of this study provide evidence of ageing processes in frozen-thawed boar spermatozoa following prolonged cryostorage. It seems that, even though cryopreservation allows long-term semen storage in liquid nitrogen, spermatozoa from individual boars are more susceptible to cryo-induced damage. Copyright © 2014 Elsevier B.V. All rights reserved.

DNA DAMAGE AND EXTERNAL LESIONS IN BROWN BULLHEAD FROM CONTAMINATED HABITATS

EPA Science Inventory

The single cell gel electrophoresis ("Comet") assay was used to compare levels of DNA damage in brown bullheads (Ameiurus nebulosus) collected from three known contaminated locations, the Cuyahoga River, Ashtabula River, and Ashumet Pond (Cape Cod), with brown bullheads collected...

The effects of urbanization on Lepomis macrochirus using the comet assay

EPA Science Inventory

Urbanization has been linked to increased concentrations of polycyclic aromatic hydrocarbons in natural waterways. This study was designed to examine the impact of urbanization and a wastewater treatment plant by investigating the impact on field-collected bluegill (Lepomis macr...

A Microplate Growth Inhibition Assay for Screening Bacteriocins against Listeria monocytogenes to Differentiate Their Mode-of-Action.

PubMed

Vijayakumar, Paul Priyesh; Muriana, Peter M

2015-06-11

Lactic acid bacteria (LAB) have historically been used in food fermentations to preserve foods and are generally-recognized-as-safe (GRAS) by the FDA for use as food ingredients. In addition to lactic acid; some strains also produce bacteriocins that have been proposed for use as food preservatives. In this study we examined the inhibition of Listeria monocytogenes 39-2 by neutralized and non-neutralized bacteriocin preparations (Bac+ preps) produced by Lactobacillus curvatus FS47; Lb. curvatus Beef3; Pediococcus acidilactici Bac3; Lactococcus lactis FLS1; Enterococcus faecium FS56-1; and Enterococcus thailandicus FS92. Activity differences between non-neutralized and neutralized Bac+ preps in agar spot assays could not readily be attributed to acid because a bacteriocin-negative control strain was not inhibitory to Listeria in these assays. When neutralized and non-neutralized Bac+ preps were used in microplate growth inhibition assays against L. monocytogenes 39-2 we observed some differences attributed to acid inhibition. A microplate growth inhibition assay was used to compare inhibitory reactions of wild-type and bacteriocin-resistant variants of L. monocytogenes to differentiate bacteriocins with different modes-of-action (MOA) whereby curvaticins FS47 and Beef3, and pediocin Bac3 were categorized to be in MOA1; enterocins FS92 and FS56-1 in MOA2; and lacticin FLS1 in MOA3. The microplate bacteriocin MOA assay establishes a platform to evaluate the best combination of bacteriocin preparations for use in food applications as biopreservatives against L. monocytogenes.

A Microplate Growth Inhibition Assay for Screening Bacteriocins against Listeria monocytogenes to Differentiate Their Mode-of-Action

PubMed Central

Vijayakumar, Paul Priyesh; Muriana, Peter M.

2015-01-01

Lactic acid bacteria (LAB) have historically been used in food fermentations to preserve foods and are generally-recognized-as-safe (GRAS) by the FDA for use as food ingredients. In addition to lactic acid; some strains also produce bacteriocins that have been proposed for use as food preservatives. In this study we examined the inhibition of Listeria monocytogenes 39-2 by neutralized and non-neutralized bacteriocin preparations (Bac+ preps) produced by Lactobacillus curvatus FS47; Lb. curvatus Beef3; Pediococcus acidilactici Bac3; Lactococcus lactis FLS1; Enterococcus faecium FS56-1; and Enterococcus thailandicus FS92. Activity differences between non-neutralized and neutralized Bac+ preps in agar spot assays could not readily be attributed to acid because a bacteriocin-negative control strain was not inhibitory to Listeria in these assays. When neutralized and non-neutralized Bac+ preps were used in microplate growth inhibition assays against L. monocytogenes 39-2 we observed some differences attributed to acid inhibition. A microplate growth inhibition assay was used to compare inhibitory reactions of wild-type and bacteriocin-resistant variants of L. monocytogenes to differentiate bacteriocins with different modes-of-action (MOA) whereby curvaticins FS47 and Beef3, and pediocin Bac3 were categorized to be in MOA1; enterocins FS92 and FS56-1 in MOA2; and lacticin FLS1 in MOA3. The microplate bacteriocin MOA assay establishes a platform to evaluate the best combination of bacteriocin preparations for use in food applications as biopreservatives against L. monocytogenes. PMID:26111195

DNA damage evaluation of hydroxyapatite on fibroblast cell L929 using the single cell gel electrophoresis assay.

PubMed

Rajab, N F; Yaakob, T A; Ong, B Y; Hamid, M; Ali, A M; Annuar, B O; Inayat-Hussain, S H

2004-05-01

Hydroxyapatite is the main component of the bone which is a potential biomaterial substance that can be applied in orthopaedics. In this study, the biocompatibility of this biomaterial was assessed using an in vitro technique. The cytotoxicity and genotoxicity effect of HA2 and HA3 against L929 fibroblast cell was evaluated using the MTT Assay and Alkaline Comet Assay respectively. Both HA2 and HA3 compound showed low cytotoxicity effect as determined using MTT Assay. Cells viability following 72 hours incubation at maximum concentration of both HA2 and HA3 (200 mg/ml) were 75.3 +/- 8.8% and 86.7 +/- 13.1% respectively. However, the cytotoxicity effect of ZnSO4.7H2O as a positive control showed an IC50 values of 46 mg/ml (160 microM). On the other hand, both HA2 and HA3 compound showed a slight genotoxicity effect as determined using the Alkaline Comet Assay following incubation at the concentration 200 mg/ml for 72 hours. This assay has been widely used in genetic toxicology to detect DNA strand breaks and alkali-labile site. The percentage of the cells with DNA damage for both substance was 27.7 +/- 1.3% and 15.6 +/- 1.0% for HA2 and HA3 respectively. Incubation of the cells for 24 hours with 38 microg/ml (IC25) of positive control showed an increase in percentage of cells with DNA damage (67.5 +/- 0.7%). In conclusion, our study indicated that both hydroxyapatite compounds showed a good biocompatibility in fibroblast cells.

HPLC-DAD-ESI-MS/MS analysis of fruits from Firmiana simplex (L.) and evaluation of their antioxidant and antigenotoxic properties.

PubMed

Ghareeb, Mosad Ahmed; Mohamed, Tamer; Saad, Amal Mohamed; Refahy, Laila Abdel-Ghany; Sobeh, Mansour; Wink, Michael

2018-01-01

The secondary metabolites of the fruits of Firmiana simplex (L.) were analysed by LC-DAD-ESI-MS/MS; furthermore, we evaluated their antioxidant and antigenotoxic properties. The antioxidant activity was investigated using the 2,2'-diphenyl-1-picrylhydrazyl radical (DPPH), the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) and the ferric reducing antioxidant power (FRAP) assays. The antigenotoxic potential was determined via the comet assay. The ethyl acetate fraction (EtOAc) was analysed by LC-DAD-ESI-MS/MS: phenolic acids and flavonoids were the main polyphenols of the fruits. The EtOAc fraction yielded the highest content of polyphenols with 314.61 mg GAE/g extract, followed by 297.51, 153.75, 101.47, 97.19 for dichloromethane, butanol, methanol and water extracts, respectively. As expected, a strong correlation exists between the antioxidant activity of the investigated extracts and their total phenolic content. In the DPPH assay, the IC 50 value of the most active EtOAc fraction was 6.79 μg/ml, relative to 2.92 μg/ml of the standard ascorbic acid. ABTS and FRAP assays supported the results of DPPH assay. Moreover, using the comet assay, we could show that the phenol-rich EtOAc extract exhibits an antigenotoxic potential in human liver cancer cells (Hep-G2) treated with hydrogen peroxide (H 2 O 2 ) as a genotoxic agent. The fruits of Firmiana simplex may be a good natural source of antioxidant and antigenotoxic agents. © 2017 Royal Pharmaceutical Society.

Assessment of the in vitro cytotoxicity and in vivo anti-tumor activity of the alcoholic stem bark extract/fractions of Mimusops elengi Linn.

PubMed

Kumar, Harish; Savaliya, Mihir; Biswas, Subhankar; Nayak, Pawan G; Maliyakkal, Naseer; Manjunath Setty, M; Gourishetti, Karthik; Pai, K Sreedhara Ranganath

2016-08-01

Various parts of Mimusops elengi Linn. (Sapotaceae) have been used widely in traditional Indian medicine for the treatment of pain, inflammation and wounds. The study was conducted to explore the use of stem bark of M. elengi on pharmacological grounds and to evaluate the scientific basis of cytotoxic and anti-tumor activity. Extract/fractions were prepared and in vitro cytotoxicity was assessed using SRB assay. Most effective fractions were subjected to fluorescence microscopy based acridine orange/ethidium bromide (AO/EB) and Hoechst 33342 staining to determine apoptosis induction and DNA fragmentation assay. Comet and micronuclei assay were performed to assess genotoxicity. Cell cycle analysis was also performed. In vivo anti-tumor potential was evaluated by Ehrlich ascites carcinoma (EAC) model in mice. The alcoholic stem bark extract of M. elengi along with four fractions showed potential in vitro cytotoxicity in SRB assay. Of these, dichloromethane and ethyl acetate fractions were selected for further studies. The fractions revealed apoptosis inducing potential in AO/EB and Hoechst 33342 staining, which was further confirmed by DNA fragmentation assay. Genotoxic potential was revealed by comet and micronuclei assay. Fractions also exhibited specific cell cycle inhibition in G0/G1 phase. In EAC model, ethyl acetate fraction along with the standard (cisplatin) effectively reduced the increase in body weight compared to control and improved mean survival time. Both fractions were able to restore the altered hematological and biochemical parameters. Hence, M. elengi stem bark may be a possible therapeutic candidate having cytotoxic and anti-tumor potential.

Genotoxic effect of Physalis angulata L. (Solanaceae) extract on human lymphocytes treated in vitro.

PubMed

Alves dos Santos, Raquel; Cabral, Teresinha Rosa; Cabral, Isabel Rosa; Antunes, Lusânia Maria; Pontes Andrade, Cristiane; Cerqueira dos Santos Cardoso, Plínio; de Oliveira Bahia, Marcelo; Pessoa, Claudia; Martins do Nascimento, José Luis; Rodríguez Burbano, Rommel; Takahashi, Catarina Satie

2008-08-01

Physalis angulata L (Solanaceae) is a medicinal plant from North of Brazil, whose different extracts and infusions are commonly used in the popular medicine for the treatment of malaria, asthma, hepatitis, dermatitis and rheumatism. However, the genotoxic effects of P. angulata on human cells is not well known. The main purpose of the present study was to evaluate the in vitro genotoxic effects of aqueous extract of P. angulata using the comet assay and the micronucleus assay in human lymphocytes provided from 6 healthy donors. Treatments with P. angulata extracts were performed in vitro in order to access the extent of DNA damage. The comet assay has shown that treatments with P. angulata at 0.5, 1.0, 2.0, 3.0 and 6.0 microg/mL in culture medium were genotoxic. Lymphocytes treated with P. angulata at the concentrations of 3.0 and 6.0 microg/mL in culture medium showed a statistically significant increase in the frequency of micronucleus (p<0.05), however, the cytokinesis blocked proliferation index (CBPI) was not decreased after P. angulata treatment. In conclusion, the present work demonstrated the genotoxic effects of P. angulata extract on human lymphocytes in vitro.

Seahorse (Hippocampus reidi) as a bioindicator of crude oil exposure.

PubMed

Delunardo, Frederico Augusto Cariello; de Carvalho, Luciano Rodrigues; da Silva, Bruno Ferreira; Galão, Michel; Val, Adalberto Luís; Chippari-Gomes, Adriana R

2015-07-01

This study explored the suitability of the seahorse Hippocampus reidi (Ginsburg, 1933) for assessing biomarkers of genotoxic effects and its use as a sentinel organism to detect the effects of acute exposure to petroleum hydrocarbons. Fish were exposed to three concentrations of crude oil (10, 20 and 30 g/kg) for 96 h, and the activity of phase II biotransformation enzyme glutathione S-transferase (GST) was measured. In addition, we performed genotoxicity assays, such as comet assay, micronucleus (MN) test and nuclear abnormalities (NA) induction, on the erythrocytes of the fish species. Our results revealed that the inhibition of hepatic GST activity in H. reidi was dependent on increasing crude oil concentrations. In contrast, an increase in the damage index (DI) and MN frequency were observed with increased crude oil concentrations. These results indicate that the alkaline comet assay and micronucleus test were suitable and useful in the evaluation of the genotoxicity of crude oil, which could improve determinations of the impact of oil spills on fish populations. In addition, H. reidi is a promising "sentinel organism" to detect the genotoxic impact of petroleum hydrocarbons. Copyright © 2015 Elsevier Inc. All rights reserved.

Evaluation of genotoxic potential of avarol, avarone, and its methoxy and methylamino derivatives in prokaryotic and eukaryotic test models.

PubMed

Kolarević, Stoimir; Milovanović, Dragana; Kračun-Kolarević, Margareta; Kostić, Jovana; Sunjog, Karolina; Martinović, Rajko; Đorđević, Jelena; Novaković, Irena; Sladić, Dušan; Vuković-Gačić, Branka

2018-01-04

In this study, mutagenic and genotoxic potential of anti-tumor compounds avarol, avarone, and its derivatives 3'-methoxyavarone, 4'-(methylamino)avarone and 3'-(methylamino)avarone was evaluated and compared to cytostatics commonly used in chemotherapy (5-fluorouracil, etoposid, and cisplatin). Mutagenic potential of selected hydroquinone and quinones was assessed in prokaryotic model by the SOS/umuC assay in Salmonella typhimurium TA1535/pSK1002. Genotoxic potential was also assessed in eukaryotic models using comet assay in human fetal lung cell line (MRC-5), human adenocarcinoma epithelial cell line (A549), and in human peripheral blood cells (HPBC). The results indicated that avarol and avarone do not exert mutagenic/genotoxic potential. Among the studied avarone derivatives, mutagenic potential was detected by SOS/umuC test for 3'-(methylamino)avarone, but only after metabolic activation. The results of comet assay indicated that 3'-methoxyavarone and 3'-(methylamino)avarone have a significant impact on the level of DNA damage in the MRC-5 cell line. Genotoxic potential was not observed in A549 cells or HPBC probably due to a different uptake rate for the compounds and lower in metabolism rate within these cells.

First cytotoxic, genotoxic, and antigenotoxic assessment of Euterpe oleracea fruit oil (açaí) in cultured human cells.

PubMed

Marques, E S; Tsuboy, M S F; Carvalho, J C T; Rosa, P C P; Perazzo, F F; Gaivão, I O M; Maistro, E L

2017-08-17

Euterpe oleracea Mart., popularly known as "açaí", is a tropical fruit from the Amazon region where it has considerable economic importance. Açaí has been used as food and for several medicinal purposes. Despite the widespread use of this fruit, there is a lack of data regarding the safety of using this fruit oil exclusively. Therefore, we evaluated the in vitro cytotoxic, genotoxic, and antigenotoxic effects of E. oleracea fruit oil (EOO) in cultured human lymphocytes (non-metabolizing cells) and HepG2 cell line (human hepatoma) (metabolizing cells) by using MTT, comet, and micronucleus assays. A wide range of EOO concentrations was tested with a preliminary MTT assay, which allowed selecting five concentrations for comet and micronucleus assays: 2.5, 10, 100, 500, and 1000 µg/mL. The results showed that none of the EOO tested concentrations presented cytotoxic effects. The genotoxic assessment revealed an absence of significant DNA and chromosome damage in human lymphocytes and HepG2 cells but did not show chemoprotection against the DNA damage induced by methyl methanesulfonate and benzo[a]pyrene, used as DNA-damaging agents.

Sleep duration is associated with sperm chromatin integrity among young men in Chongqing, China.

PubMed

Wang, Xiaogang; Chen, Qing; Zou, Peng; Liu, Taixiu; Mo, Min; Yang, Huan; Zhou, Niya; Sun, Lei; Chen, Hongqiang; Ling, Xi; Peng, Kaige; Ao, Lin; Yang, Huifang; Cao, Jia; Cui, Zhihong

2017-10-09

This study explores whether sleep duration is associated with sperm chromatin integrity. To do so, we conducted a three-phase panel study of 796 male volunteers from colleges in Chongqing (China) from 2013 to 2015. Sleep duration was measured using a modified Munich Chronotype Questionnaire. Sperm DNA integrity was examined via Sperm Chromatin Structure Assay and Comet assay. Setting 7-7.5 h day -1 of sleep duration as a reference, either longer or shorter sleep duration was associated negatively with high DNA stainability (HDS) (P = 0.009), which reflected the immaturity of sperm chromatin. The volunteers with > 9.0 h day -1 sleep and those with ≤ 6.5 h day -1 sleep had 40.7 and 30.3% lower HDS than did volunteers with 7-7.5 h day -1 sleep. No association was found between sleep duration and DNA fragmentation index or Comet assay parameters. This study suggests that sleep duration is associated with sperm chromatin integrity. Further studies are required to validate these findings and investigate the mechanism underlying this association. © 2017 European Sleep Research Society.

Evaluation the urban atmospheric conditions in different cities using comet and micronuclei assay in Tradescantia pallida.

PubMed

Sposito, Juliana Caroline Vivian; Crispim, Bruno do Amaral; Romãn, Amanda Izadora; Mussury, Rosilda Mara; Pereira, Joelson Gonçalves; Seno, Leonardo Oliveira; Grisolia, Alexeia Barufatti

2017-05-01

In the present study, genotoxicity and mutagenicity were investigated in Tradescantia pallida exposed to vehicular traffic at different sites in a high-altitude tropical climate. During March, May, July, September, and November 2014, a comet assay and micronucleus bioassays were conducted on young inflorescences and leaves of T. pallida collected from twelve towns in the southern region of Mato Grosso do Sul with different amounts of vehicular traffic. Weather parameters (temperature, relative humidity and rainfall) were measured and vehicles were counted to determine traffic levels in each town. A higher frequency of genotoxic and mutagenic damage was observed in the municipality of Dourados. The highest frequency of genetic damage was observed in September and November according to both assays. Relative humidity and rainfall were inversely proportional to the frequency of genetic damage in T. pallida during the collection period. Based on these results, we conclude that the bioassays are efficient for assessing the effects of vehicular traffic in these towns with respect to weather conditions over time. These bioassays can be applied to identify risk areas, which are determined by climatic conditions and air pollutants released. Copyright © 2017 Elsevier Ltd. All rights reserved.

Genotoxic risk identification of soil contamination at a major industrialized city in northeast China by a combination of in vitro and in vivo bioassays.

PubMed

Xiao, Rui-Yang; Wang, Zijian; Wang, Chun-Xia; Yu, Guo; Zhu, Yong-Guan

2006-10-01

The present study evaluated the genotoxicity of field soils in the Tianjin area, one of the most industrialized contaminated areas in northeast China. The genotoxicity of organic extracts of 41 soils was assayed by an in vitro SOS/ umu bioassay with Salmonella typhimurium TA 1535/pSK 1002. From the 41 soil samples, 11 samples were selected to confirm the genotoxic effect by in vivo single-cell gel electrophoresis (comet assay) using earthworms (Eisenia fetida). The results obtained demonstrated that, in the in vitro assay, genotoxicity expressed as induction ratios (IR) ranged from 1.00 to 4.60, and in the in vivo assay, the genotoxicity expressed as tail moment (TM) varied from 14.6 to 57.8 microm. All samples with high genotoxicity assessed by the SOS/umu bioassay possessed significantly high genotoxic effects in the comet assay, and there was a correlation (R2 = 0.736, p < 0.05) between IR and TM in both bioassays. It is concluded that soils in the Tianjin area were seriously contaminated by organic genotoxicants and higher levels of genotoxic effects existed in soils in the urban area of Tianjin as well as in areas near the coastal towns in the northeast part of the city. It can be concluded that a combination of in vivo and in vitro bioassays as a powerful and efficient genotoxicity-assessing tool could facilitate the assessment of genotoxic risk at a regional scale.

Effect of drinking water disinfection by-products in human peripheral blood lymphocytes and sperm.

PubMed

Ali, Aftab; Kurzawa-Zegota, Malgorzata; Najafzadeh, Mojgan; Gopalan, Rajendran C; Plewa, Michael J; Anderson, Diana

2014-12-01

Drinking water disinfection by-products (DBPs) are generated by the chemical disinfection of water and may pose hazards to public health. Two major classes of DBPs are found in finished drinking water: haloacetic acids (HAAs) and trihalomethanes (THMs). HAAs are formed following disinfection with chlorine, which reacts with iodide and bromide in the water. Previously the HAAs were shown to be cytotoxic, genotoxic, mutagenic, teratogenic and carcinogenic. To determine the effect of HAAs in human somatic and germ cells and whether oxidative stress is involved in genotoxic action. In the present study both somatic and germ cells have been examined as peripheral blood lymphocytes and sperm. The effects of three HAA compounds: iodoacetic acid (IAA), bromoacetic acid (BAA) and chloroacetic acid (CAA) were investigated. After determining appropriate concentration responses, oxygen radical involvement with the antioxidants, butylated hydroxanisole (BHA) and the enzyme catalase, were investigated in the single cell gel electrophoresis (Comet) assay under alkaline conditions, >pH 13 and the micronucleus assay. In the Comet assay, BHA and catalase were able to reduce DNA damage in each cell type compared to HAA alone. In the micronucleus assay, micronuclei (MNi) were found in peripheral lymphocytes exposed to all three HAAs and catalase and BHA were in general, able to reduce MNi induction, suggesting oxygen radicals play a role in both assays. These observations are of concern to public health since both human somatic and germ cells show similar genotoxic responses. Copyright © 2014. Published by Elsevier B.V.

Production of Furin-cleaved Papillomavirus Pseudovirions and their use for in vitro neutralization assays of L1 or L2-specific antibodies

PubMed Central

Wang, Joshua W; Matsui, Ken; Pan, Yuanji; Kwak, Kihyuck; Peng, Shiwen; Kemp, Troy; Pinto, Ligia; Roden, Richard B.S

2015-01-01

Immunization with Human Papillomavirus (HPV) L1 virus-like particles or L2 capsid protein elicits neutralizing antibodies that mediate protection. A high throughput and sensitive in vitro neutralization assay is therefore valuable for prophylactic HPV vaccine studies. Over several hours during infection of the genital tract, virions take on a distinct intermediate conformation, including a required furin cleavage of L2 at its N-terminus. This intermediate is an important target for neutralization by L2-specific antibody, but it is very transiently exposed during in vitro infection of most cell lines resulting in insensitive measurement for L2, but not L1-specific neutralizing antibodies. To model this intermediate, we describe a protocol to generate furin-cleaved HPV pseudovirions (fc-PsV) which deliver an encapsidated reporter plasmid to facilitate infectivity measurements. We also describe a protocol for use of fc-PsV in a high throughput in vitro neutralization assay for the sensitive measurement of both L1 and L2-specific neutralizing antibodies. PMID:26237105

An innovative and highly drug-tolerant approach for detecting neutralizing antibodies directed to therapeutic antibodies.

PubMed

Sloan, John H; Conway, Richard G; Pottanat, Thomas G; Troutt, Jason S; Higgs, Richard E; Konrad, Robert J; Qian, Yue-Wei

2016-10-01

Immunogenicity testing of biotherapeutic drugs is a regulatory requirement. Herein, we describe a drug-tolerant assay for detecting neutralizing antibodies against a therapeutic antibody. Excess target of the therapeutic antibody was incorporated into the detection step of an affinity capture elution assay. Signal generated from binding of antidrug antibody (ADA) to the therapeutic antibody was compared with signal from binding of ADA to the therapeutic antibody preincubated with its target. The results demonstrated that the target blocked binding of the therapeutic antibody to neutralizing monkey ADA and to two anti-idiotypic antibodies. This highly drug-tolerant novel approach enables the detection of neutralizing antibodies and allows for one basic assay format to achieve complete characterization of ADA responses.

Optimization and Validation of a Plaque Reduction Neutralization Test for the Detection of Neutralizing Antibodies to Four Serotypes of Dengue Virus Used in Support of Dengue Vaccine Development

PubMed Central

Timiryasova, Tatyana M.; Bonaparte, Matthew I.; Luo, Ping; Zedar, Rebecca; Hu, Branda T.; Hildreth, Stephen W.

2013-01-01

A dengue plaque reduction neutralization test (PRNT) to measure dengue serotype–specific neutralizing antibodies for all four virus serotypes was developed, optimized, and validated in accordance with guidelines for validation of bioanalytical test methods using human serum samples from dengue-infected persons and persons receiving a dengue vaccine candidate. Production and characterization of dengue challenge viruses used in the assay was standardized. Once virus stocks were characterized, the dengue PRNT50 for each of the four serotypes was optimized according to a factorial design of experiments approach for critical test parameters, including days of cell seeding before testing, percentage of overlay carboxymethylcellulose medium, and days of incubation post-infection to generate a robust assay. The PRNT50 was then validated and demonstrated to be suitable to detect and measure dengue serotype-specific neutralizing antibodies in human serum samples with acceptable intra-assay and inter-assay precision, accuracy/dilutability, specificity, and with a lower limit of quantitation of 10. PMID:23458954

Cytotoxicity and Genotoxicity Assessment of Sandalwood Essential Oil in Human Breast Cell Lines MCF-7 and MCF-10A.

PubMed

Ortiz, Carmen; Morales, Luisa; Sastre, Miguel; Haskins, William E; Matta, Jaime

2016-01-01

Sandalwood essential oil (SEO) is extracted from Santalum trees. Although α-santalol, a main constituent of SEO, has been studied as a chemopreventive agent, the genotoxic activity of the whole oil in human breast cell lines is still unknown. The main objective of this study was to assess the cytotoxic and genotoxic effects of SEO in breast adenocarcinoma (MCF-7) and nontumorigenic breast epithelial (MCF-10A) cells. Proteins associated with SEO genotoxicity were identified using a proteomics approach. Commercially available, high-purity, GC/MS characterized SEO was used to perform the experiments. The main constituents reported in the oil were (Z)-α-santalol (25.34%), (Z)-nuciferol (18.34%), (E)-β-santalol (10.97%), and (E)-nuciferol (10.46%). Upon exposure to SEO (2-8 μg/mL) for 24 hours, cell proliferation was determined by the MTT assay. Alkaline and neutral comet assays were used to assess genotoxicity. SEO exposure induced single- and double-strand breaks selectively in the DNA of MCF-7 cells. Quantitative LC/MS-based proteomics allowed identification of candidate proteins involved in this response: Ku70 (p = 1.37E - 2), Ku80 (p = 5.8E - 3), EPHX1 (p = 3.3E - 3), and 14-3-3ζ (p = 4.0E - 4). These results provide the first evidence that SEO is genotoxic and capable of inducing DNA single- and double-strand breaks in MCF-7 cells.

Cytotoxicity and Genotoxicity Assessment of Sandalwood Essential Oil in Human Breast Cell Lines MCF-7 and MCF-10A

PubMed Central

Ortiz, Carmen; Morales, Luisa; Sastre, Miguel; Haskins, William E.; Matta, Jaime

2016-01-01

Sandalwood essential oil (SEO) is extracted from Santalum trees. Although α-santalol, a main constituent of SEO, has been studied as a chemopreventive agent, the genotoxic activity of the whole oil in human breast cell lines is still unknown. The main objective of this study was to assess the cytotoxic and genotoxic effects of SEO in breast adenocarcinoma (MCF-7) and nontumorigenic breast epithelial (MCF-10A) cells. Proteins associated with SEO genotoxicity were identified using a proteomics approach. Commercially available, high-purity, GC/MS characterized SEO was used to perform the experiments. The main constituents reported in the oil were (Z)-α-santalol (25.34%), (Z)-nuciferol (18.34%), (E)-β-santalol (10.97%), and (E)-nuciferol (10.46%). Upon exposure to SEO (2–8 μg/mL) for 24 hours, cell proliferation was determined by the MTT assay. Alkaline and neutral comet assays were used to assess genotoxicity. SEO exposure induced single- and double-strand breaks selectively in the DNA of MCF-7 cells. Quantitative LC/MS-based proteomics allowed identification of candidate proteins involved in this response: Ku70 (p = 1.37E − 2), Ku80 (p = 5.8E − 3), EPHX1 (p = 3.3E − 3), and 14-3-3ζ (p = 4.0E − 4). These results provide the first evidence that SEO is genotoxic and capable of inducing DNA single- and double-strand breaks in MCF-7 cells. PMID:27293457

Sodium hypochlorite-, chlorine dioxide- and peracetic acid-induced genotoxicity detected by the Comet assay and Saccharomyces cerevisiae D7 tests.

PubMed

Buschini, Annamaria; Carboni, Pamela; Furlini, Mariangela; Poli, Paola; Rossi, Carlo

2004-03-01

Mutagenicity of drinking water is due not only to industrial, agricultural and urban pollution but also to chlorine disinfection by-products. Furthermore, residual disinfection is used to provide a partial safeguard against low level contamination and bacterial re-growth within the distribution system. The aims of this study were to further evaluate the genotoxic potential of the world wide used disinfectants sodium hypochlorite and chlorine dioxide in human leukocytes by the Comet assay and in Saccharomyces cerevisiae strain D7 (mitotic gene conversion, point mutation and mitochondrial DNA mutability, with and without endogenous metabolic activation) and to compare their effects with those of peracetic acid, proposed as an alternative disinfectant. All three disinfectants are weakly genotoxic in human leukocytes (lowest effective dose 0.2 p.p.m. for chlorine dioxide, 0.5 p.p.m. for sodium hypochlorite and peracetic acid). The results in S.cerevisiae show a genotoxic response on the end-points considered with an effect only at doses higher (5- to 10-fold) than the concentration normally used for water disinfection; sodium hypochlorite and peracetic acid are able to induce genotoxic effects without endogenous metabolic activation (in stationary phase cells) whereas chlorine dioxide is effective in growing cells. The Comet assay was more sensitive than the yeast tests, with effective doses in the range normally used for water disinfection processes. The biological effectiveness of the three disinfectants on S.cerevisiae proved to be strictly dependent on cell-specific physiological/biochemical conditions. All the compounds appear to act on the DNA and peracetic acid shows effectiveness similar to sodium hypochlorite and chlorine dioxide.

Comet assay and micronucleus test in circulating erythrocytes of Cyprinus carpio specimens exposed in situ to lake waters treated with disinfectants for potabilization.

PubMed

Buschini, A; Martino, A; Gustavino, B; Monfrinotti, M; Poli, P; Rossi, C; Santoro, M; Dörr, A J M; Rizzoni, M

2004-02-14

The detection of a possible genotoxic effect of surface water treated with disinfectants for potabilization is the aim of the present work. The Comet assay and the micronucleus test were applied in circulating erythrocytes of Cyprinus carpio. Young specimens (20-30 g) were exposed in experimental basins, built within the potabilization plant of Castiglione del Lago (Perugia, Italy). In this plant the water of the Trasimeno Lake is treated and disinfected for potabilization before it is distributed to the people in the net of drinkable water. A continuous flow of water at a constant rate was supplied to basins; the water was continuously treated at a constant concentration with one of the three tested disinfectants (sodium hypochlorite, peracetic acid and chloride dioxide), one control basin being supplied with untreated water. Three sampling campaigns were performed: October 2000, February 2001 and June 2001. Repeated blood samplings through intracardiac punctures allowed to follow the same fish populations after different exposure times: before introduction of the disinfectant, and 10 or 20 days afterwards. An additional blood sampling was performed 3 h after addition of the disinfectant in other, simultaneously exposed, fish populations. Genotoxic damage was shown in fish exposed to water disinfected with sodium hypochlorite and chloride dioxide. The Comet assay showed an immediate response, i.e. DNA damage that was induced directly in circulating erythrocytes, whereas micronuclei reached their highest frequencies at later sampling times, when a genotoxic damage in stem cells of the cephalic kidney is expressed in circulating erythrocytes. The quality of the untreated surface water seems to be the most important parameter for the long-term DNA damage in circulating erythrocytes.

Explanation for excessive DNA single-strand breaks and endogenous repair foci in pluripotent mouse embryonic stem cells.

PubMed

Banáth, J P; Bañuelos, C A; Klokov, D; MacPhail, S M; Lansdorp, P M; Olive, P L

2009-05-01

Pluripotent mouse embryonic stem cells (mES cells) exhibit approximately 100 large gammaH2AX repair foci in the absence of measurable numbers of DNA double-strand breaks. Many of these cells also show excessive numbers of DNA single-strand breaks (>10,000 per cell) when analyzed using the alkaline comet assay. To understand the reasons for these unexpected observations, various methods for detecting DNA strand breaks were applied to wild-type mES cells and to mES cells lacking H2AX, ATM, or DNA-PKcs. H2AX phosphorylation and expression of other repair complexes were measured using flow and image analysis of antibody-stained cells. Results indicate that high numbers of endogenous gammaH2AX foci and single-strand breaks in pluripotent mES cells do not require ATM or DNA-PK kinase activity and appear to be associated with global chromatin decondensation rather than pre-existing DNA damage. This will limit applications of gammaH2AX foci analysis in mES cells to relatively high levels of initial or residual DNA damage. Excessive numbers of single-strand breaks in the alkaline comet assay can be explained by the vulnerability of replicating chromatin in mES cells to osmotic shock. This suggests that caution is needed in interpreting results with the alkaline comet assay when applied to certain cell types or after treatment with agents that make chromatin vulnerable to osmotic changes. Differentiation of mES cells caused a reduction in histone acetylation, gammaH2AX foci intensity, and DNA single-strand breakage, providing a link between chromatin structural organization, excessive gammaH2AX foci, and sensitivity of replicating mES cell chromatin to osmotic shock.

Biomonitoring of genotoxicity of industrial fertilizer pollutants in Aiolopus thalassinus (Orthoptera: Acrididae) using alkaline comet assay.

PubMed

Abdelfattah, Eman A; Augustyniak, Maria; Yousef, Hesham A

2017-09-01

Phosphate fertilizer industry is considered as one of the main sources of environmental pollutants. Besides solid waste products, e.g. phosphates, sulphates, and heavy metals, also atmospheric pollutants, such as hydrofluoric acid fumes (HF), sulphur dioxide (SO 2 ), nitrogen oxides (NO 2 ), and particulate matter with diameter up to 10 μm (PM 10 ) can be dangerous. Genotoxic effect of these pollutants was monitored by assessing the DNA damage using alkaline comet assay on cells from brain, thoracic muscles and gut of Aiolopus thalassinus collected at three sites (A-C) located at 1, 3, and 6 km away from Abu-Zaabal Company for Fertilizers and Chemical Industries. Control site was established 32 km from the source of pollution, at the Cairo University Campus. The level of the DNA damage was significantly higher in insects from polluted sites comparing to that from the control site. A strong negative correlation between percentage of cells with visible DNA damage (% of severed cells) and the distance of the sites from Abu-Zaabal Company was found. The best parameter for monitoring of fertilizer pollutants is % of severed cells. Possible impact of Abu-Zaabal Company (extremely high concentration of phosphates and sulphates in all the polluted sites) on DNA integrity in A. thalassinus tissues was discussed. The potential use of the comet assay as a biomonitoring method of the environmental pollution caused by fertilizer industry was proposed. Specific pollution resulting from the activity of the fertilizer industry can cause comparable adverse effects in the organisms inhabiting areas up to 6 km from the source of contamination. Copyright © 2017 Elsevier Ltd. All rights reserved.

ORIGIN OF MOLECULAR OXYGEN IN COMET 67P/CHURYUMOV–GERASIMENKO

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mousis, O.; Ronnet, T.; Brugger, B.

2016-06-01

Molecular oxygen has been detected in the coma of comet 67P/Churyumov–Gerasimenko with abundances in the 1%–10% range by the Rosetta Orbiter Spectrometer for Ion and Neutral Analysis-Double Focusing Mass Spectrometer instrument on board the Rosetta spacecraft. Here we find that the radiolysis of icy grains in low-density environments such as the presolar cloud may induce the production of large amounts of molecular oxygen. We also show that molecular oxygen can be efficiently trapped in clathrates formed in the protosolar nebula (PSN), and that its incorporation as crystalline ice is highly implausible, because this would imply much larger abundances of Armore » and N{sub 2} than those observed in the coma. Assuming that radiolysis has been the only O{sub 2} production mechanism at work, we conclude that the formation of comet 67P/Churyumov–Gerasimenko is possible in a dense and early PSN in the framework of two extreme scenarios: (1) agglomeration from pristine amorphous icy grains/particles formed in ISM and (2) agglomeration from clathrates that formed during the disk’s cooling. The former scenario is found consistent with the strong correlation between O{sub 2} and H{sub 2}O observed in comet 67P/Churyumov-Gerasimenko’s coma while the latter scenario requires that clathrates formed from ISM icy grains that crystallized when entering the PSN.« less

Measurement of neutralizing serum antibodies of patients vaccinated with human papillomavirus L1 or L2-based immunogens using furin-cleaved HPV Pseudovirions.

PubMed

Wang, Joshua W; Jagu, Subhashini; Wang, Chenguang; Kitchener, Henry C; Daayana, Sai; Stern, Peter L; Pang, Susana; Day, Patricia M; Huh, Warner K; Roden, Richard B S

2014-01-01

Antibodies specific for neutralizing epitopes in either Human papillomavirus (HPV) capsid protein L1 or L2 can mediate protection from viral challenge and thus their accurate and sensitive measurement at high throughput is likely informative for monitoring response to prophylactic vaccination. Here we compare measurement of L1 and L2-specific neutralizing antibodies in human sera using the standard Pseudovirion-Based Neutralization Assay (L1-PBNA) with the newer Furin-Cleaved Pseudovirion-Based Neutralization Assay (FC-PBNA), a modification of the L1-PBNA intended to improve sensitivity towards L2-specific neutralizing antibodies without compromising assay of L1-specific responses. For detection of L1-specific neutralizing antibodies in human sera, the FC- PBNA and L1-PBNA assays showed similar sensitivity and a high level of correlation using WHO standard sera (n = 2), and sera from patients vaccinated with Gardasil (n = 30) or an experimental human papillomavirus type 16 (HPV16) L1 VLP vaccine (n = 70). The detection of L1-specific cross-neutralizing antibodies in these sera using pseudovirions of types phylogenetically-related to those targeted by the L1 virus-like particle (VLP) vaccines was also consistent between the two assays. However, for sera from patients (n = 17) vaccinated with an L2-based immunogen (TA-CIN), the FC-PBNA was more sensitive than the L1-PBNA in detecting L2-specific neutralizing antibodies. Further, the neutralizing antibody titers measured with the FC-PBNA correlated with those determined with the L2-PBNA, another modification of the L1-PBNA that spacio-temporally separates primary and secondary receptor engagement, as well as the protective titers measured using passive transfer studies in the murine genital-challenge model. In sum, the FC-PBNA provided sensitive measurement for both L1 VLP and L2-specific neutralizing antibody in human sera. Vaccination with TA-CIN elicits weak cross-protective antibody in a subset of patients, suggesting the need for an adjuvant.

Genotoxicity of drinking water disinfection by-products (bromoform and chloroform) by using both Allium anaphase-telophase and comet tests.

PubMed

Khallef, Messaouda; Liman, Recep; Konuk, Muhsin; Ciğerci, İbrahim Hakkı; Benouareth, Djameleddine; Tabet, Mouna; Abda, Ahlem

2015-03-01

Genotoxic effects of bromoform and chloroform, disinfection by-products of the chlorination of drinking water, were examined by using mitotic index (MI), mitotic phase, chromosome aberrations (CAs) and comet assay on root meristematic cells of Allium cepa. Different concentrations of bromoform (25, 50, 75 and 100 μg/mL) and chloroform (25, 50, 100 and 200 μg/mL) were introduced to onion tuber roots. Distilled water was used as a negative control and methyl methansulfonate (MMS-10 μg/mL) as positive control. All obtained data were subjected to statistical analyses by using SPSS 15.0 for Windows software. For comparison purposes, Duncan multiple range tests by using one-way analysis of variance were employed and p < 0.05 was accepted as significant value. Exposure of both chemicals (except 25 μg/mL applications of bromoform) significantly decreased MI. Bromoform and chloroform (except 25 μg/mL applications) increased total CAs in Allium anaphase-telophase test. A significant increase in DNA damage was also observed at all concentrations of both bromoform and chloroform examined by comet assay. The damages were higher than that of positive control especially at 75-100 μg/mL for bromoform and 100-200 μg/mL for chloroform.

Exposure to non-ionizing electromagnetic fields emitted from mobile phones induced DNA damage in human ear canal hair follicle cells.

PubMed

Akdag, Mehmet; Dasdag, Suleyman; Canturk, Fazile; Akdag, Mehmet Zulkuf

2018-01-01

The aim of this study was to investigate effect of radiofrequency radiation (RFR) emitted from mobile phones on DNA damage in follicle cells of hair in the ear canal. The study was carried out on 56 men (age range: 30-60 years old)in four treatment groups with n = 14 in each group. The groups were defined as follows: people who did not use a mobile phone (Control), people use mobile phones for 0-30 min/day (second group), people use mobile phones for 30-60 min/day (third group) and people use mobile phones for more than 60 min/day (fourth group). Ear canal hair follicle cells taken from the subjects were analyzed by the Comet Assay to determine DNA damages. The Comet Assay parameters measured were head length, tail length, comet length, percentage of head DNA, tail DNA percentage, tail moment, and Olive tail moment. Results of the study showed that DNA damage indicators were higher in the RFR exposure groups than in the control subjects. In addition, DNA damage increased with the daily duration of exposure. In conclusion, RFR emitted from mobile phones has a potential to produce DNA damage in follicle cells of hair in the ear canal. Therefore, mobile phone users have to pay more attention when using wireless phones.

Pharmacological and Genotoxic Properties of Polyphenolic Extracts of Cedrela odorata L. and Juglans regia L. Barks in Rodents

PubMed Central

Almonte-Flores, Dulce Carolina; Paniagua-Castro, Norma; Escalona-Cardoso, Gerardo; Rosales-Castro, Martha

2015-01-01

Evaluation of the phenolic compounds and antioxidant activity of Cedrela odorata L. and Juglans regia L. bark extracts was performed in vitro. Juglans regia showed greater extract concentration and higher antioxidant activity. Hypoglycemic activity in rats was assessed by generating a glucose tolerance curve and determining the area under the curve (AUC). Diabetes was later induced by an injection with streptozotocin (65 mg/kg of b.w.) and confirmed after 24 hours. The extract was administered (200 mg/kg b.w.) over 10 days, and blood glucose was monitored and compared with a control group. The glucose AUC showed a hypoglycemic effect of J. regia and C. odorata in normal rats. Both extracts reduced hepatic lipid peroxidation in diabetic rats. Polyphenolic extracts reduced cholesterol levels in a hypercholesterolemic mouse model and decreased hepatic lipid peroxidation. Polyphenolic extract doses of 100 and 200 mg/kg b.w. were administered alone or with cyclophosphamide (CPA) 50 mg/kg ip, which was used as a positive control. Analyses were performed using leukocytes in a comet assay after 4 and 24 h of treatment. Genotoxic effects were evaluated by the comet assay, which showed that while J. regia extract had no effect, C. odorata extract induced slight damage at 200 mg/kg, with the formation of type 0 and 1 comets. PMID:25945104

Serologic evidence of Lyssavirus infections among bats, the Philippines.

PubMed

Arguin, Paul M; Murray-Lillibridge, Kristy; Miranda, Mary E G; Smith, Jean S; Calaor, Alan B; Rupprecht, Charles E

2002-03-01

Active surveillance for lyssaviruses was conducted among populations of bats in the Philippines. The presence of past or current Lyssavirus infection was determined by use of direct fluorescent antibody assays on bat brains and virus neutralization assays on bat sera. Although no bats were found to have active infection with a Lyssavirus, 22 had evidence of neutralizing antibody against the Australian bat lyssavirus (ABLV). Seropositivity was statistically associated with one species of bat, Miniopterus schreibersi. Results from the virus neutralization assays are consistent with the presence in the Philippines of a naturally occurring Lyssavirus related to ABLV.

Serologic Evidence of Lyssavirus Infections among Bats, the Philippines

PubMed Central

Murray-Lillibridge, Kristy; Miranda, Mary E.G.; Smith, Jean S.; Calaor, Alan B.; Rupprecht, Charles E.

2002-01-01

Active surveillance for lyssaviruses was conducted among populations of bats in the Philippines. The presence of past or current Lyssavirus infection was determined by use of direct fluorescent antibody assays on bat brains and virus neutralization assays on bat sera. Although no bats were found to have active infection with a Lyssavirus, 22 had evidence of neutralizing antibody against the Australian bat lyssavirus (ABLV). Seropositivity was statistically associated with one species of bat, Miniopterus schreibersi. Results from the virus neutralization assays are consistent with the presence in the Philippines of a naturally occurring Lyssavirus related to ABLV. PMID:11927022