Science.gov

Sample records for comet assay analysis

  1. OpenComet: an automated tool for comet assay image analysis.

    PubMed

    Gyori, Benjamin M; Venkatachalam, Gireedhar; Thiagarajan, P S; Hsu, David; Clement, Marie-Veronique

    2014-01-01

    Reactive species such as free radicals are constantly generated in vivo and DNA is the most important target of oxidative stress. Oxidative DNA damage is used as a predictive biomarker to monitor the risk of development of many diseases. The comet assay is widely used for measuring oxidative DNA damage at a single cell level. The analysis of comet assay output images, however, poses considerable challenges. Commercial software is costly and restrictive, while free software generally requires laborious manual tagging of cells. This paper presents OpenComet, an open-source software tool providing automated analysis of comet assay images. It uses a novel and robust method for finding comets based on geometric shape attributes and segmenting the comet heads through image intensity profile analysis. Due to automation, OpenComet is more accurate, less prone to human bias, and faster than manual analysis. A live analysis functionality also allows users to analyze images captured directly from a microscope. We have validated OpenComet on both alkaline and neutral comet assay images as well as sample images from existing software packages. Our results show that OpenComet achieves high accuracy with significantly reduced analysis time.

  2. OpenComet: An automated tool for comet assay image analysis

    PubMed Central

    Gyori, Benjamin M.; Venkatachalam, Gireedhar; Thiagarajan, P.S.; Hsu, David; Clement, Marie-Veronique

    2014-01-01

    Reactive species such as free radicals are constantly generated in vivo and DNA is the most important target of oxidative stress. Oxidative DNA damage is used as a predictive biomarker to monitor the risk of development of many diseases. The comet assay is widely used for measuring oxidative DNA damage at a single cell level. The analysis of comet assay output images, however, poses considerable challenges. Commercial software is costly and restrictive, while free software generally requires laborious manual tagging of cells. This paper presents OpenComet, an open-source software tool providing automated analysis of comet assay images. It uses a novel and robust method for finding comets based on geometric shape attributes and segmenting the comet heads through image intensity profile analysis. Due to automation, OpenComet is more accurate, less prone to human bias, and faster than manual analysis. A live analysis functionality also allows users to analyze images captured directly from a microscope. We have validated OpenComet on both alkaline and neutral comet assay images as well as sample images from existing software packages. Our results show that OpenComet achieves high accuracy with significantly reduced analysis time. PMID:24624335

  3. 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

  4. Aspects of design and statistical analysis in the Comet assay.

    PubMed

    Wiklund, Stig Johan; Agurell, Eva

    2003-03-01

    Some aspects of the statistical design and analysis of the Comet (single cell gel electrophoresis) assay have been evaluated by means of a simulation study. The tail length and tail moment were selected for the quantification of DNA migration. Results from the simulation study showed that the choice of measure to summarize the cells on each slide is extremely important in order to facilitate an efficient analysis. For tail moment, the mean of log transformed data is clearly superior to the other evaluated measures, whereas using the mean of raw data without transformation can lead to very inefficient analyses. The 90th percentile, capturing the upper tail of the distribution, performs well for the tail length, with a slight improvement obtained by applying a log transformation prior to calculations. Furthermore, the simulation study has been used to assess the appropriateness of some models for statistical analysis and to address the issue of design (i.e. number of cultures or animals in each group, number of slides per animal/culture and number of cells scored per slide). Combining the results from the simulations with practical experience from the pharmaceutical industry, we conclude the paper by providing concise recommendations regarding the design and statistical analysis in the Comet assay.

  5. 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.

  6. Analysis of genotoxic potentiality of stevioside by comet assay.

    PubMed

    Nunes, A P M; Ferreira-Machado, S C; Nunes, R M; Dantas, F J S; De Mattos, J C P; Caldeira-de-Araújo, A

    2007-04-01

    Stevioside is a natural non-caloric sweetener extracted from Stevia rebaudiana (Bertoni) leaves. It has been widely used in many countries, including Japan, Korea, China, Brazil and Paraguay, either as a substitute for sucrose in beverages and foods or as a household sweetener. The aim of this work was to study its genotoxic potentiality in eukaryotic cells. Wistar rats were treated with stevioside solution (4mg/mL) through oral administration (ad libitum) and the DNA-induced damage was evaluated using the single cell gel electrophoresis (comet assay). The results showed that treatment with stevioside generates lesions in peripheral blood, liver, brain and spleen cells in different levels, the largest effect being in liver. Therefore, these undesired effects must be better understood, once the data present here point to possible stevioside mutagenic properties.

  7. 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.

  8. 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.

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

    PubMed Central

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

    2016-01-01

    Introduction 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. Aim 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). Materials and Methods 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. Results 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. Conclusion The current investigation unraveled increased levels of DNA damage in children with non syndromic developmental delay when compared to the controls. PMID:27437200

  10. Comprehensive analysis of sperm DNA fragmentation by five different assays: TUNEL assay, SCSA, SCD test and alkaline and neutral Comet assay.

    PubMed

    Ribas-Maynou, J; García-Peiró, A; Fernández-Encinas, A; Abad, C; Amengual, M J; Prada, E; Navarro, J; Benet, J

    2013-09-01

    Sperm DNA fragmentation (SDF) is becoming an important test to assess male infertility. Several different tests are available, but no consensus has yet been reached as to which tests are most predictive of infertility. Few publications have reported a comprehensive analysis comparing these methods within the same population. The objective of this study was to analyze the differences between the five most common methodologies, to study their correlations and to establish their cut-off values, sensitivity and specificity in predicting male infertility. We found differences in SDF between fertile donors and infertile patients in TUNEL, SCSA, SCD and alkaline Comet assays, but none with the neutral Comet assay. The alkaline COMET assay was the best in predicting male infertility followed by TUNEL, SCD and SCSA, whereas the neutral COMET assay had no predictive power. For our patient population, threshold values for infertility were 20.05% for TUNEL assay, 18.90% for SCSA, 22.75% for the SCD test, 45.37% for alkaline Comet and 34.37% for neutral Comet. This work establishes in a comprehensive study that the all techniques except neutral Comet are useful to distinguish fertile and infertile men.

  11. The Comet-FISH assay for the analysis of DNA damage and repair.

    PubMed

    Spivak, Graciela

    2010-01-01

    In this chapter, I describe the alkaline single-cell gel electrophoresis (Comet assay) combined with fluorescence in situ hybridization (FISH) technology, used in our laboratory, to study the incidence and repair of lesions induced in human cells by ultraviolet light. The Comet-FISH method permits the simultaneous and comparative analysis of DNA damage and its repair throughout the genome and in defined chromosomal regions. This very sensitive approach can be applied to any lesion, such as those induced by chemical carcinogens and products of cellular metabolism that can be converted to DNA single- or double-strand breaks. The unique advantages and limitations of the method for particular applications are discussed.

  12. 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.

  13. 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

  14. Establishment of experimental conditions for preserving samples of fish blood for analysis with both comet assay and flow cytometry.

    PubMed

    Ramsdorf, Wanessa A; Guimarães, Fernando de S F; Ferraro, Marcos V M; Gabardo, Juarez; Trindade, Edvaldo da Silva; Cestari, Marta Margarete

    2009-02-19

    When environmental analysis is performed, the high number of samples required and handling conditions during the transport of these samples to the laboratory are common problems. The comet assay is a useful, highly sensitive tool in biomonitoring. Some studies in the literature aim to preserve slides in lysis solution for use in the comet assay. Until now, however, no efficient methodology for preserving blood samples for this assay has been described. Because of this, the present report aimed to establish the proper conditions for samples maintenance prior to comet assay analysis. Samples were conserved in three different solutions: a high protein concentration solution (fetal bovine serum-FBS), an anticoagulant agent (a calcium chelator - ethylenediaminetetracetic acid - EDTA), and a salt buffered solution (phosphate buffered saline-PBS). Therefore, peripheral blood samples of Rhamdia quelen specimens were collected and maintained in these solutions until testing at 72h. Analyses of DNA fragmentation via the comet assay and cell viability via flow cytometry were performed at intervals of 24h. The results showed that samples maintained in FBS were preserved better; this was followed by those preserved in PBS and then last by those preserved in EDTA. In conclusion, blood samples from freshwater fish can be preserved up to 48h in fetal bovine serum at 4 degrees C in the absence of light. In this period, no DNA fragmentation occurs. We thus describe an excellent method of sample conservation for subsequent analysis in the laboratory.

  15. 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.

  16. The Comet Assay: Automated Imaging Methods for Improved Analysis and Reproducibility

    PubMed Central

    Braafladt, Signe; Reipa, Vytas; Atha, Donald H.

    2016-01-01

    Sources of variability in the comet assay include variations in the protocol used to process the cells, the microscope imaging system and the software used in the computerized analysis of the images. Here we focus on the effect of variations in the microscope imaging system and software analysis using fixed preparations of cells and a single cell processing protocol. To determine the effect of the microscope imaging and analysis on the measured percentage of damaged DNA (% DNA in tail), we used preparations of mammalian cells treated with etoposide or electrochemically induced DNA damage conditions and varied the settings of the automated microscope, camera, and commercial image analysis software. Manual image analysis revealed measurement variations in percent DNA in tail as high as 40% due to microscope focus, camera exposure time and the software image intensity threshold level. Automated image analysis reduced these variations as much as three-fold, but only within a narrow range of focus and exposure settings. The magnitude of variation, observed using both analysis methods, was highly dependent on the overall extent of DNA damage in the particular sample. Mitigating these sources of variability with optimal instrument settings facilitates an accurate evaluation of cell biological variability. PMID:27581626

  17. Analysis of DNA damage using the comet assay in infants fed cow's milk.

    PubMed

    Dündaröz, Ruşen; Ulucan, Hakan; Aydin, Halil Ibrahim; Güngör, Tayfun; Baltaci, Volkan; Denli, Metin; Sanisoğlu, Yavuz

    2003-01-01

    It has been hypothesized that non-human milk feeding may increase the risk for cancer or for a specific cancer or group of cancers as well as the risk for diseases such as type-1 diabetes mellitus and Crohn's disease. Regarding DNA damage leading to cancer development in the absence of human milk protection, a comparison between infants fed human milk and cow's milk has been performed. Each group consisted of 35 infants, whose ages ranged from 9 to 12 months. The level of DNA damage in the peripheral blood lymphocytes of infants has been studied by the comet assay. A significant increase has been found in the number of limited DNA-damaged (p < 0.001) and extensive DNA-damaged (p < 0.001) cells of infants fed cow's milk. To our knowledge, this is the first study using the comet assay on infants not breast-fed. Supporting our previous SCE study, these results suggest that there is some level of DNA damage in the lymphocytes of infants not breast-fed and this may lead to malignancy in childhood or later in life.

  18. 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.

  19. Acellular comet assay: a tool for assessing variables influencing the alkaline comet assay.

    PubMed

    Kennedy, Erin K; McNamee, James P; Prud'homme Lalonde, Louise; Jones, Trevor; Wilkinson, Diana

    2012-01-01

    In this study, an acellular modification to the alkaline comet assay to further evaluate key variables within the assay that may influence the outcome of genotoxicity studies is described. This acellular comet assay can detect differences of 0.2 Gy of (60)Co gamma-ray radiation between 0 and 1 Gy and differences of 1 Gy between 0 and 8 Gy; thus, this assay is applicable for a wide range of DNA damage levels. It is also shown that DNA damage from different radiation energies was not significantly different from (60)Co gamma-ray. This assay displayed a statistical increase in DNA damage due to uncontrolled exposure to natural light; however, the slope of the dose-response curve for light-exposed samples was similar to that for samples protected from light. A comparison of the alkaline comet assay with the acellular comet assay allowed for the intrinsic repair capacity of the alkaline comet assay to be quantified.

  20. Genetic Alterations in Pesticide Exposed Bolivian Farmers: An evaluation by analysis of chromosomal aberrations and the comet assay.

    PubMed

    Jørs, Erik; Gonzáles, Ana Rosa; Ascarrunz, Maria Eugenia; Tirado, Noemi; Takahashi, Catharina; Lafuente, Erika; Dos Santos, Raquel A; Bailon, Natalia; Cervantes, Rafael; O, Huici; Bælum, Jesper; Lander, Flemming

    2007-11-12

    Pesticides are of concern in Bolivia because of increasing use. Frequent intoxications have been demonstrated due to use of very toxic pesticides, insufficient control of distribution and sale and little knowledge among farmers of protective measures and hygienic procedures. Questionnaires were applied and blood tests taken from 81 volunteers from La Paz County, of whom 48 were pesticide exposed farmers and 33 non-exposed controls. Sixty males and 21 females participated with a mean age of 37.3 years (range 17-76). Data of exposure and possible genetic damage were collected and evaluated by well known statistical methods, controlling for relevant confounders. To measure genetic damage chromosomal aberrations and the comet assay analysis were performed. Pesticide exposed farmers had a higher degree of genetic damage compared to the control group. The number of chromosomal aberrations increased with the intensity of pesticide exposure. Females had a lower number of chromosomal aberrations than males, and people living at altitudes above 2500 metres seemed to exhibit more DNA damage measured by the comet assay. Bolivian farmers showed signs of genotoxic damage, probably related to exposure to pesticides. Due to the potentially negative long term health effects of genetic damage on reproduction and the development of cancer, preventive measures are recommended. Effective control with imports and sales, banning of the most toxic pesticides, education and information are possible measures, which could help preventing the negative effects of pesticides on human health and the environment.

  1. The comet assay: a heavenly method!

    PubMed

    Collins, Andrew R

    2015-01-01

    The contributions to this special issue of Mutagenesis have been selected to cover the main research areas served by the comet assay, namely genotoxicology, environmental toxicology, human biomonitoring and fundamental investigations into mechanisms of DNA damage and repair. Innovative methods are described, technical issues are explored, and guidelines are given for venturing into relatively new or unexploited areas of research. The popularity of the comet assay in a historical context is illustrated by a bibliometric survey. © 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.

  2. New Applications of the Comet Assay: Comet-FISH and Transcription-Coupled DNA Repair

    PubMed Central

    Cox, Rachel A.; Hanawalt, Philip C.

    2009-01-01

    Transcription-coupled repair (TCR) is a pathway dedicated to the removal of damage from the template strands of actively transcribed genes. Although the detailed mechanism of TCR is not yet understood, it is believed to be triggered when a translocating RNA polymerase is arrested at a lesion or unusual structure in the DNA. Conventional assays for TCR require high doses of DNA damage for the statistical analysis of repair in the individual strands of DNA sequences ranging in size from a few hundred bases to 30 kb. The single cell gel electrophoresis (Comet) assay allows detection of single-or double-strand breaks at a 10 to 100-fold higher level of resolution. Fluorescence in situ hybridization (FISH) combined with the Comet assay (Comet-FISH) affords a heightened level of sensitivity for the assessment of repair in defined DNA sequences of cells treated with physiologically relevant doses of genotoxins. This approach also reveals localized susceptibility to chromosomal breakage in cells from individuals with hypersensitivity to radiation or chemotherapy. Several groups have reported preferential repair in transcriptionally active genes or chromosomal domains using Comet-FISH. The prevailing interpretation of the behavior of DNA in the Comet assay assumes that the DNA is arranged in loops and matrix-attachment sites; that supercoiled, undamaged loops are contained within the nuclear matrix and appear in Comet “heads”, and that Comet “tails” consist of relaxed DNA loops containing one or more breaks. According to this model, localization of FISH probes in Comet heads signifies that loops containing the targeted sequences are free of damage. This implies that preferential repair as detected by Comet-FISH might encompass large chromosomal domains containing both transcribed and non-transcribed sequences. We review the existing evidence and discuss the implications in relation to current models for the molecular mechanism of TCR. PMID:18291710

  3. 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.

  4. 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

  5. Cytogentic analysis of human dermal fibroblasts (HDFs) in early and late passages using both karyotyping and comet assay techniques.

    PubMed

    Allahbakhshian-Farsani, Mehdi; Abdian, Narges; Ghasemi-Dehkordi, Payam; Sadeghiani, Marzieh; Saffari-Chaleshtori, Javad; Hashemzadeh-Chaleshtori, Morteza; Khosravi-Farsani, Somayeh

    2014-10-01

    Human dermal fibroblasts (HDFs) are a potential source of somatic cells for genetic manipulation and tissue engineering. Confirmation of cytogenetic stability of these cells is an essential step for cell nuclear transfer and generation of a suitable and functional induced pluripotent stem cells line. HDF cells were isolated and cultured from human foreskin samples. Cytogenetic stability of these cells was evaluated in early (3-4) and late (10-15) passages using karyotype test and alkaline comet assay techniques. HDF cells in early and late passages showed normal karyotype but by comet assay abnormality and DNA damages in late passages of HDFs were observed. Also, the parameters of alkaline comet assay in early passages of HDFs compared with late passages and positive control groups more significantly were different (p < 0.05). These findings indicate that single-strand breaks or DNA damage after many passages may have occurred in HDF cells. Our results demonstrate that only early passages of HDF cells maintain cytogenetic stability and are good candidates for gene reprogramming. In conclusion, karyotype testing alone can not be used for detection of all signs of cytogenetic abnormality and DNA damages of cells. So, for precise evaluation of DNA damage and cytogenetic instability of fibroblast cells comet assay and karyotype techniques could complement each other.

  6. 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...

  7. 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...

  8. 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

  9. 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.

  10. Reference cells and ploidy in the comet assay.

    PubMed

    Brunborg, Gunnar; Collins, Andrew; Graupner, Anne; Gutzkow, Kristine B; Olsen, Ann-Karin

    2015-01-01

    In the comet assay single cells are analyzed with respect to their level of DNA damage. Discrimination of the individual cell or cell type based on DNA content, with concomitant scoring of the DNA damage, is useful since this may allow analysis of mixtures of cells. Different cells can then be characterized based on their ploidy, cell cycle stage, or genome size. We here describe two applications of such a cell type-specific comet assay: (i) Testicular cell suspensions, analyzed on the basis of their ploidy during spermatogenesis; and (ii) reference cells in the form of fish erythrocytes which can be included as internal standards to correct for inter-assay variations. With standard fluorochromes used in the comet assay, the total staining signal from each cell - whether damaged or undamaged - was found to be associated with the cell's DNA content. Analysis of the fluorescence intensity of single cells is straightforward since these data are available in scoring systems based on image analysis. The analysis of testicular cell suspensions provides information on cell type specific composition, susceptibility to genotoxicants, and DNA repair. Internal reference cells, either untreated or carrying defined numbers of lesions induced by ionizing radiation, are useful for investigation of experimental factors that can cause variation in comet assay results, and for routine inclusion in experiments to facilitate standardization of methods, and comparison of comet assay data obtained in different experiments or in different laboratories. They can also be used - in combination with a reference curve - to quantify the DNA lesions induced by a certain treatment. Fish cells of a range of genome sizes, both greater and smaller than human, are suitable for this purpose, and they are inexpensive.

  11. Is the Comet Assay a Sensitive Procedure for Detecting Genotoxicity?

    PubMed Central

    Kawaguchi, Satomi; Nakamura, Takanori; Yamamoto, Ayumi; Honda, Gisho; Sasaki, Yu F.

    2010-01-01

    Although the Comet assay, a procedure for quantitating DNA damage in mammalian cells, is considered sensitive, it has never been ascertained that its sensitivity is higher than the sensitivity of other genotoxicity assays in mammalian cells. To determine whether the power of the Comet assay to detect a low level of genotoxic potential is superior to those of other genotoxicity assays in mammalian cells, we compared the results of Comet assay with those of micronucleus test (MN test). WTK1 human lymphoblastoid cells were exposed to methyl nitrosourea (MNU), ethyl nitrosourea (ENU), methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), bleomycin (BLM), or UVC. In Comet assay, cells were exposed to each mutagen with (Comet assay/araC) and without (Comet assay) DNA repair inhibitors (araC and hydroxyurea). Furthermore, acellular Comet assay (acellular assay) was performed to determine how single-strand breaks (SSBs) as the initial damage contributes to DNA migration and/or to micronucleus formation. The lowest genotoxic dose (LGD), which is defined as the lowest dose at which each mutagen causes a positive response on each genotoxicity assay, was used to compare the power of the Comet assay to detect a low level of genotoxic potential and that of MN test; that is, a low LGD indicates a high power. Results are summarized as follows: (1) for all mutagens studied, LGDs were MN test ≦ Comet assay; (2) except for BLM, LGDs were Comet assay/araC ≦ MN test; (3) except for UVC and MNU, LGDs were acellular assayComet assay/araC ≦ MN test ≦ Comet assay. The following is suggested by the present findings: (1) LGD in the Comet assay is higher than that in MN test, which suggests that the power of the MN test to detect a low level of genotoxic potential is superior to that of the Comet assay; (2) for the studied mutagens, all assays were able to detect all mutagens correctly, which suggests that the sensitivity of the Comet assay and that of the MN test were

  12. Controlling variation in the comet assay

    PubMed Central

    Collins, Andrew R.; El Yamani, Naouale; Lorenzo, Yolanda; Shaposhnikov, Sergey; Brunborg, Gunnar; Azqueta, Amaya

    2014-01-01

    Variability of the comet assay is a serious issue, whether it occurs from experiment to experiment in the same laboratory, or between different laboratories analysing identical samples. Do we have to live with high variability, just because the comet assay is a biological assay rather than analytical chemistry? Numerous attempts have been made to limit variability by standardizing the assay protocol, and the critical steps in the assay have been identified; agarose concentration, duration of alkaline incubation, and electrophoresis conditions (time, temperature, and voltage gradient) are particularly important. Even when these are controlled, variation seems to be inevitable. It is helpful to include in experiments reference standards, i.e., cells with a known amount of specific damage to the DNA. They can be aliquots frozen from a single large batch of cells, either untreated (negative controls) or treated with, for example, H2O2 or X-rays to induce strand breaks (positive control for the basic assay), or photosensitiser plus light to oxidize guanine (positive control for Fpg- or OGG1-sensitive sites). Reference standards are especially valuable when performing a series of experiments over a long period—for example, analysing samples of white blood cells from a large human biomonitoring trial—to check that the assay is performing consistently, and to identify anomalous results necessitating a repeat experiment. The reference values of tail intensity can also be used to iron out small variations occurring from day to day. We present examples of the use of reference standards in human trials, both within one laboratory and between different laboratories, and describe procedures that can be used to control variation. PMID:25368630

  13. 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

  14. Toxicity effect of dichlorvos on loach (Misgurnus anguillicaudatus) assessed by micronucleus test, hepatase activity analysis and comet assay.

    PubMed

    Nan, Ping; Yan, Shuaiguo; Li, Li; Chen, Jianjun; Du, Qiyan; Chang, Zhongjie

    2015-06-01

    Pesticides and other chemicals at environmental concentrations often have detrimental effects. Many aquatic species are particularly threatened because of their susceptibility and also because water environment are often polluted. This study preliminarily evaluated the toxicity effect of dichlorvos (DDVP) on loach (Misgurnus anguillicaudatus) using the methods of micronucleus (MN) test, hepatase activity and comet assay. The tested results showed that indeed very little DDVP had strong toxicity effect on loach and its 50% lethal concentration (LC50) at 24 h, 48 h and 96 h was 8.38 μg l(-1), 7.168 μg l(-1) and 6.411 μg l(-1), respectively; The glutamic-pyruvic transaminase (GPT) and glutamic-oxalacetic transaminase (GOT) activity of loach liver decreased; meanwhile, the GPT and GOT activity of loach serum, the MN rate (‰) and three comet parameters of tested fish increased with the increase in the treatment concentration and treatment time of DDVP, and there was significant difference between control group and each treatment group (p < 0.05). These results suggested that DDVP residues might become toxic chemical contaminant in environment and would threaten aquatic and other organisms.

  15. 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

    2017-03-04

    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.

  16. 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

  17. 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

  18. 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.

  19. Single Cell Analysis of Human RAD18-Dependent DNA Post-Replication Repair by Alkaline Bromodeoxyuridine Comet Assay

    PubMed Central

    Mórocz, Mónika; Gali, Himabindu; Raskó, István; Downes, C. Stephen; Haracska, Lajos

    2013-01-01

    Damage to DNA can block replication progression resulting in gaps in the newly synthesized DNA. Cells utilize a number of post-replication repair (PRR) mechanisms such as the RAD18 controlled translesion synthesis or template switching to overcome the discontinuities formed opposite the DNA lesions and to complete DNA replication. Gaining more insights into the role of PRR genes promotes better understanding of DNA damage tolerance and of how their malfunction can lead to increased genome instability and cancer. However, a simple and efficient method to characterise gene specific PRR deficiencies at a single cell level has not been developed. Here we describe the so named BrdU comet PRR assay to test the contribution of human RAD18 to PRR at a single cell level, by which we kinetically characterized the consequences of the deletion of human RAD18 on the replication of UV-damaged DNA. Moreover, we demonstrate the capability of our method to evaluate PRR at a single cell level in unsynchronized cell population. PMID:23936422

  20. 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.

  1. 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.

  2. 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.

  3. 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

  4. The study of comets, part 1. [conference on photometry and spectrum analysis of Kohoutek comet and comet tails

    NASA Technical Reports Server (NTRS)

    Donn, B. (Editor); Mumma, M. J. (Editor); Jackson, W. M. (Editor); Ahearn, M. (Editor); Harrington, R. (Editor)

    1976-01-01

    Papers are presented dealing with observations of comets. Topic discussed include: photometry, polarimetry, and astrometry of comets; detection of water and molecular transitions in comets; ion motions in comet tails; determination of comet brightness and luminosity; and evolution of cometary orbits. Emphasis is placed on analysis of observations of comet Kohoutek.

  5. Alkaline and neutral Comet assay profiles of sperm DNA damage in clinical groups.

    PubMed

    Ribas-Maynou, J; García-Peiró, A; Abad, C; Amengual, M J; Navarro, J; Benet, J

    2012-03-01

    The analysis of sperm DNA fragmentation has become a new marker to predict male infertility, and many techniques have been developed. The sperm Comet assay offers the possibility of differentiating single- and double-stranded DNA (ssDNA and dsDNA) breaks, which could have different effects on fertility. The objective of this study was to perform a descriptive characterization of different groups of patients, such as those with asthenoteratozoospermic (ATZ) with or without varicocele, oligoasthenoteratozoospermic (OATZ) or balanced chromosome rearrangements, as compared with fertile donors. The Comet assay was used to investigate sperm samples for ssDNA and dsDNA breaks. The analysis of alkaline and neutral Comet assays in different groups of patients showed different sperm DNA damage profiles. Most fertile donors presented low values for ssDNA and dsDNA fragmentation (low-equivalent Comet profile), which would be the best prognosis for achieving a pregnancy. OATZ, ATZ and ATZ with varicocele presented high percentages of ssDNA and dsDNA fragmentation (high-equivalent Comet assay profile), ATZ with varicocele being associated with the worst prognosis, due to higher levels of DNA fragmentation. Rearranged chromosome carriers display a very high variability and, interestingly, two different profiles were seen: a high-equivalent Comet assay profile, which could be compatible with a bad prognosis, and a non-equivalent Comet assay profile, which has also been found in three fertile donors. Comet assay profiles, applied to different clinical groups, may be useful for determining prognosis in cases of male infertility.

  6. 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

  7. Re-analysis results using medians of the data from the JaCVAM-organized international validation study of the in vivo rat alkaline comet assay.

    PubMed

    Uno, Yoshifumi; Omori, Takashi

    2015-07-01

    The data from the JaCVAM-organized international validation study of the in vivo rat alkaline comet assay were reported and analyzed statistically using the simple means of % tail DNA. However, OECD test guideline TG 489 recommends use of the median for data analysis due to the hierarchical nature of the data. Comparison between the simple mean approach and the median based approach for positive/negative/equivocal chemical calls was conducted using the % tail DNA data for the 40 chemicals tested in the JaCVAM-organized international validation study of the in vivo rat alkaline comet assay, using liver and stomach as target organs. In the liver, two genotoxic chemicals, o-anisidine and 9-aminoacridine hydrochloride monohydrate, were positive using the median based approach but negative using the simple mean approach, and two genotoxic chemicals, 2-acetylaminofluorene and busulfan were equivocal using the median based approach but negative using the simple mean approach. In contrast, cadmium chloride (genotoxic carcinogen) was equivocal in both organs using the median based approach, while positive and equivocal in liver and stomach, respectively, using the simple mean approach. Two data sets of sodium arsenite showed equivocal and negative results for liver using the median based approach, although both data sets were equivocal using the simple mean approach. Overall, there are no large differences in terms of the genotoxic call between both approaches. However, the median based approach recommended in OECD TG 489 has an advantage toward higher precision within the groups treated with a test chemical, whereas the approach might show the lower values for the effect.

  8. 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

  9. 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.

  10. 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.

  11. 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

  12. 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

  13. 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.

  14. Genotoxicity testing: Comparison of the γH2AX focus assay with the alkaline and neutral comet assays.

    PubMed

    Nikolova, Teodora; Marini, Federico; Kaina, Bernd

    2017-10-01

    Genotoxicity testing relies on the quantitative measurement of adverse effects, such as chromosome aberrations, micronuclei, and mutations, resulting from primary DNA damage. Ideally, assays will detect DNA damage and cellular responses with high sensitivity, reliability, and throughput. Several novel genotoxicity assays may fulfill these requirements, including the comet assay and the more recently developed γH2AX assay. Although they are thought to be specific for genotoxicants, a systematic comparison of the assays has not yet been undertaken. In the present study, we compare the γH2AX focus assay with the alkaline and neutral versions of the comet assay, as to their sensitivities and limitations for detection of genetic damage. We investigated the dose-response relationships of γH2AX foci and comet tail intensities at various times following treatment with four prototypical genotoxicants, methyl methanesulfonate (MMS), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), mitomycin C, and hydrogen peroxide (H2O2) and we tested whether there is a correlation between the endpoints, i.e., alkali-labile sites and DNA strand breaks on the one hand and the cell's response to DNA double-strand breaks and blocked replication forks on the other. Induction of γH2AX foci gave a linear dose response and all agents tested were positive in the assay. The increase in comet tail intensity was also a function of dose; however, mitomycin C was almost completely ineffective in the comet assay, and the doses needed to achieve a significant effect were somewhat higher for some treatments in the comet assay than in the γH2AX foci assay, which was confirmed by threshold analysis. There was high correlation between tail intensity and γH2AX foci for MMS and H2O2, less for MNNG, and none for mitomycin C. From this we infer that the γH2AX foci assay is more reliable, sensitive, and robust than the comet assay for detecting genotoxicant-induced DNA damage. Copyright © 2017 Elsevier B

  15. 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.

  16. Hummingbird Comet Nucleus Analysis Mission

    NASA Technical Reports Server (NTRS)

    Kojiro, Daniel; Carle, Glenn C.; Lasher, Larry E.

    2000-01-01

    Hummingbird is a highly focused scientific mission, proposed to NASA s Discovery Program, designed to address the highest priority questions in cometary science-that of the chemical composition of the cometary nucleus. After rendezvous with the comet, Hummingbird would first methodically image and map the comet, then collect and analyze dust, ice and gases from the cometary atmosphere to enrich characterization of the comet and support landing site selection. Then, like its namesake, Hummingbird would carefully descend to a pre-selected surface site obtaining a high-resolution image, gather a surface material sample, acquire surface temperature and then immediately return to orbit for detailed chemical and elemental analyses followed by a high resolution post-sampling image of the site. Hummingbird s analytical laboratory contains instrumentation for a comprehensive molecular and elemental analysis of the cometary nucleus as well as an innovative surface sample acquisition device.

  17. 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.

  18. 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.

  19. 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.

  20. Detection of DNA damage by the alkaline comet assay after exposure to low-dose gamma radiation.

    PubMed

    Malyapa, R S; Bi, C; Ahern, E W; Roti Roti, J L

    1998-04-01

    The alkaline comet assay as described by Olive et al. (Exp. Cell Res. 198, 259-267, 1992) was used to detect DNA damage in cells exposed to low doses (0-5 cGy) of gamma radiation. Experiments were performed using lymphocytes isolated from whole blood of rats. The comet parameters, normalized comet moment and comet length, described by Kent et al. (Int. J. Radiat. Biol. 67, 655-660, 1995), were used as measurements of DNA damage. It was observed that the alkaline comet assay can detect DNA damage at doses as low as 0.6 cGy. The results of the experiments using low-dose gamma radiation are comparable with published results obtained using the alkaline comet assay according to the method of Singh et al. (Int. J. Radiat. Biol. 66, 23-28, 1994). Based on this observation and analysis of results published previously, we conclude that the version of the alkaline comet assay described by Olive et al. is as sensitive as other modifications of the comet assay reported in literature for the detection of DNA damage in cells exposed to low doses of ionizing radiation.

  1. DNA ``comet assay'' for rapid detection of irradiated food

    NASA Astrophysics Data System (ADS)

    Delincée, H.

    1996-09-01

    Since treatment with ionizing radiation causes DNA fragmentation, microgel electrophoresis of single cell (``comet assay'') can be applied as a simple and rapid tool for identification of irradiated foods. The DNA ``comet assay'' has been employed successfully in the past to frozen meats (chicken, pork, beef) and its application is now being extended to a variety of other food items, such as fish, fruits, legumes, seeds and even spices. The electrophoretic separation requires only a few minutes, and after visualising DNA by silver staining, the DNA fragmentation pattern observed in a simple transmission microscope may indicate a possible irradiation treatment. Suspected samples may subsequently be analyzed by the more sophisticated (expensive) and validated techniques.

  2. 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

  3. Genotoxicity of environmental agents assessed by the alkaline comet assay.

    PubMed

    Møller, Peter

    2005-01-01

    Generation of DNA damage is considered to be an important initial event in carcinogenesis. A considerable battery of assays exists for the detection of different genotoxic effects of compounds in experimental systems, or for investigations of exposure to genotoxic agents in environmental or occupational settings. Some of the tests may have limited use because of complicated technical setup or because they only are applicable to a few cell types. The single cell gel electrophoresis (comet) assay is technically simple, relatively fast, cheap, and DNA damage can be investigated in virtually all mammalian cell types without requirement for cell culture. The aim of this thesis was to evaluate the comet assay as a genotoxicity test in genetic toxicology of environmental agents, encompassing both experimental animal models and biomonitoring. The comet assay detects strand breaks (SB). The cells are embedded in agarose and lysed, generating nucleus-like structures in the gel (referred to as nucleoids). Following alkaline electrophoresis, the DNA strands migrate toward the anode, and the extent of migration depends on the number of SB in the nucleoid. The migration is visualized and scored in a fluorescence microscope after staining. Broad classes of oxidative DNA damage can be detected as additional SB if nucleoids are incubated with bacterial DNA glycosylase/endonuclease enzymes. Oxidized pyrimidines and purines can be detected by incubation with endonuclease III and formamidopyrimidine DNA glycosylase, respectively. The animal experimental studies indicated that the comet assay was able to detect genotoxic effects of diesel exhaust particles in lung tissue, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)-induced DNA damage in colon epithelial cells and liver tissue, and benzene-induced damage in bone marrow and liver cells. The strength of the comet assay was further outlined by application of repair enzymes, indicating no oxidative DNA base damage following IQ treatment

  4. 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

  5. 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

  6. 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.

  7. 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.

  8. [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

  9. 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.

  10. 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.

  11. Observation of DNA damage of human hepatoma cells irradiated by heavy ions using comet assay

    PubMed Central

    Qiu, Li-Mei; Li, Wen-Jian; Pang, Xin-Yue; Gao, Qing-Xiang; Feng, Yan; Zhou, Li-Bin; Zhang, Gao-Hua

    2003-01-01

    AIM: Now many countries have developed cancer therapy with heavy ions, especially in GSI (Gesellschaft fürSchwerionenforschung mbH, Darmstadt, Germany), remarkable results have obtained, but due to the complexity of particle track structure, the basic theory still needs further researching. In this paper, the genotoxic effects of heavy ions irradiation on SMMC-7721 cells were measured using the single cell gel electrophoresis (comet assay). The information about the DNA damage made by other radiations such as X-ray, γ-ray, UV and fast neutron irradiation is very plentiful, while little work have been done on the heavy ions so far. Hereby we tried to detect the reaction of liver cancer cells to heavy ion using comet assay, meanwhile to establish a database for clinic therapy of cancer with the heavy ions. METHODS: The human hepatoma cells were chosen as the test cell line irradiated by 80Mev/u 20Ne10+ on HIRFL (China), the radiation-doses were 0, 0.5, 1, 2, 4 and 8 Gy, and then comet assay was used immediately to detect the DNA damages, 100-150 cells per dose-sample (30-50 cells were randomly observed at constant depth of the gel). The tail length and the quantity of the cells with the tail were put down. EXCEL was used for statistical analysis. RESULTS: We obtained clear images by comet assay and found that SMMC-7721 cells were all damaged apparently from the dose 0.5 Gy to 8 Gy (t-test: P < 0.001, vs control). The tail length and tail moment increased as the doses increased, and the number of cells with tails increased with increasing doses. When doses were higher than 2 Gy, nearly 100% cells were damaged. Furthermore, both tail length and tail moment, showed linear equation. CONCLUSION: From the clear comet assay images, our experiment proves comet assay can be used to measure DNA damages by heavy ions. Meanwhile DNA damages have a positive correlation with the dose changes of heavy ions and SMMC-7721 cells have a great radiosensitivity to 20Ne10+. Different

  12. 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).

  13. Recommendations for increasing alkaline comet assay reliability in plants.

    PubMed

    Pourrut, Bertrand; Pinelli, Eric; Celiz Mendiola, Vanessa; Silvestre, Jérôme; Douay, Francis

    2015-01-01

    In plants, an increasing interest for the comet assay was shown in the last decade. This versatile technique appears to be promising to detect the genotoxic effect of pollutants and to monitor the environment. However, the lack of a standardised protocol and the low throughput of the assay limit its use in plants. The aims of this paper are to identify key factors affecting comet assay performance and to improve its reliability and reproducibility. We examined the effect of varying several parameters on four different plant species: broad bean (Vicia faba), white clover (Trifolium repens), English ryegrass (Lolium perenne) and miscanthus (Miscanthus x giganteus). The influence of both internal (different nucleus isolation methods, presence or absence of filtration and lysis steps) and external (room temperature, light intensity) parameters were evaluated. Results clearly indicate that short chopping is more efficient to isolate nuclei than the standard slicing method. Filtration and lysis steps were shown to be unnecessary and thus should be skipped. Data also demonstrate that high room temperatures and light could induce DNA damage in isolated nuclei. Calibration tests with H2O2 or ethyl methanesulfonate revealed that a special attention should be paid to plant growing stage, leaf position and exposure duration. © 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.

  14. 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.

  15. Evaluation of DNA damage in Eurasian marsh frogs (Pelophylax ridibundus) by comet assay for determination of possible pollution in the different lakes in central Anatolia, Turkey.

    PubMed

    Erismis, Ugur Cengiz; Ciğerci, İbrahim Hakki; Konuk, Muhsin

    2013-06-01

    In the present study, adult Eurasian marsh frogs, Pelophylax ridibundus, and water samples were collected from a reference lake and three water bodies in central Anatolia, Turkey, to evaluate the water for chemical pollutants and possible effects of pollutants on the DNA of frog erythrocytes by using a comet assay. The results for DNA damage parameters of the comet assay (total comet length, tail intensity, and olive tail moment) and their statistical analysis by ANOVA demonstrated that P. ridibundus and the comet assay together represent an useful approach for the early detection of polluted water bodies.

  16. 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.

  17. Comet assay to measure DNA repair: approach and applications

    PubMed Central

    Azqueta, Amaya; Slyskova, Jana; Langie, Sabine A. S.; O’Neill Gaivão, Isabel; Collins, Andrew

    2014-01-01

    Cellular repair enzymes remove virtually all DNA damage before it is fixed; repair therefore plays a crucial role in preventing cancer. Repair studied at the level of transcription correlates poorly with enzyme activity, and so assays of phenotype are needed. In a biochemical approach, substrate nucleoids containing specific DNA lesions are incubated with cell extract; repair enzymes in the extract induce breaks at damage sites; and the breaks are measured with the comet assay. The nature of the substrate lesions defines the repair pathway to be studied. This in vitro DNA repair assay has been modified for use in animal tissues, specifically to study the effects of aging and nutritional intervention on repair. Recently, the assay was applied to different strains of Drosophila melanogaster proficient and deficient in DNA repair. Most applications of the repair assay have been in human biomonitoring. Individual DNA repair activity may be a marker of cancer susceptibility; alternatively, high repair activity may result from induction of repair enzymes by exposure to DNA-damaging agents. Studies to date have examined effects of environment, nutrition, lifestyle, and occupation, in addition to clinical investigations. PMID:25202323

  18. ISO's analysis of Comet Hale-Bopp

    NASA Astrophysics Data System (ADS)

    1997-03-01

    of the comet's dust and vapour, and also rates of escape of vapour, which will help in assessing the loss of material from Comet Hale-Bopp during this visit to the Sun's vicinity. "Watch out for some fascinating news," says Thijs de Graauw of Groningen University, who is in charge of the SWS instrument used in this study. "What excites me is the opportunity we shall have to compare dusty Comet Hale-Bopp, seen in the Solar System, with dusty objects far away among the stars which seem to be made of similar materials. Infrared astronomy has a special ability to unify cosmic chemistry at all scales from little dust grains in the Earth's vicinity to vast and distant galaxies." The dust itself interests the infrared astronomers, not least because their view of the Universe at large is spoiled to some extent by dust left behind by comets. Together with fine debris from asteroids, the comet dust makes a bright infrared band around the sky, which corresponds with the zodiacal light sometimes seen by eye, slanting above the horizon at twilight. ISO's predecessor, the US-Dutch-UK infrared astronomical satellite IRAS, found trails of comet dust much longer and more persistent than the familiar comet tails. ISO has seen a trail from Comet Kopff. By detecting dust grains that are typically much larger than those seen by visible light, ISO scientists hope to learn more about the dust's long-term behaviour in the Solar System. A series of images of Comet Hale-Bopp, obtained by the camera ISOCAM in October 1996, is the subject of continuing analysis. Leading this work in progress is Philippe Lamy of Marseille, France. "We hope to unveil the nucleus of the comet," Professor Lamy explains. "In principle, the Hubble Space Telescope can see finer details by visible light, but the contrast of the nucleus against the bright surrounding coma is superior at infrared wavelengths. This is because the thermal emission from the nucleus is very large and can be detected thanks to the high

  19. In vivo genotoxic effect of potassium dichromate in mice leukocytes using comet assay.

    PubMed

    Dana Devi, K; Rozati, R; Saleha Banu, B; Jamil, K; Grover, P

    2001-08-01

    Hexavalent chromium is a well-known mutagen and carcinogen. In the present investigation, single-/double-stranded DNA breaks by potassium dichromate (K2Cr2O7) in mice, a sensitive model for genotoxic effects, have been studied in vivo using alkaline single-cell gel electrophoresis (SCGE)/comet assay. Mice were administered orally with a range of doses starting from 0.59 to 76.0 mg/kg body weight of K2Cr2O7 and samples of whole blood were collected at 24, 48, 72, 96 h, week 1 and week 2 post-treatment for alkaline SCGE assay to study DNA damage. The rationale for using leukocytes was to reflect biomarker analysis in humans. Significant increase in mean comet tail length (5.7-24.25 microM) indicating DNA damage was observed at all the doses with K2Cr2O7 when compared with controls (3.26 microM). Maximum increase in mean comet tail length was observed at 9.5 mg/kg body weight at 48 h post-treatment (24.25 microM). The mean comet tail length showed a clear dose-dependent increase from 0.59 to 9.5 mg/kg body weight and a dose-dependent decrease in higher doses (19.0-76.0 mg/kg body weight). A gradual decrease in the tail lengths from 72 h post-treatment was observed by the second week, and values had returned to control levels at all doses, indicating repair of the damaged DNA and/or loss of heavily damaged cells. The study also reveals that comet assay is a sensitive and rapid method for detecting DNA damage caused by heavy metals such as chromium (Cr).

  20. 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.

  1. Inter-laboratory variation in DNA damage using a standard comet assay protocol.

    PubMed

    Forchhammer, Lykke; Ersson, Clara; Loft, Steffen; Möller, Lennart; Godschalk, Roger W L; van Schooten, Frederik J; Jones, George D D; Higgins, Jennifer A; Cooke, Marcus; Mistry, Vilas; Karbaschi, Mahsa; Collins, Andrew R; Azqueta, Amaya; Phillips, David H; Sozeri, Osman; Routledge, Michael N; Nelson-Smith, Kirsty; Riso, Patrizia; Porrini, Marisa; Matullo, Giuseppe; Allione, Alessandra; Stępnik, Maciej; Steepnik, Maciej; Komorowska, Magdalena; Teixeira, João Paulo; Costa, Solange; Corcuera, Laura-Ana; López de Cerain, Adela; Laffon, Blanca; Valdiglesias, Vanessa; Møller, Peter

    2012-11-01

    There are substantial inter-laboratory variations in the levels of DNA damage measured by the comet assay. The aim of this study was to investigate whether adherence to a standard comet assay protocol would reduce inter-laboratory variation in reported values of DNA damage. Fourteen laboratories determined the baseline level of DNA strand breaks (SBs)/alkaline labile sites and formamidopyrimidine DNA glycosylase (FPG)-sensitive sites in coded samples of mononuclear blood cells (MNBCs) from healthy volunteers. There were technical problems in seven laboratories in adopting the standard protocol, which were not related to the level of experience. Therefore, the inter-laboratory variation in DNA damage was only analysed using the results from laboratories that had obtained complete data with the standard comet assay protocol. This analysis showed that the differences between reported levels of DNA SBs/alkaline labile sites in MNBCs were not reduced by applying the standard assay protocol as compared with the laboratory's own protocol. There was large inter-laboratory variation in FPG-sensitive sites by the laboratory-specific protocol and the variation was reduced when the samples were analysed by the standard protocol. The SBs and FPG-sensitive sites were measured in the same experiment, indicating that the large spread in the latter lesions was the main reason for the reduced inter-laboratory variation. However, it remains worrying that half of the participating laboratories obtained poor results using the standard procedure. This study indicates that future comet assay validation trials should take steps to evaluate the implementation of standard procedures in participating laboratories.

  2. 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.

  3. In Vivo Autofluorescence Spectroscopic Study and Evaluation of DNA Damage By Comet Assay in Smokers

    PubMed Central

    Rajmohan, M; Thamaraiselvi, D; M, Deepasree

    2015-01-01

    Context Tobacco is known environmental factor to alter the chemical composition of cells and the structure of DNA. Cellular level changes of smoker’s mucosa are assessed by autofluorescence spectroscopy and the DNA damage can be evaluated by single cell gel electrophoresis (comet assay). Aim To substantiate the changes in the autofluorescence due to smoking with that of early DNA damage without any clinical change. Materials and Methods Group I consists of 20 individuals with normal mucosa and Group II consists of 40 individuals with smoking habit. Only males were included in this study and their age ranging from 25 to 35 years. In vivo fluorescence spectra from both groups were obtained by using hand held fiber optic probe attached to Varian Cary Eclipse fluorescence spectrophotometer and comet assay was carried out for normal and smokers by their peripheral blood. Statistical Analysis Used Independent-Samples t-test was used for statistical analysis. P-value was obtained to discriminate the statistical differences between the two groups. Results The averaged excitation and emission spectra of normal and smoker’s mucosa showed significant differences statistically. In comet assay, the mean tail length of smoker group was higher than the normal group. The results showed statistically significant differences (p ≤ 0.05). Conclusion These techniques will be very useful for monitoring of very early changes of mucosa before clinical manifestation of the lesion in high risk smokers and thus prevents the occurrence of premalignant disorders and early invasive carcinoma. PMID:26155555

  4. 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.

  5. 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.

  6. Comet assay to assess the genotoxicity of Persian walnut (Juglans regia L.) husks with statistical evaluation.

    PubMed

    Petriccione, Milena; Ciniglia, Claudia

    2012-07-01

    The aim of this study was to confirm the utility of the Comet assay as a genotoxicity screening test for evaluating the impact of walnut husk aqueous extract. Phytotoxicity assays using diluted and undiluted walnut husk aqueous extracts were performed on young roots of Raphanus sativus (radish), and the Comet assay was used to evaluate DNA integrity in isolated radish radicle nuclei. The results reveal a dose-dependent accumulation of DNA damage in radish radicles treated with walnut husks water extract and that the Kolmogorov-Smirnov test combined with Johnson SB distribution was the best approach for describing Comet assay data.

  7. 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.

  8. 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.

  9. 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.

  10. A Comparative Analysis and Taxonomy of Comets

    NASA Astrophysics Data System (ADS)

    Osip, David James

    1995-01-01

    A comparative analysis of narrowband photometry observations of 85 comets yields a wealth of information about the nature of comets. The data set consists of 2020 observations obtained over 429 nights between 1976 and 1992 that are fully reduced in a consistent manner to molecular production rates of 5 emission species (OH, NH, CN, C _3, and C_2) as well as a comparative measure of the dust production rate. Ratios of these production rates serve as a means of exploring compositional variation between comets. Vaporization models are used to estimate the active surface area for each comet, which is indicative of intrinsic differences in activity level among comets. Orbital parameters distinguish comets by dynamical age and separate the database into several dynamical classes. The current investigation confirms a number of results from earlier studies. There are no significant or systematic variations of the various abundance ratios with heliocentric distance. Neither the cometary dust -to-gas ratio nor the activity level vary systematically with dynamical age or with heliocentric distance. There is no conclusive evidence suggesting changes in composition based on dynamical evolution of the comet. Also, this investigation finds for many comets the strong degree of homogeneity in abundance ratios suggested by other studies, particularly among the carbon bearing species. Results unique to the present database include a significant correlation between the cometary dust-to -gas ratio and perihelion distance of a comet, suggesting mantling effects on the nucleus. Additional evidence for mantling is provided by the large number of comets with very low levels of activity as measured by the surface active area and the low fractional active areas calculated for those comets with nucleus size estimates available. The most important result from this study, however, is a taxonomic classification of comets separating chemically distinct compositional groupings most likely due to

  11. Titanium dioxide nanoparticles: an in vitro study of DNA binding, chromosome aberration assay, and comet assay.

    PubMed

    Patel, Suhani; Patel, Palak; Bakshi, Sonal R

    2017-04-01

    Engineered titanium dioxide nanoparticles (TiO2 NPs) are extensively used in cosmetic, pharmaceutical and other industries globally due to their unique properties, which has raised concern for biosafety. Genotoxicity assessment is an important part of biosafety evaluation; we report in vitro cytogenetic assays for NPs considering their unique physicochemical characteristics to fill the gap of laboratory data regarding biological safety along with mechanistic study for mode of interaction of NP with genetic material. Comet and chromosome aberration assay (CA assay) using short-term human peripheral blood cultures following exposure to TiO2 NPs; along with physicochemical parameters for stability of nano form in cultures; and DNA binding activity were carried out. The dynamic light scattering and zeta potential measurements revealed mono dispersion in media. The fluorescence spectroscopy for binding affinity of TiO2 NPs and human genomic DNA showed binding constant (Kb), 4.158 × 10(6) M(-1) indicating strong binding affinity and negative ΔG(0) value suggesting spontaneous DNA binding supporting its genotoxic potential. Following in vitro exposure to TiO2 NPs for 24 h, the cultures were analyzed for comet and CA assays, which showed significant results (p < 0.05) for % DNA intensity in tail, Olive Tail Moment and frequency of Chromosomal aberrations (CA) at 75 and 125 μM but not at 25 μM.

  12. Human semen cryopreservation: a sperm DNA fragmentation study with alkaline and neutral Comet assay.

    PubMed

    Ribas-Maynou, J; Fernández-Encinas, A; García-Peiró, A; Prada, E; Abad, C; Amengual, M J; Navarro, J; Benet, J

    2014-01-01

    Sperm cryopreservation is widely used for both research and reproduction purposes, but its effect on sperm DNA damage remains controversial. Sperm DNA fragmentation (SDF) has become an important biomarker to assess male infertility. In particular, the differentiation between single- and double-stranded DNA fragmentation (ssSDF and dsSDF) has clinical implications for male infertility where ssSDF is associated with reduced fertility, whereas dsSDF is associated with increased risk of miscarriage. In this study, semen samples from 30 human males have been analysed in both fresh and cryopreserved using the alkaline and neutral Comet assays. Results show an increase of about 10% of ssSDF, assessed by the alkaline Comet assay, regardless of the male fertility status. Neutral Comet analysis of dsSDF does not show any statistical increase when comparing fresh and cryopreserved samples in any of the patient groups. Results support previous reports that oxidative stress is the major effector in DNA damage during sample cryopreservation, as, on one hand, ssSDF has previously been related to oxidative damage and, on the other hand, we have not found any effect on dsSDF. Therefore, there might be a slight risk of decreased fertility after using a freezed sample, but no evidence for increased miscarriage risk from cryopreserved spermatozoa should be expected. © 2013 American Society of Andrology and European Academy of Andrology.

  13. 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.

  14. Identification of irradiated refrigerated poultry with the DNA comet assay

    NASA Astrophysics Data System (ADS)

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

    2004-09-01

    Food irradiation could make a significant contribution to the reduction of food-borne diseases caused by harmful bacteria such as Salmonella and parasites. In fact these organisms cause an increasing number of diseases and eventually deaths all over the world, also in industrialized countries. Radiation processing has the advantage that in addition to eliminating pathogens, thereby enhancing food safety, it also extends shelf life through destruction of spoilage organisms. The DNA molecule because of its big size is an easy target for ionizing radiation, therefore, changes in DNA offer potential to be used as a detection method for the irradiation treatment. In our study, poultry has been irradiated and changes in DNA analyzed by the Comet Assay. Samples were packed in plastic bags and irradiated. Doses were 0, 1.5, 3.0 and 4.5kGy. Immediately after irradiation the samples were returned to the refrigerator (4°C). Samples were analyzed 1 and 10 days after irradiation. This method proved to be an inexpensive and rapid screening technique for qualitative detection of irradiation treatment.

  15. The low molecular weight DNA diffusion assay as an indicator of cytotoxicity for the in vitro comet assay.

    PubMed

    Speit, Günter; Vesely, Alexandra; Schütz, Petra; Linsenmeyer, Regina; Bausinger, Julia

    2014-07-01

    The low molecular weight DNA diffusion assay (LMW assay) has been recommended as a measure for cytotoxicity for the in vivo comet assay. To better understand the relationship between effects in the LMW assay, DNA migration in the comet assay and effects in established cytotoxicity tests, we performed in vitro experiments with cultured human cell lines (TK6, A549) and comparatively investigated five test substances (methyl methanesulfonate, (±)-benzo[a]pyrene diol epoxide, sodium dodecyl sulphate, menthol and sodium arsenite). We measured DNA migration (tail intensity) in the comet assay and the frequency of 'hedgehogs' (cells with almost all DNA in the tail), DNA diffusion in the LMW assay, cell viability (trypan blue and fluorescein diacetate/ethidium bromide staining) and inhibition of proliferation (relative cell counts). Our in vitro experiments indicate that effects in the LMW assay occur independently from DNA effects in the comet assay and are not related to the occurrence of hedgehogs. Results from the LMW assay are in good agreement with results from viability assays and seem to allow discriminating genotoxic from non-genotoxic substances when appropriate preparation times are considered. Measurements of cytotoxicity by these methods only at an early preparation time after exposure to genotoxic substances may lead to erroneous results.

  16. 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.

  17. 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.

  18. The relationship between environmental exposures to phthalates and DNA damage in human sperm using the neutral comet assay.

    PubMed Central

    Duty, Susan M; Singh, Narendra P; Silva, Manori J; Barr, Dana B; Brock, John W; Ryan, Louise; Herrick, Robert F; Christiani, David C; Hauser, Russ

    2003-01-01

    Phthalates are industrial chemicals widely used in many commercial applications. The general population is exposed to phthalates through consumer products as well as through diet and medical treatments. To determine whether environmental levels of phthalates are associated with altered DNA integrity in human sperm, we selected a population without identified sources of exposure to phthalates. One hundred sixty-eight subjects recruited from the Massachusetts General Hospital Andrology Laboratory provided a semen and a urine sample. Eight phthalate metabolites were measured in urine by using high-performance liquid chromatography and tandem mass spectrometry; data were corrected for urine dilution by adjusting for specific gravity. The neutral single-cell microgel electrophoresis assay (comet assay) was used to measure DNA integrity in sperm. VisComet image analysis software was used to measure comet extent, a measure of total comet length (micrometers); percent DNA in tail (tail%), a measure of the proportion of total DNA present in the comet tail; and tail distributed moment (TDM), an integrated measure of length and intensity (micrometers). For an interquartile range increase in specific gravity-adjusted monoethyl phthalate (MEP) level, the comet extent increased significantly by 3.6 micro m [95% confidence interval (95% CI), 0.74-6.47]; the TDM also increased 1.2 micro m (95% CI, -0.05 to 2.38) but was of borderline significance. Monobutyl, monobenzyl, monomethyl, and mono-2-ethylhexyl phthalates were not significantly associated with comet assay parameters. In conclusion, this study represents the first human data to demonstrate that urinary MEP, at environmental levels, is associated with increased DNA damage in sperm. PMID:12842768

  19. The comet assay, DNA damage, DNA repair and cytotoxicity: hedgehogs are not always dead.

    PubMed

    Lorenzo, Yolanda; Costa, Solange; Collins, Andrew R; Azqueta, Amaya

    2013-07-01

    DNA damage is commonly measured at the level of individual cells using the so-called comet assay (single-cell gel electrophoresis). As the frequency of DNA breaks increases, so does the fraction of the DNA extending towards the anode, forming the comet tail. Comets with almost all DNA in the tail are often referred to as 'hedgehog' comets and are widely assumed to represent apoptotic cells. We review the literature and present theoretical and empirical arguments against this interpretation. The level of DNA damage in these comets is far less than the massive fragmentation that occurs in apoptosis. 'Hedgehog' comets are formed after moderate exposure of cells to, for example, H2O2, but if the cells are incubated for a short period, 'hedgehogs' are no longer seen. We confirm that this is not because DNA has degraded further and been lost from the gel, but because the DNA is repaired. The comet assay may detect the earliest stages of apoptosis, but as it proceeds, comets disappear in a smear of unattached DNA. It is clear that 'hedgehogs' can correspond to one level on a continuum of genotoxic damage, are not diagnostic of apoptosis and should not be regarded as an indicator of cytotoxicity.

  20. Fluorescence in situ hybridization in combination with the comet assay and micronucleus test in genetic toxicology

    PubMed Central

    2010-01-01

    Comet assay and micronucleus (MN) test are widely applied in genotoxicity testing and biomonitoring. While comet assay permits to measure direct DNA-strand breaking capacity of a tested agent MN test allows estimating the induced amount of chromosome and/or genome mutations. The potential of these two methods can be enhanced by the combination with fluorescence in situ hybridization (FISH) techniques. FISH plus comet assay allows the recognition of targets of DNA damage and repairing directly. FISH combined with MN test is able to characterize the occurrence of different chromosomes in MN and to identify potential chromosomal targets of mutagenic substances. Thus, combination of FISH with the comet assay or MN test proved to be promising techniques for evaluation of the distribution of DNA and chromosome damage in the entire genome of individual cells. FISH technique also permits to study comet and MN formation, necessary for correct application of these methods. This paper reviews the relevant literature on advantages and limitations of Comet-FISH and MN-FISH assays application in genetic toxicology. PMID:20840797

  1. 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

  2. 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.

  3. Genotoxicity evaluation of drinking water sources in human peripheral blood lymphocytes using the comet assay.

    PubMed

    Wu, Yulin; Chen, Haigang; Li, Zhaoli; Sun, Liwei; Qu, Mengmeng; Li, Mei; Kong, Zhiming

    2008-01-01

    The potential harm of organic pollutants in drinking water to human health is widely focused on in the world; more and more pollutants with genotoxic substances are released into the aquatic environment. Water source samples were collected from 7 different localities of Nanjing City. The potential genotoxicity of organic extracts from drinking water sources were investigated by means of the comet assay in human peripheral lymphocytes. The results showed that all the organic extracts from all the water source samples could induce DNA damages of human peripheral blood lymphocytes at different levels. A significant difference (P < 0.01) was observed when compared with the solvent control. The DNA damage increased with the increase of the dosage of the original water source. Significant differences of DNA damage were observed in different drinking water sources, as shown by the multiple comparisons analysis at the dosage of 100x; the degree of DNA damage treated by Hushu waterworks (at town level) was the most serious, the arbitrary units (AU) was 141.62 +/- 6.96, however, that of Shangyuanmen waterworks (at city level) was only 109.64 +/- 2.97. The analysis also revealed that the genotoxicity of town's water sources was higher than that of the city. The results demonstrated that the comet assay can be successfully applied to the genotoxicity monitoring programs of drinking water sources.

  4. DNA damage in birds after the mining waste spill in southwestern Spain: a Comet assay evaluation.

    PubMed

    Pastor, N; López-Lázaro, M; Tella, J L; Baos, R; Forrero, M G; Hiraldo, F; Cortés, F

    2001-01-01

    In April 1998, an ecological disaster resulting from a massive toxic spill of mining acid waste rich in heavy metals posed a serious threat to the Doñana National Park in southwestern Spain. This especially important protected area is the nesting and breeding site for many endangered bird species; white storks (Ciconia ciconia) and black kites (Milvus migrans) are considered the more representative. The suitability of the Comet assay as a biomarker for genotoxic analysis in environmental biomonitoring has been recently validated in studies using different sentinel organisms such as fish, amphibians, rodents, or mollusks. Birds preying on a variety of invertebrate and vertebrate species in the marshlands are appropriate for evaluating the potential deleterious effects of the toxic spill on wildlife of the Dofiana area. Our study on wetland birds high on the aquatic trophic chain sampled within a few months after the toxic spill in the area around Doñana National Park has shown the accumulation of heavy metals. Fourteen months after the mine waste spill, blood samples from white storks and kites collected in the neighborhood of the park and from control birds at reference areas for comparison were examined by fluorescence image analysis after lymphocyte isolation, and by subsequent alkaline single-cell gel (SCG) electrophoresis, known as the Comet assay. Our results indicate that the exposed birds had a significantly increased level of genotoxic damage compared with control animals from noncontaminated locations.

  5. Evaluation of the DNA damaging effects of amitraz on human lymphocytes in the Comet assay.

    PubMed

    Radakovic, Milena; Stevanovic, Jevrosima; Djelic, Ninoslav; Lakic, Nada; Knezevic-Vukcevic, Jelena; Vukovic-Gacic, Branka; Stanimirovic, Zoran

    2013-03-01

    Amitraz is formamidine pesticide widely used as insecticide and acaricide. In veterinary medicine, amitraz has important uses against ticks, mites and lice on animals. Also, amitraz is used in apiculture to control Varroa destructor. It this study, the alkaline Comet assay was used to evaluate DNA damaging effects of amitraz in human lymphocytes. Isolated human lymphocytes were incubated with varying concentrations of amitraz (0.035, 0.35, 3.5, 35 and 350 mu g/mL). The Comet assay demonstrated that all concentrations of amitraz caused statistically significant increase in the level of DNA damage, thus indicating that amitraz possesses genotoxic potential. The concentration of amitraz that produced the highest DNA damage (3.5 mu g/mL) was chosen for further analysis with the antioxidant catalase. The obtained results showed that co-treatment with antioxidant catalase (100 IU/mL or 500 IU/mL) significantly reduced the level of DNA damage, indicating the possible involvement of reactive oxygen species in DNA damaging effects of amitraz. Flow cytometric analysis revealed increase of the apoptotic index following treatment with amitraz. However, co-treatment with catalase reduced the apoptotic index, while treatment with catalase alone reduced the percentage of apoptotoc cells even in comparison with the negative control. Therefore, catalase had protective effects against ROS-mediated DNA damage and apoptosis.

  6. 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.

  7. Assessment of DNA interstrand crosslinks using the modified alkaline comet assay.

    PubMed

    Wu, Jian Hong; Jones, Nigel J

    2012-01-01

    The single cell gel electrophoresis (SCGE) assay, more commonly known as the comet assay, due to the "comet-like" appearance of the cells, was originally developed as a technique to measure the presence of DNA single-strand breaks. The assay is performed on single cells embedded in agar and placed in an electrical field at alkaline pH, so that fragments of negatively charged single-stranded DNA move through the gel toward the positively charged anode. Undamaged DNA moves relatively slowly, forming the head of the comet, while DNA fragmented due to the presence of single-strand breaks, moves more quickly giving the appearance of the tail. The extent of DNA migration is a measure of the DNA damage present. Since it was first developed, the comet assay has been adapted for measuring other types of DNA damage. The neutral comet assay has been employed for DNA double-strand breaks, while techniques using DNA repair enzymes to cleave specific adducts, UvrABC for ultraviolet radiation induced adducts, for example, have also been described. Here, we describe a modified version of the comet assay for the measurement of interstrand crosslinks (ICLs). Interstrand crosslinking agents include the chemotherapeutic agents mitomycin C and cis-platin, psoralen plus UVA light (PUVA) used to treat hyperproliferative skin disorders and diepoxybutane, a metabolite of 1,3-butadiene used in industrial processes and an environmental pollutant. ICLs are a potent and cytotoxic form of DNA damage as they prevent DNA strand separation, thereby preventing DNA replication. Their removal requires several different DNA repair processes including translesion synthesis and homologous recombination. As ICLs prevent separation of the DNA strands, their presence results in less DNA migration in the comet assay. To successfully measure ICLs, it is necessary to incorporate a step that induces single-strand breaks (using a defined dose of ionizing radiation) that allows the crosslinked DNA to migrate.

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

    PubMed

    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.

  9. 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

  10. Combination of physico-chemical analysis, Allium cepa test system and Oreochromis niloticus erythrocyte based comet assay/nuclear abnormalities tests for cyto-genotoxicity assessments of treated effluents discharged from textile industries.

    PubMed

    Hemachandra, Chamini K; Pathiratne, Asoka

    2016-09-01

    Bioassays for cyto-genotoxicity assessments are generally not required in current textile industry effluent discharge management regulations. The present study applied in vivo plant and fish based toxicity tests viz. Allium cepa test system and Oreochromis niloticus erythrocyte based comet assay and nuclear abnormalities tests in combination with physico-chemical analysis for assessing potential cytotoxic/genotoxic impacts of treated textile industry effluents reaching a major river (Kelani River) in Sri Lanka. Of the treated effluents tested from two textile industries, color in the Textile industry 1 effluents occasionally and color, biochemical oxygen demand and chemical oxygen demand in the Textile industry 2 effluents frequently exceeded the specified Sri Lankan tolerance limits for discharge of industrial effluents into inland surface waters. Exposure of A. cepa bulbs to 100% and 12.5% treated effluents from both industries resulted in statistically significant root growth retardation, mito-depression, and induction of chromosomal abnormalities in root meristematic cells in comparison to the dilution water in all cases demonstrating cyto-genotoxicity associated with the treated effluents. Exposure of O. niloticus to the 100% and 12.5% effluents, resulted in erythrocytic genetic damage as shown by elevated total comet scores and induction of nuclear abnormalities confirming the genotoxicity of the treated effluents even with 1:8 dilution. The results provide strong scientific evidence for the crucial necessity of incorporating cyto-genotoxicity impact assessment tools in textile industry effluent management regulations considering human health and ecological health of the receiving water course under chronic exposure. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Cytotoxicity and genotoxicity assessment of Euphorbia hirta in MCF-7 cell line model using comet assay

    PubMed Central

    Ping, Kwan Yuet; Darah, Ibrahim; Chen, Yeng; Sasidharan, Sreenivasan

    2013-01-01

    Objective To evaluate the cytotoxicity and genotoxicity activity of Euphorbia hirta (E. hirta) in MCF-7 cell line model using comet assay. Methods The cytotoxicity of E. hirta extract was investigated by employing brine shrimp lethality assay and the genotoxicity of E. hirta was assessed by using Comet assay. Results Both toxicity tests exhibited significant toxicity result. In the comet assay, the E. hirta extract exhibited genotoxicity effects against MCF-7 DNA in a time-dependent manner by increasing mean percentage of DNA damage. The extract of E. hirta showed significant toxicity against brine shrimp with an LC50 value of 620.382 µg/mL (24 h). Comparison with positive control potassium dichromate signifies that cytotoxicity exhibited by the methanol extract might have moderate activity. Conclusion The present work confirmed the cytotoxicity and genotoxicity of E. hirta. However, the observed toxicity of E. hirta extracts needs to be confirmed in additional studies. PMID:23998008

  12. What is Comet assay not telling us: AFLP reveals wider aspects of genotoxicity.

    PubMed

    Šrut, Maja; Štambuk, Anamaria; Klobučar, Göran I V

    2013-06-01

    DNA damage detected by genotoxicity biomarkers such as the Comet assay is not always a reliable indicator of the consequences that genotoxic agents can have on the genome integrity of the exposed organisms. Therefore, to reveal the existence of more permanent alterations of DNA structure after genotoxic stress, the RTG-2 rainbow trout cell line was exposed for 3 days to benzo[a]pyrene (B[a]P, 0.1-10 μM) and ethyl methanesulfonate (EMS, 0.1-1mM) followed by 3 days of recovery period. Primary DNA damage was evaluated by the Comet assay and DNA alterations were assessed using AFLP (amplified fragment length polymorphism). Qualitative and quantitative modifications in AFLP profiles were analyzed in order to detect genetic alterations arising from mutation events and/or DNA damage. Significant induction in DNA damage measured by the Comet assay was noticed after B[a]P treatment at all concentrations but values returned to the control level after recovery. Exposure to EMS induced significant DNA damage only at the highest concentration and damage persisted after the recovery period. AFLP profiles detected DNA alterations even when Comet assay indicated complete DNA repair, revealing more persistent damage. Since such DNA damage can impair its structure and function, Comet assay results should preferably be supplemented with other methods in order to predict the consequences of genotoxic insult more accurately.

  13. 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.

  14. 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.

  15. 15 years of comet photometry: A comparative analysis of 80 comets

    NASA Technical Reports Server (NTRS)

    Osip, David J.; Schleicher, David G.; Millis, Robert L.; Hearn, M. F. A.; Birch, P. V.

    1992-01-01

    In 1976 we began a program of narrowband photometry of comets that has encompassed well over 400 nights of observations. To date, the program has provided detailed information on 80 comets, 11 of which have been observed on multiple apparitions. In this paper we present the observed range of compositions (molecular production rate ratios) and dustiness (gas production compared with AF-rho) for a well sampled group of comets. Based on these results we present preliminary analysis of taxonomic groupings as well as the abundance ratios we associate with a 'typical' comet.

  16. Detection of Hypoxia in Human Brain Tumor Xenografts Using a Modified Comet Assay1

    PubMed Central

    Wang, Jingli; Klem, Jack; Wyrick, Jan B; Ozawa, Tomoko; Cunningham, Erin; Golinveaux, Jay; Allen, Max J; Lamborn, Kathleen R; Deen, Dennis F

    2003-01-01

    Abstract We used the standard comet assay successfully to generate in vitro dose-response curves under oxic and hypoxic conditions. We then made mixtures of cells that had been irradiated with 3 and 9 Gy of X-rays to simulate two subpopulations in a tumor, but efforts to accurately detect and quantify the subpopulations using the standard comet assay were unsuccessful. Therefore, we investigated a modified comet assay to determine whether it could be used for measuring hypoxia in our model systems. U251 MG cells were grown as subcutaneous tumors in athymic mice; U251 MG and U87 MG cells were grown as intracerebral (i.c.) tumors in athymic rats. Animals were injected with RSU 1069, irradiated, and euthanized. Tumors and normal brains were removed, and the cells were analyzed using a modified comet assay. Differences in comet tail moment distributions between tumor and contralateral normal brain, using tail moments at either the 25th or 50th percentile in each distribution, were taken as measures of the degree of tumor hypoxia. For U251 MG tumors, there was a positive relationship between tumor size and the degree of hypoxia, whereas preliminary data from U87 MG i.c. tumors showed less hypoxia and no apparent relationship between tumor size and hypoxia. PMID:14511400

  17. 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.

  18. 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.

  19. Evaluation of sperm DNA quality in men presenting with testicular cancer and lymphoma using alkaline and neutral Comet assays.

    PubMed

    Kumar, K; Lewis, S; Vinci, S; Riera-Escamilla, A; Fino, M-G; Tamburrino, L; Muratori, M; Larsen, P; Krausz, C

    2017-09-26

    Despite more cancers in young men over the past two decades, improvements in therapies give a greater chance to live full lives following treatment. Sperm genomic quality is variable following cancer diagnosis, so its assessment is important if sperm cryopreservation is being considered. Here, we evaluated DNA damage using two DNA damage assays: an alkaline and for the first time, a neutral Comet assays in men presenting with testicular cancer (n = 19 for alkaline and 13 for neutral group) and lymphoma (n = 13 for alkaline and 09 for neutral group) compared with fertile donors (n = 20 for alkaline and 14 for neutral group). No significant differences were observed in any semen analysis parameters. In contrast, sperm DNA damage was higher in men with testicular cancer than in donors as assessed by both the alkaline (12.4% vs. 37.4%, p < 0.001) and neutral (7.5% vs. 13.4%; p < 0.05) Comet assays. Similar trends were observed in men with lymphoma. Here, sperm DNA damage was higher using both the alkaline (35.0% vs. 12.4%) and neutral (10.7% against 7.5% (p < 0.05) Comet assays. Moreover, the DNA strand breaks (particularly double-strand breaks) were significantly more prominent in men with cancer having abnormal seminal parameters than normozoospermic ones. This study showed that sperm DNA testing using alkaline and neutral Comet assays is more sensitive than semen analysis in detecting impaired sperm quality in men presenting with cancer. It may provide a useful adjunct when considering storage prior to cancer investigations and assisted reproductive techniques (ART)-based treatment. © 2017 American Society of Andrology and European Academy of Andrology.

  20. Comet assays to assess DNA damage and repair in grass shrimp embryos exposed to phototoxicants.

    PubMed

    Lee, R; Kim, G B

    2002-01-01

    Exposure of grass shrimp (Palaemonetes pugio) embryos to four compounds (anthracene, pyrene, alpha-terthienyl, methylene blue) along with solar exposure resulted in extensive DNA strand damage using the comet assay. DNA tail moments of embryos exposed to these chemicals in the dark ranged from 1.8 to 4.3, while exposure to chemicals and solar resulted in tail moments of 14.3-15.3. Reduction of DNA tail moments when solar exposed embryos were transferred to the dark, suggested DNA repair systems were active. The comet assay can be used to follow both DNA damage and repair following exposure to phototoxic chemicals.

  1. Comets

    NASA Image and Video Library

    Did you know that comets seen streaking across the night sky may have brought the building blocks of life to our planet billions of years ago? Join NASA in learning more about these fascinating obj...

  2. Genotoxicity evaluation of hydroalcoholic and aqueous extracts of Dorema aucheri by the comet assay

    PubMed Central

    Etebari, Mahmoud; Sajjadi, Seyed Ebrahim; Jafarian-Dehkordi, Abbas; Nazmakanipour, Sajjad

    2016-01-01

    Background: Dorema aucheri is a plant of Apiaceae family which is used widely in some states of Iran. Different extracts and essential oil of Dorema species contain flavonoids and cumarin compounds which have anti-hypertensive, cholesterol- and triglycerides-lowering properties. This study was undertaken to evaluate the genotoxic properties of hydroalcoholic and aqueous extracts of D. aucheri on human hepatoma cells using the comet assay method for safety evaluation. Materials and Methods: In this method, after incubation of cells with different concentrations of extracts, cell suspensions were added to pre-coated normal agarose slides. After lysis, electrophoresis and neutralization process, staining was done by ethidium bromide and comets were observed using a fluorescence microscope. Tail length, percentage of DNA in tail and tail moment parameters were measured. Results: Statistical analysis of the results demonstrated that concentrations more than 500 μg/ml of hydroalcoholic and aqueous extract of D. aucheri were genotoxic. Conclusion: It can be concluded from the results that taking the concentrations less than these dosages of extracts are safe but more studies are required to determine genotoxic mechanisms of this plant. PMID:28217637

  3. Estimates of DNA strand breakage in bottlenose dolphin (Tursiops truncatus) leukocytes measured with the Comet and DNA diffusion assays

    PubMed Central

    2009-01-01

    The analysis of DNA damage by mean of Comet or single cell gel electrophoresis (SCGE) assay has been commonly used to assess genotoxic impact in aquatic animals being able to detect exposure to low concentrations of contaminants in a wide range of species. The aims of this work were 1) to evaluate the usefulness of the Comet to detect DNA strand breakage in dolphin leukocytes, 2) to use the DNA diffusion assay to determine the amount of DNA strand breakage associated with apoptosis or necrosis, and 3) to determine the proportion of DNA strand breakage that was unrelated to apoptosis and necrosis. Significant intra-individual variation was observed in all of the estimates of DNA damage. DNA strand breakage was overestimated because a considerable amount (~29%) of the DNA damage was derived from apoptosis and necrosis. The remaining DNA damage in dolphin leukocytes was caused by factors unrelated to apoptosis and necrosis. These results indicate that the DNA diffusion assay is a complementary tool that can be used together with the Comet assay to assess DNA damage in bottlenose dolphins. PMID:21637693

  4. Estimates of DNA strand breakage in bottlenose dolphin (Tursiops truncatus) leukocytes measured with the Comet and DNA diffusion assays.

    PubMed

    Díaz, Adriana; Carro, Sandra; Santiago, Livia; Estévez, Juan; Guevara, Celia; Blanco, Miriam; Sánchez, Laima; Sánchez, Liena; López, Nirka; Cruz, Danilo; López, Ronar; Cuetara, Elizabeth B; Fuentes, Jorge Luis

    2009-04-01

    The analysis of DNA damage by mean of Comet or single cell gel electrophoresis (SCGE) assay has been commonly used to assess genotoxic impact in aquatic animals being able to detect exposure to low concentrations of contaminants in a wide range of species. The aims of this work were 1) to evaluate the usefulness of the Comet to detect DNA strand breakage in dolphin leukocytes, 2) to use the DNA diffusion assay to determine the amount of DNA strand breakage associated with apoptosis or necrosis, and 3) to determine the proportion of DNA strand breakage that was unrelated to apoptosis and necrosis. Significant intra-individual variation was observed in all of the estimates of DNA damage. DNA strand breakage was overestimated because a considerable amount (~29%) of the DNA damage was derived from apoptosis and necrosis. The remaining DNA damage in dolphin leukocytes was caused by factors unrelated to apoptosis and necrosis. These results indicate that the DNA diffusion assay is a complementary tool that can be used together with the Comet assay to assess DNA damage in bottlenose dolphins.

  5. Genotoxicity assessment of mouse oocytes by comet assay before vitrification and after warming with three vitrification protocols.

    PubMed

    Berthelot-Ricou, Anais; Perrin, Jeanne; di Giorgio, Carole; de Meo, Michel; Botta, Alain; Courbiere, Blandine

    2013-09-01

    To assess the genotoxicity of three oocyte vitrification protocols. Murine assay. Biogenotoxicology research laboratory. CD1 female mice. Three mouse oocyte groups were exposed to three commercialized human oocyte vitrification protocols. Protocols 1 and 2 contained dimethyl sulfoxide and ethylene glycol (EG), and protocol 3 contained EG and 1,2-propanediol (PrOH). DNA damage was first evaluated by comet assay after oocyte exposure to the three different equilibration and vitrification solutions. Comet assay was also performed after full vitrification and warming procedure and compared with a negative control group (oocytes stored in medium culture only) and a positive control group (oocytes exposed to hydrogen peroxide just before comet assay). DNA damage was quantified as Olive tail moment (OTM). Statistical analysis consisted of a Shapiro-Wilk test. Then, median protocol OTM was compared with the negative control group with the Mann-Whitney U test. The difference was considered to be statistically significant if the P value was <.05. In both parts of our study, protocols 1 and 2 did not induce significant DNA damage, whereas protocol 3 induced statistically higher DNA damage compared with the negative control group. Vitrification protocols containing PrOH induced significant DNA damage on mouse oocytes, both before cooling and after warming. Therefore, for the moment, we prefer vitrification techniques without PrOH while we await more studies on PrOH toxicity and long-term evaluation. Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  6. 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

  7. 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.

  8. Comparative investigations of genotoxic activity of five nitriles in the comet assay and the Ames test.

    PubMed

    Wu, Jong-C; Hseu, You C; Chen, Chin-H; Wang, Shu-H; Chen, Ssu C

    2009-09-30

    Two short-term assays, the modified Ames test and the comet assay, were carried out to evaluate the genotoxicity of five nitriles (acetonitrile, propionitrile, methacrylonitrile, butyronitrile, and benzonitrile). With the comet assay, all the nitriles studied were found to induce the genotoxicity in human lymphocytes and Hep G2 cells. Except for butyronitrile, the genotoxic potency in lymphocytes was more pronounced than that in Hep G2 cells, and the rank order of genotoxicity induced by these five nitriles in lymphocytes was different from that in Hep G2 cells, indicating that the pathways leading to genotoxicity in both types of cells were different. In the modified Ames test, no tested nitriles showed mutagenic activity on Salmonella typhimurium strain TA 98 and TA 100 with and without metabolic activation. Comparing the results obtained from both tests in this study, the comet assay seems to be more sensitive than the modified Ames test. Thus, the comet assay can be used to detect the genotoxicity of all nitriles.

  9. 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.

  10. Sperm DNA quality evaluated by comet assay and sperm chromatin structure assay in stallions after unilateral orchiectomy.

    PubMed

    Serafini, R; Varner, D D; Bissett, W; Blanchard, T L; Teague, S R; Love, C C

    2015-09-15

    Unilateral orchiectomy (UO) may interfere with thermoregulation of the remaining testis caused by inflammation surrounding the incision site, thus altering normal spermatogenesis and consequently sperm quality. Two measures of sperm DNA quality (neutral comet assay and the sperm chromatin structure assay [SCSA]) were compared before UO (0 days) and at 14, 30, and 60 days after UO to determine whether sperm DNA changed after a mild testis stress (i.e., UO). The percent DNA in the comet tail was higher at 14 and 60 days compared to 0 days (P < 0.05) after UO. All other comet tail measures (i.e., length, moment, migration) were higher at all time periods after UO compared to 0 days (P < 0.05). Two SCSA measures (mean-αt, mode-αt) increased at 14 days after UO (P < 0.05), whereas two measures (SD-αt and COMP-αt) did not change. This study identified a decrease in sperm DNA quality using both the neutral comet assay and the SCSA, which was not identified using traditional measures of sperm quality.

  11. 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.

  12. 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.

  13. Genotoxicity evaluation of titanium dioxide nanoparticles using the Ames test and Comet assay.

    PubMed

    Woodruff, Robert S; Li, Yan; Yan, Jian; Bishop, Michelle; Jones, M Yvonne; Watanabe, Fumiya; Biris, Alexandru S; Rice, Penelope; Zhou, Tong; Chen, Tao

    2012-11-01

    Titanium dioxide nanoparticles (TiO2-NPs) are being used increasingly for various industrial and consumer products, including cosmetics and sunscreens because of their photoactive properties. Therefore, the toxicity of TiO2-NPs needs to be thoroughly understood. In the present study, the genotoxicity of 10nm uncoated sphere TiO2-NPs with an anatase crystalline structure, which has been well characterized in a previous study, was assessed using the Salmonella reverse mutation assay (Ames test) and the single-cell gel electrophoresis (Comet) assay. For the Ames test, Salmonella strains TA102, TA100, TA1537, TA98 and TA1535 were preincubated with eight different concentrations of the TiO2-NPs for 4 h at 37 °C, ranging from 0 to 4915.2 µg per plate. No mutation induction was found. Analyses with transmission electron microscopy (TEM) and energy-dispersive X-ray spectroscopy (EDS) showed that the TiO2-NPs were not able to enter the bacterial cell. For the Comet assay, TK6 cells were treated with 0-200 µg ml(-1) TiO2-NPs for 24 h at 37 °C to detect DNA damage. Although the TK6 cells did take up TiO2-NPs, no significant induction of DNA breakage or oxidative DNA damage was observed in the treated cells using the standard alkaline Comet assay and the endonuclease III (EndoIII) and human 8-hydroxyguanine DNA-glycosylase (hOGG1)-modified Comet assay, respectively. These results suggest that TiO2-NPs are not genotoxic under the conditions of the Ames test and Comet assay. Published 2012. This article is a US Government work and is in the public domain in the USA.

  14. 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.

  15. The Comet assay for detection of DNA damage in canine sperm.

    PubMed

    Pereira, A F; Borges, P; Fontbonne, A; Cardoso, L; Gaivão, I; Martins-Bessa, A

    2017-08-13

    Sperm DNA integrity is a fundamental prerequisite in fertilization and embryo development. Among DNA integrity tests, the Comet assay is an accurate and sensitive test for the detection of sperm oxidative damage. The aim of this work was to evaluate sperm oxidative damage using the Comet assay and to study the correlation between Comet and routine assays for the evaluation of semen quality. Dogs were divided in two groups: group A (n = 6), comprising dogs with abnormal spermiogram, that is astheno-, terato- or oligoasthenoteratozoospermic (OAT); and group B (n = 8), comprising normospermic dogs. The distribution of sperm oxidative damage was significantly different between the two groups (p = .001): group A-median: 31.55%, interquartile range (IQR): 30.18-38.01; group B-median: 0.90%, IQR: 0.65-1.96. The correlation between oxidative damage and abnormal morphology was high (r = .846; p < .001). There was a negative correlation between progressive motility and oxidative damage (r = -.792; p = .001). Basal and oxidative DNA damage of spermatozoa are increased in dogs with non-normospermic semen. In conclusion, and considering the elevated correlation with classical tests of sperm quality, the Comet assay has ample potential for clinical and research purposes in dogs. © 2017 Blackwell Verlag GmbH.

  16. [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.

  17. 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).

  18. Comets

    NASA Astrophysics Data System (ADS)

    Barbieri, Cesare; Bertini, Ivano

    2017-08-01

    The paper reviews properties of comets, from historical sightings and interpretations, to contemporary ground- and space-based knowledge. The importance of comets in understanding the present Solar System and its dynamical, physical and chemical evolution, their relationship with other minor bodies, their possible role for the very early phases of our Earth, will be examined. Emphasis will be on the results of the recently completed European Rosetta mission to comet 67P/Churyumov-Gerasimenko, in particular those by OSIRIS, its imaging system. It is fair to say that Rosetta's results represent a most important step in the development of cometary science, whose full implications start just to surface and will be fully appreciated over several more years.

  19. Comets

    NASA Technical Reports Server (NTRS)

    Feldman, P. D.

    2006-01-01

    Spectroscopy of comets, in the X-ray and far-ultraviolet from space, and in the near infrared and millimeter from the ground, have revealed a wealth of new information, particularly about the molecular constituents that make up the volatile fraction of the comet s nucleus. Interpretation of these data requires not only proper wavelengths for identification but also information about the photolytic and excitation processes at temperatures typical of the inner coma (70-100 K) that lead to the observed spectral signatures. Several examples, mainly from Far Ultraviolet Spectroscopic Explorer and Hubble Space Telescope spectra of comets observed during the last few years, will be given to illustrate some of the current issues.

  20. Application of the comet and micronucleus assays to butterfish (Pholis gunnellus) erythrocytes from the Firth of Forth, Scotland.

    PubMed

    Bombail, V; Aw, D; Gordon, E; Batty, J

    2001-07-01

    This report describes an investigation of genotoxic effects in an inter-tidal fish species sampled along a pollution gradient in the Firth of Forth, Scotland, UK. The comet assay is an electrophoretic technique for measuring DNA breakage in nuclei from individual cells and has only recently been applied to field investigations of genotoxicity. The measurement of nuclear anomalies (NA), such as the presence of micronuclei (MN) and 'lobes', has been successfully utilised in many field studies of genotoxic effects of contaminated sediments. These two techniques were applied to nucleated red blood cells (RBC) from the butterfish, Pholis gunnellus. The comet assay was adapted and validated for use in this species. Fish were sampled from the inner Firth of Forth, which has a legacy of industrial contamination and the outer Firth of Forth which is comparatively clean. The analysis of DNA strand breakage using this technique did not reveal any significant differences between animals sampled from inner and outer zones of the Firth. In contrast, MN and NA frequencies were elevated in the inner polluted zone of the Firth compared to the outer zone. This study suggests: (1) there are genotoxic effects associated with contaminants in the inner Firth of Forth, and (2) the comet assay may not be a suitable genotoxicity biomarker in fish.

  1. Comets

    NASA Astrophysics Data System (ADS)

    Brownlee, D. E.

    2003-12-01

    Comets are surviving members of a formerly vast distribution of solid bodies that formed in the cold regions of the solar nebula. Cometary bodies escaped incorporation into planets and ejection from the solar system and they have been stored in two distant reservoirs, the Oort cloud and the Kuiper Belt, for most of the age of the solar system. Observed comets appear to have formed between 5 AU and 55 AU. From a cosmochemical viewpoint, comets are particularly interesting bodies because they are preserved samples of the solar nebula's cold ice-bearing regions that occupied 99% of the areal extent of the solar nebula disk. All comets formed beyond the "snow line" of the nebula, where the conditions were cold enough for water ice to condense, but they formed from environments that significantly differed in temperature. Some formed in the comparatively "warm" regions near Jupiter where the nebular temperature may have been greater than 120 K and others clearly formed beyond Neptune where temperatures may have been less than 30 K (Bell et al., 1997). Although comets are the best-preserved materials from the early solar system, they should be a mix of nebular and presolar materials that accreted over a vast range of distances from the Sun in environments that differed in temperature, pressure, and accretional conditions such as impact speed.Comets, by conventional definition, are unstable near the Sun; they contain highly volatile ices that vigorously sublime within 2-3 AU of the Sun. When heated, they release gas and solids due to "cometary activity," a series of processes usually detected from afar by the presence of a coma of gas and dust surrounding the cometary nucleus and or elongated tails composed of dust and gas. Active comets clearly have not been severely modified by the moderate to extreme heating that has affected all other solar system materials, including planets, moons, and even the asteroids that produced the most primitive meteorites. Comets have been

  2. Long-term biomonitoring of breast cancer patients under adjuvant chemotherapy: the comet assay as a possible predictive factor.

    PubMed

    Uriol, E; Sierra, M; Comendador, M A; Fra, J; Martínez-Camblor, P; Lacave, A J; Sierra, L M

    2013-01-01

    Most chemotherapy treatments induce DNA damage in the exposed patients. Using the comet assay and peripheral blood mononuclear cells (PBMC), we have quantified this induced DNA damage and studied its relationship with GSTM1 and GSTT1 polymorphisms, and clinical parameters. For this purpose, 29 Caucasian women, breast cancer patients under CMF or CEF adjuvant chemotherapy were included in the study. The clinical parameters considered were (i) therapies side effects, like haematological and biochemical toxicities, (ii) prognostic and predictive factors, like hormonal receptor expression, tumour differentiation degree, sickness stage, and nodal status, and (iii) the effectiveness of the chemotherapy measured as five years relapse probability. The results were also related to the confounding factor age. Comet assay results indicate that 13 patients were characterised by absence of induced DNA strand breaks, and 16 patients presented induced DNA strand breaks along the treatment. Relationships between comet variables and clinical parameters, found with principal component analysis, correlations, one-way ANOVA and multivariate logistic regression analyses revealed that: (1) baseline levels of DNA damage are related to GSTM1 genotype and to hormonal receptor expression; (2) GSTM1 genotype also influences comet results after chemotherapy, as it does the AST level; (3) the tail moment values of the cycle 6.1 and the sickness stage might predict cancer relapse at five years: for the Stage, OR = 13.8 (IIB versus I+IIA), 95% CI 0.80-238.97, and for 6.1 cycle TM, OR = 1.3, 95%, CI 0.97-1.79, with a potential model (10* Stage (I-IIA = 0, IIB = 1) + 6.1 cycle), that has a good predictive capacity, with an area under ROC curve of 0.872 (CI 0.62-1.00). To our knowledge, this is the first time such a predictive value is found for the comet assay. Nevertheless, before the comet assay could be used as a tool for oncologists, this relationship should be confirmed in more patients, and

  3. 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.

  4. 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

  5. Analysis of possible genotoxicity of the herbicide flurochloridone and its commercial formulations: Endo III and Fpg alkaline comet assays in Chinese hamster ovary (CHO-K1) cells.

    PubMed

    Soloneski, Sonia; Nikoloff, Noelia; Larramendy, Marcelo L

    2016-02-01

    Cytotoxic and genotoxic effects of flurochloridone (FLC) and its formulations Twin Pack Gold(®) and Rainbow(®) were evaluated in CHO-K1 cells. Using the alkaline single-cell gel electrophoresis (SCGE) assay, we observed that FLC (15 μg/ml), Twin Pack Gold(®) or Rainbow(®) induced primary DNA damage, increasing the frequency of damaged nucleoids. Vitamin E pretreatment did not modify the effect. Decreased cell viability was observed only in Twin Pack Gold(®)-treated cultures and was significantly ameliorated by vitamin E. Post-treatment of herbicide-damaged CHO-K1 cells with the enzymes Endo III or Fpg did not increase FLC-, Twin Pack Gold(®)-, or Rainbow(®)-induced DNA damage. These results demonstrate that neither FLC nor FLC-based formulations induce DNA damage through hydroxyl radical or lipid alkoxyl radical production, and that the induced DNA lesions were not related to oxidative damage at the purine/pyrimidine level. Our observations strongly suggest that the cytotoxic effects observed after Twin Pack Gold(®) exposure are due to the excipients contained within the technical formulation rather than FLC itself.

  6. Towards a more reliable comet assay: optimising agarose concentration, unwinding time and electrophoresis conditions.

    PubMed

    Azqueta, Amaya; Gutzkow, Kristine B; Brunborg, Gunnar; Collins, Andrew R

    2011-09-18

    The comet assay is now the method of choice for measuring most kinds of DNA damage in cells. However, due to the lack of a standardised protocol inter-laboratory comparisons are of limited value. The aim of this paper is to demonstrate how small changes in comet-assay variables may significantly affect the results. We examined the effect of varying agarose concentrations, alkaline unwinding time, electrophoresis time, voltage and current, by use of two cell types, viz. human peripheral blood lymphocytes and the lymphoblastoid cell line TK-6. All these variables have marked effects on assay performance and, therefore, on the determination of DNA damage. Here we identify factors of particular importance.

  7. Eco-genotoxicity of six anticancer drugs using comet assay in daphnids.

    PubMed

    Parrella, Alfredo; Lavorgna, Margherita; Criscuolo, Emma; Russo, Chiara; Isidori, Marina

    2015-04-09

    The eco-genotoxicity of six anti-neoplastic drugs, 5-fluorouracil, capecitabine, cisplatin, doxorubicin, etoposide, and imatinib, belonging to five classes of anatomical therapeutic classification (ATC), was studied applying the in vivo comet assay on cells from whole organisms of Daphnia magna and Ceriodaphnia dubia. For the first time, this test was performed in C. dubia. In addition, to have a wider genotoxic/mutagenic profile of the anticancer drugs selected, SOS chromotest and Salmonella mutagenicity assay were performed. The comet results showed that all drugs induced DNA damage, in both Cladocerans, with environmental concern; indeed Doxorubicin induced DNA damage in the order of tens of ng L(-1) in both crustaceans, as well as 5-flurouracil in C. dubia and cisplatin in D. magna. In the SOS Chromotest all drugs, except imatinib, were able to activate the repair system in Escherichia coli PQ37 while in the Salmonella mutagenicity assay, doxorubicin was the only drug able to cause direct and indirect frameshift and base-pair substitution mutations. Comet assay was the most sensitive tool of genotoxic exposure assessment, able to detect in vivo the adverse effects at concentration lower than those evaluated in vitro by bacterial assays.

  8. Comets

    NASA Technical Reports Server (NTRS)

    Wilkening, L. L. (Editor); Matthews, M. S. (Editor)

    1982-01-01

    Vacuum ultraviolet observations from sounding rockets and satellite observatories of the gaseous comae of several comets are reviewed. The earliest of these led to discovery of the hydrogen envelope extending for millions of km from the nucleus. Subsequent observations of H I Lyman alpha, the OH (0,0 band and the oxygen resonance triplet provided strong evidence for the water-ice model of the cometary nucleus. Several species were discovered in the coma including C, C(+), CO, S, and CS. High resolution spectroscopy and the spatial variation of the observed emissions provide means to elucidate the production and excitation mechanisms of these species. The similarity of the spectra of the half dozen comets observed to date argues for a common, homogeneous composition (with the exception of dust and CO) of the cometary ice and a minimal effect on the neutral species due to molecular collisions in the inner coma.

  9. Antigenotoxicity of artepillin C in vivo evaluated by the micronucleus and comet assays.

    PubMed

    de Azevedo Bentes Monteiro Neto, Moacir; de Souza Lima, Ildercílio Mota; Furtado, Ricardo Andrade; Bastos, Jairo Kenupp; da Silva Filho, Ademar Alves; Tavares, Denise Crispim

    2011-11-01

    Artepillin C (3,5-diprenyl-p-coumaric acid), a major compound found in Brazilian green propolis and Baccharis dracunculifolia, shows anti-inflammatory, antibacterial, antiviral, antioxidant and antitumoral activities, among others. The aim of this study was to evaluate the genotoxic potential of artepillin C and its ability to prevent the chemically induced chromosome breakage or loss and the primary DNA damage using the micronucleus and comet assays in male Swiss mice, respectively. The animals were treated by gavage with different doses of artepillin C (0.4, 0.8 and 1.6 mg kg(-1) b.w.). For the antigenotoxicity assays, the different doses of artepillin C were administered simultaneously to doxorubicin (DXR; micronucleus test; 15 mg kg(-1) b.w.) and to methyl methanesulfonate (MMS; comet assay; 40 mg kg(-1) b.w.). The results showed that artepillin C itself was not genotoxic in the mouse micronucleus and comet assays. In the animals treated with artepillin C and DXR, the number of micronucleated reticulocytes was significantly lower in comparison with the animals treated only with DXR. Regarding antigenotoxicity, artepillin C at the tested doses significantly reduced the extent of DNA damage in liver cells induced by MMS.

  10. In vivo genotoxic effect of nickel chloride in mice leukocytes using comet assay.

    PubMed

    Danadevi, K; Rozati, R; Saleha Banu, B; Grover, P

    2004-05-01

    DNA damage induced by nickel chloride (NiCl2) in leucocytes of Swiss albino mice has been studied in vivo. The comet assay or the alkaline single cell gel electrophoresis (SCGE) assay was used to measure the DNA damage. The mice were administered orally with acute doses of 3.4, 6.8, 13.6, 27.2, 54.4 and 108.8 mg/kg body weight (b.wt.) NiCl2. Samples of whole blood were collected at 24, 48 and 72 h, first week and second week post-treatment for alkaline SCGE assay to study single/double strand breaks in DNA. A significant increase in mean comet tail length indicating DNA damage was observed with NiCl2 at 24, 48 and 72 h post-treatment (P<0.05). A gradual decrease in the mean tail length was observed at 72 h post-treatment indicating repair of the damaged DNA. The mean tail length showed a dose-related increase and time dependent decrease after treatment with NiCl2 when compared to controls. The study also confirms that the comet assay is a sensitive and rapid method to detect DNA damage caused by heavy metals like nickel (Ni).

  11. Does the duration of lysis affect the sensitivity of the in vitro alkaline comet assay?

    PubMed

    Enciso, José Manuel; Sánchez, Oscar; López de Cerain, Adela; Azqueta, Amaya

    2015-01-01

    The alkaline comet assay is now the method of choice for measuring different kinds of DNA damage in cells. Several attempts have been made to identify and evaluate the critical points affecting the comet assay outcome, highlighting the requirement of arriving at a standardised protocol in order to be able to compare the results obtained in different laboratories. However, reports on the effect of modifying the time of lysis are lacking. Here we tested different times of lysis (from no lysis to 1 week) in control HeLa cells and HeLa cells treated with different concentrations of methyl methanesulfonate (MMS) or H2O2. We also tested different times of lysis in the comet assay combined with formamidopyrimidine DNA glycosylase (FPG) in untreated and Ro 19-8022 plus light-treated HeLa cells. The same DNA damage levels were detected in the absence of lysis or after 1h of lysis when the standard comet assay was used to detect the MMS- and H2O2-induced lesions; the response increased when longer lysis was used, up to at least 1 week. When FPG was used, a minimum lysis period of 5 min was necessary to allow the enzyme to reach the DNA; the same DNA damage levels were detected after 5 min or 1h of lysis and the response increased up to 24h. In conclusion, the time of lysis can be varied depending on the sensitivity needed in both versions of the assay, and a constant time of lysis should be used if results from different experiments or laboratories are to be compared.

  12. Genotoxicity of nano/microparticles in in vitro micronuclei, in vivo comet and mutation assay systems

    PubMed Central

    Totsuka, Yukari; Higuchi, Takashi; Imai, Toshio; Nishikawa, Akiyoshi; Nohmi, Takehiko; Kato, Tatsuya; Masuda, Shuich; Kinae, Naohide; Hiyoshi, Kyoko; Ogo, Sayaka; Kawanishi, Masanobu; Yagi, Takashi; Ichinose, Takamichi; Fukumori, Nobutaka; Watanabe, Masatoshi; Sugimura, Takashi; Wakabayashi, Keiji

    2009-01-01

    Background Recently, manufactured nano/microparticles such as fullerenes (C60), carbon black (CB) and ceramic fiber are being widely used because of their desirable properties in industrial, medical and cosmetic fields. However, there are few data on these particles in mammalian mutagenesis and carcinogenesis. To examine genotoxic effects by C60, CB and kaolin, an in vitro micronuclei (MN) test was conducted with human lung cancer cell line, A549 cells. In addition, DNA damage and mutations were analyzed by in vivo assay systems using male C57BL/6J or gpt delta transgenic mice which were intratracheally instilled with single or multiple doses of 0.2 mg per animal of particles. Results In in vitro genotoxic analysis, increased MN frequencies were observed in A549 cells treated with C60, CB and kaolin in a dose-dependent manner. These three nano/microparticles also induced DNA damage in the lungs of C57BL/6J mice measured by comet assay. Moreover, single or multiple instillations of C60 and kaolin, increased either or both of gpt and Spi- mutant frequencies in the lungs of gpt delta transgenic mice. Mutation spectra analysis showed transversions were predominant, and more than 60% of the base substitutions occurred at G:C base pairs in the gpt genes. The G:C to C:G transversion was commonly increased by these particle instillations. Conclusion Manufactured nano/microparticles, CB, C60 and kaolin, were shown to be genotoxic in in vitro and in vivo assay systems. PMID:19725983

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

    PubMed

    Zhang, Huifeng; Spitz, Margaret R; Tomlinson, Gail E; Schabath, Matthew B; Minna, John D; Wu, Xifeng

    2002-01-01

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

  14. Genotoxic effects of Bismuth (III) oxide nanoparticles by Allium and Comet assay.

    PubMed

    Liman, Recep

    2013-09-01

    Genotoxic effects of Bismuth (III) oxide nanoparticles (BONPs) were investigated on the root cells of Allium cepa by Allium and Comet assay. A. cepa roots were treated with the aqueous dispersions of BONPs at five different concentrations (12.5, 25, 50, 75, and 100ppm) for 4h. Exposure of BONPs significantly increased mitotic index (MI) except 12.5ppm, total chromosomal aberrations (CAs) in Allium test. While stickiness chromosome laggards, disturbed anaphase-telophase and anaphase bridges were observed in anaphase-telophase cells, pro-metaphase and c-metaphase in other cells. A significant increase in DNA damage was also observed at all concentrations of BONPs except 12.5ppm by Comet assay. The results were also analyzed statistically by using SPSS for Windows; Duncan's multiple range test was performed. These results indicate that BONPs exhibit genotoxic activity in A. cepa root meristematic cells.

  15. DNA strand breaks (comet assay) in blood lymphocytes from wild bottlenose dolphins.

    PubMed

    Lee, Richard F; Bulski, Karrie; Adams, Jeffrey D; Peden-Adams, Margie; Bossart, Gregory D; King, Lydia; Fair, Patricia A

    2013-12-15

    The comet assay was carried out on blood lymphocytes from a large number of wild dolphins (71 from Indian River Lagoon, FL, USA; 51 from Charleston Harbor, SC, USA) and provides a baseline study of DNA strand breaks in wild dolphin populations. There were no significant differences in the comet assay (% DNA in tail) results between the different age and sex categories. Significant difference in DNA strand breaks were found between Charleston Harbor dolphins (median--17.4% DNA in tail) and Indian River Lagoon dolphins (median--14.0% DNA in tail). A strong correlation found between T-cell proliferation and DNA strand breaks in dolphin lymphocytes suggests that dolphins with a high numbers of DNA strand breaks have a decreased ability to respond to infection. Higher concentrations of genotoxic agents in Charleston Harbor compared with Indian River lagoon may have been one of the causes of higher DNA strand breaks in these dolphins. Copyright © 2013. Published by Elsevier Ltd.

  16. Lack of genotoxicity of formocresol, paramonochlorophenol, and calcium hydroxide on mammalian cells by comet assay.

    PubMed

    Ribeiro, Daniel Araki; Marques, Mariângela Esther Alencar; Salvadori, Daisy Maria Fávero

    2004-08-01

    Formocresol, paramonochlorophenol, and calcium hydroxide are widely used in dentistry because of their antibacterial activities in root canal disinfection. However, the results of genotoxicity studies using these materials are inconsistent in literature. The goal of this study was to examine the genotoxic potential of formocresol, paramonochlorophenol, and calcium hydroxide using mouse lymphoma cells and human fibroblasts cells in vitro by the comet assay. Data were assessed by Kruskal-Wallis nonparametric test. The results showed that all compounds tested did not cause DNA damage for the tail moment or tail intensity parameters. These findings suggest that formocresol, paramonochlorophenol, and calcium hydroxide do not promote DNA damage in mammalian cells and that the comet assay is a suitable tool to investigate genotoxicity.

  17. Genotoxicity of Aflatoxin B1 and Ochratoxin A after simultaneous application of the in vivo micronucleus and comet assay.

    PubMed

    Corcuera, Laura-Ana; Vettorazzi, Ariane; Arbillaga, Leire; Pérez, Noemí; Gil, Ana Gloria; Azqueta, Amaya; González-Peñas, Elena; García-Jalón, Jose Antonio; López de Cerain, Adela

    2015-02-01

    Aflatoxin B1 (AFB1) and Ochratoxin A (OTA) are genotoxic mycotoxins that can contaminate a variety of foodstuffs, the liver and the kidney being their target organs, respectively. The micronucleus (MN) assay (bone marrow) and the comet assay (liver and kidney) were performed simultaneously in F344 rats, treated with AFB1 (0.25 mg/kg b.w.), OTA (0.5 mg/kg b.w.) or both mycotoxins. After AFB1 treatment, histopathology and biochemistry analysis showed liver necrosis, focal inflammation and an increase in Alanine Aminotransferase and Aspartate Aminotransferase. OTA alone did not cause any alteration. The acute hepatotoxic effects caused by AFB1 were less pronounced in animals treated with both mycotoxins. With regard to the MN assay, after 24 h, positive results were obtained for AFB1 and negative results were obtained for OTA, although both toxins caused bone marrow toxicity. In the combined treatment, OTA reduced the toxicity and the number of MN produced by AFB1. In the comet assay, after 3 h, positive results were obtained for AFB1 in the liver and for OTA in the kidney. The combined treatment reduced DNA damage in the liver and had no influence in the kidney. Altogether, these results may be indicative of an antagonistic relationship regarding the genotoxicity of both mycotoxins.

  18. Orbital error analysis for comet Encke, 1980

    NASA Technical Reports Server (NTRS)

    Yeomans, D. K.

    1976-01-01

    Before a particular comet is selected as a flyby target, the following criteria should be considered in determining its ephemeris uncertainty: (1) A target comet should have good observability during the apparition of the proposed intercept; and (2) A target comet should have a good observational history. Several well observed and consecutive apparitions allow an accurate determination of a comet's mean motion and nongravitational parameters. Using these criteria, along with statistical and empirical error analyses, it has been demonstrated that the 1980 apparition of comet Encke is an excellent opportunity for a cometary flyby space probe. For this particular apparition, a flyby to within 1,000 km of comet Encke seems possible without the use of sophisticated and expensive onboard navigation instrumentation.

  19. 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.

  20. 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

  1. 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 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. © The Author 2016. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society.

  2. 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

  3. Evaluation of effect during cell isolation process in alkaline comet assay using epidermal skin cells.

    PubMed

    Toyoizumi, Tomoyasu; Watanabe, Mika; Sui, Hajime; Nakagawa, Yuzuki; Ohta, Ryo; Yamakage, Kohji

    2012-01-01

    The aim of the present study was to evaluate the effect of the cell isolation process in the alkaline comet assay using epidermal skin cells. When we explored the cell isolation method for the alkaline comet assay using the 3-dimensional (3D) human epidermal skin model, we found that DNA damage and cytotoxicity were induced during the cell isolation process. In particular, trypsin 5 min treatment with ethylene diamine tetraacetic acid (EDTA) showed about 5 times %DNA in the tail value compared to without EDTA treatment. In general, EDTA is commonly used for cell isolation, but it is known to induce genotoxicity due to secondary effects. We therefore evaluated the effect of EDTA and pH in the alkaline comet assay on a monolayer culture of rat keratinocytes. As a result, there was a significant increase of %DNA in tail values by treatment with 0.1 w/v% EDTA for 60 min; however, there was no difference in the %DNA in tail values between 0.1 w/v% EDTA/PBS(-) (pH 6.8) and 0.1 w/v% EDTA/PBS(-) (pH 7.4). These data imply that there is a need to control the EDTA conditions for cell isolation in the epidermal skin cells.

  4. Genotoxicity evaluation of dental restoration nanocomposite using comet assay and chromosome aberration test

    NASA Astrophysics Data System (ADS)

    Musa, Marahaini; Thirumulu Ponnuraj, Kannan; Mohamad, Dasmawati; Rahman, Ismail Ab

    2013-01-01

    Nanocomposite is used as a dental filling to restore the affected tooth, especially in dental caries. The dental nanocomposite (KelFil) for tooth restoration used in this study was produced by the School of Dental Sciences, Universiti Sains Malaysia, Malaysia and is incorporated with monodispersed, spherical nanosilica fillers. The aim of the study was to determine the genotoxic effect of KelFil using in vitro genotoxicity tests. The cytotoxicity and genotoxicity of KelFil was evaluated using MTT assay, comet assay and chromosome aberration tests with or without the addition of a metabolic activation system (S9 mix), using the human lung fibroblast cell line (MRC-5). Concurrent negative and positive controls were included. In the comet assay, no comet formation was found in the KelFil groups. There was a significant difference in tail moment between KelFil groups and positive control (p < 0.05). Similarly, no significant aberrations in chromosomes were noticed in KelFil groups. The mitotic indices of treatment groups and negative control were significantly different from positive controls. Hence, it can be concluded that the locally produced dental restoration nanocomposite (KelFil) is non-genotoxic under the present test conditions.

  5. 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.

  6. In vivo comet assay of a novel galacto-oligosaccharide in rats.

    PubMed

    Narumi, K; Takasawa, H; Ohyama, W; Kaneko, K

    2014-05-01

    A novel galacto-oligosaccharide (GOS) manufactured by a two-step enzyme reaction of lactose was examined in a comet assay for its potential to induce DNA damage in vivo by estimating the DNA fragmentation level in the cellular nuclei of the glandular stomach, colon, and peripheral blood. GOS was orally administered at doses of 0 (vehicle alone), 500, 1000, and 2000 mg/kg/day to five male Crl: CD(Sprague Dawley) rats per group three times (48, 24, and 3 h before the animals were terminated). The specimens were prepared in accordance with the standard protocol (version 14.2) of the "International Validation of the In Vivo Rodent Alkaline Comet Assay for the Detection of Genotoxic Carcinogens" organized by the Japanese Center for the Validation of Alternative Methods. No significant differences in the percentage of DNA in the tail were observed between the GOS-treated groups and vehicle controls in any of the organs evaluated. Additionally, no GOS-related clinical signs or effects on body weight were seen. Based on these results, the comet assay of GOS in the glandular stomach, colon, and peripheral blood using rats was judged negative. Therefore, it is concluded that GOS did not induce DNA damage in vivo under the conditions employed in this study.

  7. Oxygenated water does not induce genotoxic effects in the comet assay.

    PubMed

    Speit, Günter; Schütz, Petra; Trenz, Kristina; Rothfuss, Andreas

    2002-07-21

    Drinking of oxygenated water (i.e. water with increased concentration of physically dissolved oxygen) is said to improve oxygen availability of the body and will do the consumer good. However, increased oxygen concentrations can also lead to an increased production of reactive oxygen species (ROS). If antioxidant defences are not completely efficient, ROS can cause cell injury including DNA damage. We therefore investigated whether drinking of oxygenated water can lead to increased DNA damage in peripheral blood cells of test subjects. We also tested whether direct exposure of V79 Chinese hamster cells to oxygenated medium or oxygenated Hank's solution for various time periods induces DNA damage. Induction of DNA damage was measured with the alkaline comet assay (single cell gel electrophoresis). The comet assay, in particular the modification with FPG post-treatment for the determination of oxidative DNA base damage, has been proven to be extremely sensitive for the detection of oxygen-induced DNA damage. However, both the in vivo and the in vitro studies with the comet assay in the absence and presence of FPG post-treatment did not provide evidence for a genotoxic effect of oxygenated water.

  8. 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.

  9. 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.

  10. Analysis of Returned Comet Nucleus Samples

    NASA Technical Reports Server (NTRS)

    Chang, Sherwood (Compiler)

    1997-01-01

    This volume contains abstracts that have been accepted by the Program Committee for presentation at the Workshop on Analysis of Returned Comet Nucleus Samples, held in Milpitas, California, January 16-18, 1989. Conveners are Sherwood Chang (NASA Ames Research Center) and Larry Nyquist (NASA Johnson Space Center). Program Committee members are Thomas Ahrens (ex-officio; California Institute of Technology), Lou Allamandola (NASA Ames Research Center), David Blake (NASA Ames Research Center), Donald Brownlee (University of Washington, Seattle), Theodore E. Bunch (NASA Ames Research Center), Humberto Campins (Planetary Science Institute), Jeff Cuzzi (NASA Ames Research Center), Eberhard Griin (Max-Plank-Institut fiir Kemphysik), Martha Hanner (Jet Propulsion Laboratory), Alan Harris (Jet Propulsion Laboratory), John Kerrid-e (University of Califomia, Los Angeles), Yves Langevin (University of Paris), Gerhard Schwehm (ESTEC), and Paul Weissman (Jet Propulsion Laboratory). Logistics and administrative support for the workshop were provided by the Lunar and Planetary Institute Projects Office.

  11. Analysis of Returned Comet Nucleus Samples

    NASA Astrophysics Data System (ADS)

    Chang, Sherwood

    1997-12-01

    This volume contains abstracts that have been accepted by the Program Committee for presentation at the Workshop on Analysis of Returned Comet Nucleus Samples, held in Milpitas, California, January 16-18, 1989. Conveners are Sherwood Chang (NASA Ames Research Center) and Larry Nyquist (NASA Johnson Space Center). Program Committee members are Thomas Ahrens (ex-officio; California Institute of Technology), Lou Allamandola (NASA Ames Research Center), David Blake (NASA Ames Research Center), Donald Brownlee (University of Washington, Seattle), Theodore E. Bunch (NASA Ames Research Center), Humberto Campins (Planetary Science Institute), Jeff Cuzzi (NASA Ames Research Center), Eberhard Griin (Max-Plank-Institut fiir Kemphysik), Martha Hanner (Jet Propulsion Laboratory), Alan Harris (Jet Propulsion Laboratory), John Kerrid-e (University of Califomia, Los Angeles), Yves Langevin (University of Paris), Gerhard Schwehm (ESTEC), and Paul Weissman (Jet Propulsion Laboratory). Logistics and administrative support for the workshop were provided by the Lunar and Planetary Institute Projects Office.

  12. Measurement of DNA damage in individual cells using the Single Cell Gel Electrophoresis (Comet) assay.

    PubMed

    Hartley, Janet M; Spanswick, Victoria J; Hartley, John A

    2011-01-01

    The Single Cell Gel Electrophoresis (Comet) assay is a simple, versatile and sensitive method for measuring DNA damage in individual cells, allowing the determination of heterogeneity of response within a cell population. The basic alkaline technique described is for the determination of DNA strand break damage and its repair at a single cell level. Specific modifications to the method use a lower pH ('neutral' assay), or allow the measurement of DNA interstrand cross-links. It can be further adapted to, for example, study specific DNA repair mechanisms, be combined with fluorescent in situ hybridisation, or incorporate lesion specific enzymes.

  13. 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.

  14. 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

  15. Chaga mushroom extract inhibits oxidative DNA damage in human lymphocytes as assessed by comet assay.

    PubMed

    Park, Yoo Kyoung; Lee, Hyang Burm; Jeon, Eun-Jae; Jung, Hack Sung; Kang, Myung-Hee

    2004-01-01

    The Chaga mushroom (Inonotus obliquus) is claimed to have beneficial properties for human health, such as anti-bacterial, anti-allergic, anti-inflammatory and antioxidant activities. The antioxidant effects of the mushroom may be partly explained by protection of cell components against free radicals. We evaluated the effect of aqueous Chaga mushroom extracts for their potential for protecting against oxidative damage to DNA in human lymphocytes. Cells were pretreated with various concentrations (10, 50, 100 and 500 microg/mL) of the extract for 1 h at 37 degrees C. Cells were then treated with 100 microM of H2O2 for 5 min as an oxidative stress. Evaluation of oxidative damage was performed using single-cell gel electrophoresis for DNA fragmentation (Comet assay). Using image analysis, the degree of DNA damage was evaluated as the DNA tail moment. Cells pretreated with Chaga extract showed over 40% reduction in DNA fragmentation compared with the positive control (100 micromol H2O2 treatment). Thus, Chaga mushroom treatment affords cellular protection against endogenous DNA damage produced by H2O2.

  16. 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.

  17. A modified alkaline Comet assay for in vivo detection of oxidative DNA damage in Drosophila melanogaster.

    PubMed

    Shukla, A K; Pragya, P; Chowdhuri, D Kar

    2011-12-24

    Modifications to the alkaline Comet assay by using lesion-specific endonucleases, such as formamidopyrimidine-DNA glycosylase (FPG) and endonuclease III (ENDOIII, also known as Nth), can detect DNA bases with oxidative damage. This modified assay can be used to assess the genotoxic/carcinogenic potential of environmental chemicals. The goal of this study was to validate the ability of this modified assay to detect oxidative stress-induced genotoxicity in Drosophila melanogaster (Oregon R(+)). In this study, we used three well known chemical oxidative stress inducers: hydrogen peroxide (H(2)O(2)), cadmium chloride (CdCl(2)) and copper sulfate (CuSO(4)). Third instar larvae of D. melanogaster were fed various concentrations of the test chemicals (50-200μM) mixed with a standard Drosophila food for 24h. Alkaline Comet assays with and without the FPG and ENDOIII enzymes were performed with midgut cells that were isolated from the control and treated larvae. Our results show a concentration-dependent increase (p<0.05-0.001) in the migration of DNA from the treated larvae. ENDOIII treatment detected more oxidative DNA damage (specifically pyrimidine damage) in the H(2)O(2) exposed larvae compared to FPG or no enzyme treatment (buffer only). In contrast, FPG treatment detected more oxidative DNA damage (specifically purine damage) in CuSO(4) exposed larvae compared to ENDOIII. Although previously reported to be a potent genotoxic agent, CdCl(2) did not induce more oxidative DNA damage than the other test chemicals. Our results show that the modified alkaline Comet assay can be used to detect oxidative stress-induced DNA damage in D. melanogaster and thus may be applicable for in vivo genotoxic assessments of environmental chemicals.

  18. A new approach for the oocyte genotoxicity assay: adaptation of comet assay on mouse cumulus-oocyte complexes.

    PubMed

    Greco, F; Perrin, J; Auffan, M; Tassistro, V; Orsière, T; Courbiere, B

    2015-07-01

    Conventional genotoxicity tests are technically difficult to apply to oocytes, and results obtained on somatic cells cannot be extrapolated to gametes. We have previously described a comet assay (original-CA) on denuded mouse oocytes, but, in vivo, oocytes are not isolated from their surrounding follicular cells. Our objective was to develop a comet assay on cumulus-oocyte complexes (COC-CA) for a more physiological approach to study the genotoxicity of environmental factors on oocytes. For COC-CA, whole COC were exposed directly to exogenous agents after ovulation and removal from oviducts. Three conditions were studied: a negative control group, and two positive control groups, one of which was exposed to hydrogen peroxide (H2O2) and the other group was incubated with cerium dioxide nanoparticles (CeO2 NPs). With both tests, DNA damage was significant in the presence of both H2O2 and CeO2 NPs compared with the negative control. COC-CA offers an interesting tool for assaying the genotoxicity of environmental agents towards germinal cells. Furthermore, COC-CA is less time-consuming and simplifies the protocol of the original-CA, because COC-CA is easier to perform without the washing-out procedure.

  19. Evaluation of pH effects on genomic integrity in adipose-derived mesenchymal stem cells using the comet assay.

    PubMed

    Hermeto, L C; Oliveira, R J; Matuo, R; Jardim, P H A; DeRossi, R; Antoniolli, A C M B; Deffune, E; Evaristo, T C; Santana, Á E

    2015-01-23

    The use of mesenchymal stem cells (MSCs) in experimental, clinical, and therapeutic trials has grown in recent years. However, the issue remains of whether these procedures are completely safe for transplant patients. Therefore, this study was designed and carried out with the aim of evaluating two different comet assay protocols for genomic damage pattern analysis in MSCs derived from adipose tissue. The analyzed and interpreted results suggest that genetic testing is needed to support clonal expansion safety in cell therapy procedures with MSCs. Furthermore, they also suggest that if the comet assay technique would be used as a genomic integrity screening assay, the protocol performed at pH = 12 (that yielded a frequency of damaged cells: tail intensity = 9.50 ± 0.60, tail moment = 0.0122 ± 0.0007; results are reported as means ± standard deviation) would be indicated as genomic damage, and that subsequent single-strand breaks occur at pH > 13 (frequency of damaged cells: tail intensity = 30.71 ± 4.23, tail moment = 0.0447 ± 0.0073). Our study demonstrates that, in the era of regenerative medicine, it is necessary to standardize and establish a battery of tests in order to identify genomic damage prior to MSC transplantation.

  20. The comet assay in higher terrestrial plant model: Review and evolutionary trends.

    PubMed

    Lanier, Caroline; Manier, Nicolas; Cuny, Damien; Deram, Annabelle

    2015-12-01

    The comet assay is a sensitive technique for the measurement of DNA damage in individual cells. Although it has been primarily applied to animal cells, its adaptation to higher plant tissues significantly extends the utility of plants for environmental genotoxicity research. The present review focuses on 101 key publications and discusses protocols and evolutionary trends specific to higher plants. General consensus validates the use of the percentage of DNA found in the tail, the alkaline version of the test and root study. The comet protocol has proved its effectiveness and its adaptability for cultivated plant models. Its transposition in wild plants thus appears as a logical evolution. However, certain aspects of the protocol can be improved, namely through the systematic use of positive controls and increasing the number of nuclei read. These optimizations will permit the increase in the performance of this test, namely when interpreting mechanistic and physiological phenomena.

  1. Application of the comet assay in erythrocytes of Oreochromis niloticus (Pisces): A methodological comparison

    PubMed Central

    2009-01-01

    The present study applied the comet assay to erythrocytes of Oreochromis niloticus with the aim of improving protocols to detect DNA damage in these cells, by using two distinct pHs (pH = 12.1 and pH > 13) and evaluating whether there is a correspondence between silver and ethidium bromide staining. Comets were visually examined and, the frequency of cells with and without damage was obtained, as well as the distribution of classes and scores. By using the Kruskal-Wallis test, our results revealed that pH 12.1 is more effective, although both pHs can be used. Our findings also suggest that silver staining can substitute ethidium bromide, an expensive and highly toxic stain that requires specific equipment for examination. PMID:21637662

  2. DNA damage in grasshoppers' larvae--comet assay in environmental approach.

    PubMed

    Augustyniak, Maria; Orzechowska, Helena; Kędziorski, Andrzej; Sawczyn, Tomasz; Doleżych, Bogdan

    2014-02-01

    The comet assay that provides a quantitative measure of the DNA-strand breaks may be used for assessing the 'genotoxic potential' of the environment. Young adults of Chorthippus brunneus (Orthoptera), collected at three sites in Southern Poland, differing in the level of pollution, particularly with heavy metals: Pilica (reference), Olkusz (moderately polluted) and Szopienice (heavily polluted) - were allowed to mate under laboratory conditions that were free from any pollution. Egg-pods were collected and, after diapause, brain cells from one-day old larvae were used for the comet assay. We compared the level of DNA damage in the larvae originating from these sites and also measured time-dependent DNA repair after single 10min. application of H2O2 (20μM final concentration). The DNA damage was relatively low in larval cells irrespectively of the site pollution their parents came from. However, measured comet parameters - tail DNA content (TDNA), tail length (TL), and olive tail moment (OTM) - were significantly higher in larvae originating from the Szopienice site than in those from the reference site. Incubation of cells with H2O2 resulted in significantly higher values of the comet parameters in the insects from all the study sites with the highest ones observed in the offspring of grasshoppers from Szopienice. Moreover, DNA repair, following the treatment, did not occur in the latter group. These data contribute to almost unexplored subject of genotoxic effects of environmental pollutants in insects. They are discussed in the light of the concept of adaptive strategies in energy allocation depending on the level of biotope pollution.

  3. 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.

  4. A combination of in vitro comet assay and micronucleus test using human lymphoblastoid TK6 cells.

    PubMed

    Kimura, Aoi; Miyata, Atsuro; Honma, Masamitsu

    2013-09-01

    The comet assay has been widely used as a genotoxicity test for detecting primary DNA damage in individual cells. The micronucleus (MN) test is also a well-established assay for detecting clastogenicity and aneugenicity. A combination of the comet assay (COM) and MN test is capable of detecting a variety of genotoxic potentials as an in vitro screening system. Although the in vitro MN test has a robust protocol and Organisation for Economic Co-operation and Development (OECD) test guideline, the in vitro COM does not. To establish a robust protocol for the COM and to compare its sensitivity with that of the MN, we conducted COM and MN concurrently for five genotoxic agents (ethyl methanesulfonate, methyl methanesulfonate, hydrogen peroxide, gamma-rays and mitomycin C) and one non-genotoxic agent (triton X-100), using human lymphoblastoid TK6 cells. Relative cell count (RCC), relative population doubling (RPD), relative increase in cell count (RICC) and relative cell viability determined by trypan blue dye-exclusion assay (TBDE) were employed as cytotoxic measurements. However, the relative cell viability determined by TBDE just after the treatment was not an appropriate parameter of cytotoxicity for the genotoxic agents because it remained constant even at the highest doses, which showed severe cytotoxicity by RCC, RPD and RICC. The results of the COM showed qualitative agreement (positive or negative) with those of the MN except for mitomycin C, which is an interstrand cross-linker. The COM always required higher doses than the MN to detect the genotoxic potential of the genotoxic agents under the test conditions applied here. The doses that induced a comet tail always yielded <50% RICC, and do not accord to the OECD test guideline for MN because of their high cytotoxicity. These results are helpful for interpreting the results of the COM and MN in in vitro genotoxic hazard assessments. Further investigation is required to standardise the COM.

  5. Combination comet/micronucleus assay validation performed by BioReliance under the JaCVAM initiative.

    PubMed

    Pant, Kamala; Krsmanovic, Ljubica; Bruce, Shannon Wilson; Kelley, Tawney; Arevalo, Mirna; Atta-Safoh, Samuel; Debelie, Fekadu; La Force, Michelle L Klug; Springer, Sandra; Sly, Jamie; Paranjpe, Madhav; Lawlor, Timothy; Aardema, Marilyn

    2015-07-01

    In the international validation study of the in vivo rat alkaline comet assay (comet assay), the Japanese Center for the Validation of Alternative Methods (JaCVAM) provided three coded chemicals to BioReliance, 1,3-dichloropropene, ethionamide and busulfan, to be tested in a combined in vivo comet/micronucleus assay. Induction of DNA damage (comet) in liver, stomach and jejunum (1,3-dichloropropene only) cells, and induction of MNPCEs in bone marrow, were examined in male Sprague-Dawley (Hsd:SD) rats following oral administration of the test chemical for three consecutive days. A dose range finding (DRF) test was performed with each chemical to determine the maximum tolerated dose (MTD). Based on the results of the DRF test; 1,3-dichloropropene was tested at 50, 100 and 200 mg/kg/day; ethionamide was tested at 125, 250 and 500 mg/kg/day, and busulfan was tested at 10, 20 and 40 mg/kg/day. The results indicated that 1,3-dichloropropene induced DNA damage only in liver cells at all three test article doses, while no effects were observed in the stomach and jejunum cells. Additionally, it did not increase MNPCEs in the bone marrow. 1,3-Dichloropropene was concluded to be negative in the MN assay but positive in the comet assay. Ethionamide did not induce DNA damage in liver. However, in stomach, statistically significant decreases (although still within historical range) in % tail DNA at all test article doses compared to the vehicle control were observed. There was no increase in MNPCEs in the bone marrow. Thus, ethionamide was concluded to be negative in the comet/MN combined assay. Busulfan did not induce DNA damage in any of the organs tested (liver and stomach) but it did induce a significant increase in MNPCEs in the bone marrow. Busulfan was concluded to be negative in the comet assay but positive in the MN assay.

  6. In vitro mutagenicity and genotoxicity study of a number of short-chain chlorinated hydrocarbons using the micronucleus test and the alkaline single cell gel electrophoresis technique (Comet assay) in human lymphocytes: a structure-activity relationship (QSAR) analysis of the genotoxic and cytotoxic potential.

    PubMed

    Tafazoli, M; Baeten, A; Geerlings, P; Kirsch-Volders, M

    1998-03-01

    Using the micronucleus (MN) test and the alkaline single cell gel electrophoresis (Comet) assay, potential mutagenicity (MN formation), genotoxicity (DNA breakage capacity) and cytotoxicity (cell proliferation reduction) of five chlorinated hydrocarbons (carbon tetrachloride, hexachloroethane, 1,2-dichloroethane, 1-chlorohexane and 2,3-dichlorobutane) have been evaluated in isolated human lymphocytes. With the MN test a low but statistically significant mutagenic activity was detected for all tested substances (except 2,3-dichlorobutane) with one out of the two donors and in the presence or absence of an exogenous metabolic activation system (S9 mix). However, at the concentration ranges tested none of the positive compounds induced a clear dose-dependent mutagenic effect. The Comet assay detected a strong DNA damaging effect for 1-chlorohexane, 2,3-dichlorobutane and 1,2-dichloroethane, but not for carbon tetrachloride and hexachloroethane. The influence of metabolism on the genotoxic activity of the chemicals was more clear in the Comet assay than in the MN test. The experimental genotoxicity and cytotoxicity data obtained in this study, together with data on five more related chemicals previously investigated, and their physico-chemical descriptors or electronic parameters have been used for QSAR analysis. The QSAR analysis high-lighted that the toxicity of the tested compounds was influenced by different parameters, like lipophilicity (logP), electron donor ability (charge) and longest carbon-chlorine (LBC-Cl) bond length. In addition, steric parameters, like molar refractivity (MR) and LBC-Cl, and electronic parameters, like ELUMO (energy of the lowest unoccupied molecular orbital, indicating electrophilicity), were predominant factors discriminating genotoxins from non-genotoxins in the presence but not in the absence of S9 mix. Although a limited number of compounds have been examined and cytotoxicity and genotoxicity were identified in two different

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. Genotoxic effect of lead nitrate on mice using SCGE (comet assay).

    PubMed

    Devi, K D; Banu, B S; Grover, P; Jamil, K

    2000-04-14

    Single stranded DNA breakage induced by lead nitrate in mice has been studied in vivo using alkaline single cell gel electrophoresis (comet assay). Mice were administered orally 0.7, 1.4, 2.8, 5.6, 11. 2, 22.4, 44.8 and 89.6 mg/kg body weight of lead nitrate and the assay was performed on whole blood at 24, 48, 72 h, 1st and 2nd week. Significant increase in mean tail-length of DNA was observed at all time intervals after treatment with lead nitrate when compared to controls. The mean tail-length did not show a dose-related increase and the elevation in the mean tail-length was of a fluctuating type. Increase in mean tail-lengths clearly gives evidence that lead nitrate causes DNA damage effectively. The study indicates that the alkaline comet assay is a sensitive and rapid method to detect DNA damage caused by heavy metals.

  14. 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

  15. Autonomous Onboard Science Data Analysis for Comet Missions

    NASA Technical Reports Server (NTRS)

    Thompson, David R.; Tran, Daniel Q.; McLaren, David; Chien, Steve A.; Bergman, Larry; Castano, Rebecca; Doyle, Richard; Estlin, Tara; Lenda, Matthew

    2012-01-01

    Coming years will bring several comet rendezvous missions. The Rosetta spacecraft arrives at Comet 67P/Churyumov-Gerasimenko in 2014. Subsequent rendezvous might include a mission such as the proposed Comet Hopper with multiple surface landings, as well as Comet Nucleus Sample Return (CNSR) and Coma Rendezvous and Sample Return (CRSR). These encounters will begin to shed light on a population that, despite several previous flybys, remains mysterious and poorly understood. Scientists still have little direct knowledge of interactions between the nucleus and coma, their variation across different comets or their evolution over time. Activity may change on short timescales so it is challenging to characterize with scripted data acquisition. Here we investigate automatic onboard image analysis that could act faster than round-trip light time to capture unexpected outbursts and plume activity. We describe one edge-based method for detect comet nuclei and plumes, and test the approach on an existing catalog of comet images. Finally, we quantify benefits to specific measurement objectives by simulating a basic plume monitoring campaign.

  16. Autonomous Onboard Science Data Analysis for Comet Missions

    NASA Technical Reports Server (NTRS)

    Thompson, David R.; Tran, Daniel Q.; McLaren, David; Chien, Steve A.; Bergman, Larry; Castano, Rebecca; Doyle, Richard; Estlin, Tara; Lenda, Matthew

    2012-01-01

    Coming years will bring several comet rendezvous missions. The Rosetta spacecraft arrives at Comet 67P/Churyumov-Gerasimenko in 2014. Subsequent rendezvous might include a mission such as the proposed Comet Hopper with multiple surface landings, as well as Comet Nucleus Sample Return (CNSR) and Coma Rendezvous and Sample Return (CRSR). These encounters will begin to shed light on a population that, despite several previous flybys, remains mysterious and poorly understood. Scientists still have little direct knowledge of interactions between the nucleus and coma, their variation across different comets or their evolution over time. Activity may change on short timescales so it is challenging to characterize with scripted data acquisition. Here we investigate automatic onboard image analysis that could act faster than round-trip light time to capture unexpected outbursts and plume activity. We describe one edge-based method for detect comet nuclei and plumes, and test the approach on an existing catalog of comet images. Finally, we quantify benefits to specific measurement objectives by simulating a basic plume monitoring campaign.

  17. Genotoxicity evaluation of benzene, di(2-ethylhexyl) phthalate, and trisodium ethylenediamine tetraacetic acid monohydrate using a combined rat comet/micronucleus assays.

    PubMed

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

    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 alkaline comet assay (comet assay), we examined DNA damage in the liver, stomach, and bone marrow of rats dosed orally three times with up to 2000 mg/kg of benzene, di(2-ethylhexyl) phthalate, and trisodium ethylenediamine tetraacetic acid monohydrate. All three compounds gave negative results in the liver and stomach. In addition, a bone marrow comet and micronucleus analysis revealed that benzene, but not di(2-ethylhexyl) phthalate or trisodium ethylenediamine tetraacetic acid monohydrate induced a significant increase in the median % tail DNA and micronucleated polychromatic erythrocytes, compared with the respective concurrent vehicle control. These results were in good agreement with the previously reported genotoxicity findings for each compound. The present study has shown that combining the micronucleus test with the comet assay and carrying out these analyses simultaneously is effective in clarifying the mechanism of action of genotoxic compounds such as benzene.

  18. The comet assay of nasal epithelia: measurement of DNA damage for the assessment of genotoxic air pollution.

    PubMed

    Glück, U; Gebbers, J O

    2000-01-01

    The alkaline single cell gel electrophoresis or "comet" assay allows measurement of DNA damage in single cells with a high degree of sensitivity, e.g., for investigations of the effect of environmental agents with DNA-damaging potential. This study aimed to adapt this test to respiratory cells of the human nasal mucosa to examine the genotoxic effect of air pollution (cigarette smoke). In a prospective study, nasal epithelia of 16 cigarette smokers were examined by the adapted comet assay and the results were correlated with the results of the Papanicolaou-stained nasal cytology, carried out in a blinded fashion. The control group comprised 20 non-smoking men. All subjects under investigation were healthy office workers. Nasal epithelia were harvested from the maxilloturbinates. One part of cells was Papanicolaou stained and evaluated by cytopathologists. The comet assay was performed on the other part of the cells. The examiners were blinded to the study and control groups. Among cigarette smokers, a significant correlation between cytopathological cell nucleus changes (metaplasia and dysplasia) and the DNA migration (tail lengths) in the comet assay was found as a sign of DNA damage. This was not found in nonsmoking control persons. These results confirm the sensitivity of the comet assay and the hypothesis that cell nucleus changes in conventional nasal cytology are associated with DNA damage.

  19. In vivo genotoxicity assessment of sertraline by using alkaline comet assay and the cytokinesis-block micronucleus assay.

    PubMed

    Battal, Dilek; Aktas, Ayca; Sungur, Mehmet Ali; Kadioglu, Ela; Eker, Ebru Derici; Sahin, Nefise Ozlen; Saygi, Sahan

    2013-11-01

    Sertraline, a leading antidepressant in the selective serotonin reuptake inhibitor (SSRI) group of medicine, is the most frequently prescribed drug. In this study, the alkaline comet assay and the cytokinesis-block micronucleus (CBMN) assay were used to investigate genotoxicity potential of sertraline in the peripheral blood lymphocytes (PBLs) of acute and chronic sertraline-treated Wistar albino rats. Male Wistar albino rats (n = 48) were administered low, medium and high doses of sertraline (10, 40, 80 mg/kg) for acute and chronic treatment by employing the gavage method to investigate genotoxicity of the administered drug. The data (tail length, tail intensity and tail moment) were analysed and indicated that there was no statistically significant difference between sertraline-treated groups and the negative control group with respect to DNA damage (p > 0.05). However, it was observed that acute sertraline administration had caused much more DNA damage in comparison with chronic treatment (p < 0.05). According to the data obtained from the CBMN test, an increase in the micronucleus (MN) frequency was detected at chronic and high-dose acute sertraline treatment. Based on the outcome of comet assay, detection of statistically insignificant DNA damage may be due to the fact that sertraline did not cause damage on DNA. Also, increase in frequency of MN in chronic sertraline treatment suggests that chronic sertraline administration might influence some mechanisms of cell division. Therefore, dose adjustment in depressed patients seems significant as it may help prevent further prognosis of the diseases.

  20. Dose-response relationship of temozolomide, determined by the Pig-a, comet, and micronucleus assay.

    PubMed

    Guérard, M; Johnson, G; Dertinger, S; Duran-Pacheco, G; Funk, J; Zeller, A

    2017-02-15

    Temozolomide (TMZ), a monofunctional alkylating agent, was selected as a model compound to determine its quantitative genotoxic dose-response relationship in different tissues (blood, liver, and jejunum) and endpoints [Pig-a-, comet-, and micronucleus assay (MNT)] in male rats. TMZ was administered p.o. over 5 consecutive days (day 1-5), followed by a treatment-free period of 50 days (day 6-56) and a final administration prior to necropsy (day 57-59). TMZ showed a dose-dependent increase in DNA damage in all interrogated endpoints. A statistically significant increase in Pig-a mutant phenotypes was observed on day 44 starting at 7.5 mg/kg/day for mutant reticulocytes (for RET(CD59-)) and at 3.75 mg/kg/day for mutant red blood cells (RBC(CD59-)), respectively. In addition, a statistically significant increase in cytogenetic damage, as measured by micronucleated reticulocytes, was observed starting at 3.75 mg/kg/day on day 3 and 1.5 mg/kg/day on day 59. DNA strand breaks, as detected by the comet assay, showed a dose-dependent and statistically significant increase in liver, blood, and jejunum starting at doses of 3.75, 3.75, and 7.5 mg/kg/day, respectively. The dose-response relationships of the Pig-a, MNT, and comet data were analyzed for possible points of departure (PoD) using the benchmark-dose (BMD) software PROAST with different critical effect sizes (CES) (BMD0.1, BMD0.5, BMD1, and BMD1SD). Overall, PoD values show a high concordance between different tissues and endpoints, underlining the suitability of this experimental design to explore quantitative dose-response relationships in a variety of different tissues and endpoints, while minimizing animal use.

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

    SciTech Connect

    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.

  2. Evaluation of genotoxic effects of lead in pottery-glaze workers using micronucleus assay, alkaline comet assay and DNA diffusion assay.

    PubMed

    Kašuba, V; Rozgaj, R; Milić, M; Zelježić, D; Kopjar, N; Pizent, A; Kljaković-Gašpić, Z; Jazbec, A

    2012-10-01

    We investigated genotoxic effects of occupational exposure to lead acetate in pottery-glaze ceramic workers. The study was carried out in 30 exposed workers and 30 matched controls, to whom several biochemical parameters-the blood lead (B-Pb; range: exposed, 41.68-404.77; controls, 12-52) and cadmium (B-Cd) level, the activity of delta-aminolevulinic acid dehydratase (ALAD), erythrocyte protoporphyrin (EP), the level of vitamin B(12) and folate in serum-were measured. The genotoxic effects were evaluated by the alkaline comet assay, the DNA diffusion assay and micronucleus test in peripheral blood lymphocytes. Subjects exposed to lead had significantly higher B-Pb level and, consequently, increased values of tail intensity (TI), frequency of apoptotic and necrotic cells, and frequency of micronuclei (MN). In contrast, their activity of ALAD, the level of vitamin B(12) and folate in serum were significantly lower compared to controls. Poisson regression analysis showed a significant correlation of profession, duration of exposure, smoking, level of cadmium in blood, ALAD and EP with primary DNA damage. A majority of primary damage repairs in a short period after exposure to a genotoxic agent. In addition, the influence of gender and level of vitamin B(12) and folate in serum MN frequency in exposed group was observed. In this study, DNA diffusion and micronucleus test showed higher influence of tested parameters to DNA damage. The results indicate a need for concomitant use of at least two different biomarkers of exposure when estimating a genetic risk of lead exposure.

  3. The 15 years of comet photometry: A comparative analysis of 80 comets

    NASA Technical Reports Server (NTRS)

    Osip, David J.; Schleicher, David G.; Millis, Robert L.; Ahearn, Michael F.; Birch, Peter V.

    1991-01-01

    In 1976, a program of narrowband photometry of comets was initiated that has encompassed well over 400 nights of observations. To date, the program has provided detailed information on 80 comets, 11 of which were observed during multiple apparitions. The filters (initially isolating CN, C2, and continuum and later including C3, OH, and NH) as well as the detectors used for the observations were changed over time, and the parameters adopted in the reduction and modeling of the data have likewise evolved. Accordingly, we have re-reduced the entire database and have derived production rates using current values for scalelengths and fluorescence efficiencies. Having completed this task, the results for different comets can now be meaningfully compared. The general characteristics that are discussed include ranges in composition (molecular production rate ratios) and dustiness (gas production compared with Af(rho)). Additionally an analysis of trends on how the production rates vary with heliocentric distance and on pre- and post-perihelion asymmetries in the production rates of individual comets. Possible taxonomic groupings are also described.

  4. 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.

  5. Genotoxicity of glyphosate assessed by the comet assay and cytogenetic tests.

    PubMed

    Mañas, Fernando; Peralta, Laura; Raviolo, José; Ovando, Hugo García; Weyers, Alicia; Ugnia, Laura; Cid, Marcela Gonzalez; Larripa, Irene; Gorla, Nora

    2009-07-01

    It was evaluated the genotoxicity of glyphosate which up to now has heterogeneous results. The comet assay was performed in Hep-2 cells. The level of DNA damage in the control group (5.42±1.83 arbitrary units) for tail moment (TM) measurements has shown a significant increase (p<0.01) with glyphosate at a range concentration from 3.00 to 7.50mM. In the chromosome aberrations (CA) test in human lymphocytes the herbicide (0.20-6.00mM) showed no significant effects in comparison with the control group. In vivo, the micronucleus test (MNT) was evaluated in mice at three doses rendering statistical significant increases at 400mg/kg (13.0±3.08 micronucleated erythrocytes/1000 cells, p<0.01). In the present study glyphosate was genotoxic in the comet assay in Hep-2 cells and in the MNT test at 400mg/kg in mice. Thiobarbituric acid reactive substances (TBARs) levels, superoxide dismutase (SOD) and catalase (CAT) activities were quantified in their organs. The results showed an increase in these enzyme activities.

  6. An evaluation of the relative sensitivity of two marine bivalve mollusc species using the Comet assay.

    PubMed

    Cheung, Victoria V; Depledge, Michael H; Jha, Awadhesh N

    2006-07-01

    The aim of this study was to (a) evaluate the potential for the 'Comet assay' to be used as a method for detecting genetic damage in the common cockle Cerastoderma edule; and (b) to compare the relative sensitivity with Mytilus edulis as the bivalve widely used as a sentinel species in biomonitoring studies. In vitro validation studies were carried out on haemocytes from each species using hydrogen peroxide (H2O2), a known oxidant and the induced DNA damage was measured using the Comet assay. On exposure to 0, 100, 500 and 1000 microM H2O2, a significant concentration-dependent increase was observed in both species. Use of an additional concentration of 5000 microM H2O2 showed that while DNA damage could be assessed in M. edulis at this concentration, only a few cells from C. edule were amenable to measurements owing to extensive DNA damage ("hedgehog cells"). The evidence also suggested that the cells from C. edule are more sensitive to oxidative damage induced by H2O2 when compared with M. edulis. Bearing in mind that sediments are the ultimate sink for many contaminants, this study demonstrates the potential application of sediment-dwelling C. edule as a useful biomonitoring species.

  7. Identification of DNA damage in marine fish Therapon jarbua by comet assay technique.

    PubMed

    Nagarani, N; Devi, V Janaki; Kumaraguru, A K

    2012-07-01

    The marine fish Therapon jarbua was exposed to acute concentration of mercuric chloride (HgCl2). In static acute toxicity bioassays at 24, 48, 72 and 96 hr LC50 values were estimated for each concentrations such as control, 2, 1, 0.5, 0.25 and 0.125 ppm, respectively. DNAdamage (single-strand break) was also studied in gill, kidney and blood tissues at single-cell levels in the specimens exposed to different acute doses of HgCl2, by applying single-cell electrophoresis (comet assay). Dose-dependent responses were observed in DNA damage in all tissues. A comparison of DNA damage in all tissue at two concentration namely, 0.125 and 0.25 ppm indicated that the gill cells (maximum damage as 249.3 and 289.7 AU) were more sensitive to the heavy metal exposure than kidney (maximum 225.17 AU) and blood cells (maximum 200.3 AU). This study explored the utility of the comet assay for in vivo laboratory studies using fish for screening the genotoxic potential for various agents.

  8. SILVER (NANO)MATERIALS CAUSE GENOTOXICITY IN ENCHYTRAEUS CRYPTICUS - AS DETERMINED BY THE COMET ASSAY.

    PubMed

    Maria, Vera L; Ribeiro, Maria João; Guilherme, Sofia; M Soares, Amadeu M V; Scott-Fordsmand, Janeck J; Amorim, Mónica J B

    2017-08-10

    Enchytraeids have been used in standard ecotoxicity testing for about 20 years now. Since adopting the standard test for survival and reproduction, a number of additional tools have been developed, including transcriptomics and enzymatic biomarkers. So far, a genotoxicity tool and endpoint have not been used; hence, the goals of the current study included the optimization of the in vivo alkaline comet assay in Enchytraeus crypticus. Further, the effect of silver nanomaterial (Ag NM300K, dispersed, 15 nm) was tested and compared with AgNO3 . Hydrogen peroxide (H2 O2 ) was used as a positive control. The various steps were optimized. The fully detailed standard operating procedure (SOP) is presented here. Ag materials caused genotoxicity, this being differentiated for the nano and non-nano forms. AgNO3 caused genotoxicity after 3 days (d) of exposure in a dose related manner, although after 7d the effects were either reduced or repaired, whereas Ag NM300K caused higher genotoxicity after 7d for the lowest concentration (EC20), highlighting a potential non-monotone dose-response effect. Overall, the comet assay showed the power to discriminate effects between materials and also toxicity at low relevant doses. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  9. Determination of genotoxicity by the Comet assay applied to murine precision-cut lung slices.

    PubMed

    Switalla, S; Knebel, J; Ritter, D; Dasenbrock, C; Krug, N; Braun, A; Sewald, K

    2013-03-01

    Precision-cut lung slices (PCLSs) are an organotypic lung model that is widely used in pharmacological, physiological, and toxicological studies. Genotoxicity testing, as a pivotal part of early risk assessment, is currently established in vivo in various organs including lung, brain, or liver, and in vitro in cell lines or primary cells. The aim of the present study was to provide the three-dimensional organ culture PCLS as a new ex vivo model for determination of genotoxicity using the Comet assay. Murine PCLS were exposed to increasing concentrations of ethyl methane sulfonate 'EMS' (0.03-0.4%) and formalin (0.5-5mM). Tissue was subsequently dissociated, and DNA single-strand breaks were quantified using the Comet assay. Number of viable dissociated lung cells was between 4×10(5) and 6.7×10(5)cells/slice. Even treatment with EMS did not induce toxicity compared to untreated tissue control. As expected, DNA single-strand breaks were increased dose-dependently and significantly after exposure to EMS. Here, tail length rose from 24μm to 75μm. In contrast, formalin resulted in a significant induction of DNA cross-links. The effects induced by EMS and formalin demonstrate the usefulness of PCLS as a new ex vivo lung model for genotoxicity testing in the early risk assessment of airborne substances in the future. Copyright © 2013 Elsevier Ltd. All rights reserved.

  10. Assessment of DNA integrity (COMET assay) in sperm cells of boron-exposed workers.

    PubMed

    Duydu, Yalçin; Başaran, Nurşen; Ustündağ, Aylin; Aydin, Sevtap; Undeğer, Ulkü; Ataman, Osman Yavuz; Aydos, Kaan; Düker, Yalçin; Ickstadt, Katja; Waltrup, Britta Schulze; Golka, Klaus; Bolt, Hermann M

    2012-01-01

    An extension of a male reproductive study conducted in a boric acid/borate production zone at Bandırma, Turkey, is presented. The relation between DNA-strand breaks (COMET assay, neutral and alkaline version) in sperm cells and previously described sperm quality parameters was investigated in boron-exposed males. A correlation between blood boron levels and mean DNA-strand breaks in sperm was weak, and DNA-strand breaks in sperm were statistically not different between control and exposed groups. Therefore, increasing boron exposures had no additional contribution in addition to already pre-existing DNA-strand breaks in the sperm cells. Weak but statistically significant correlations between DNA-strand breaks and motility/morphology parameters of sperm samples were observed in the neutral version of the COMET assay, while correlations between the same variables were statistically not significant in the alkaline version. A likely reason for these negative results, even in highly exposed humans, is that experimental exposures that had led to reproductive toxicity in animals were significantly higher than any boron exposures, which may be reached under realistic human conditions.

  11. Recent advances in in vivo genotoxicity testing: prediction of carcinogenic potential using comet and micronucleus assay in animal models.

    PubMed

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

    2013-12-01

    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.

  12. 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

  13. 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.

  14. Combining the in vivo comet and micronucleus assays: a practical approach to genotoxicity testing and data interpretation.

    PubMed

    Vasquez, Marie Z

    2010-03-01

    Despite regulatory directives requiring the reduction of animal use in safety testing, recent modifications to genotoxicity testing guidelines now propose the use of two in vivo genotoxicity assays as a follow-up to an in vitro positive (International Conference on Harmonization Consensus Draft Guidance S2[R1] released March, 2008). To address both goals, the in vivo comet and micronucleus (MN) assays can be successfully combined into one informative study. Combining these two assays with such differences in sensitivity, endpoints measured and the type of data generated significantly improves upon the current standard capabilities for detecting genotoxicity without requiring additional animals. But to take full advantage of the benefits of incorporating the comet assay in safety testing, these same differences must be recognized and considered. Developed from over 15 years experience using the in vivo comet and MN assays in genotoxicity testing of chemicals and pharmaceuticals, this paper presents guidelines for the appropriate experimental design, dose selection and data interpretation for combined in vivo comet/MN assay studies. To illustrate the approach, data from combined assay studies are presented and discussed.

  15. Genotoxicity evaluation of dimethoate to experimental mice by micronucleus, chromosome aberration tests, and comet assay.

    PubMed

    Ayed-Boussema, Imen; Rjiba, Karima; Mnasri, Nourhène; Moussa, Amal; Bacha, Hassen

    2012-01-01

    Dimethoate (DM) is an organophosphate insecticide with numerous uses on field and agricultural crops and ornamentals. Data concerning DM-acute genotoxicity are controversial and knowledge on its delayed effect is limited. For this reason, we aimed to further explore DM genotoxicity resulting from subchronic intoxication of experimental mice. Thus, DM was administered to mice at doses ranging from 1 to 30 mg/kg body weight for a period of 30 consecutive days. There was a significant increase (P < .05) in the frequency of micronucleated bone marrow cells following DM administration. Furthermore, the chromosome aberration assay revealed a significant increase in the percentage of chromosome abnormalities in a dose-dependent manner. Dimethoate was also found to induce significant DNA damage in mouse bone marrow cells as assessed by the comet assay. Altogether, our results showed that, after a subchronic exposure, DM was a genotoxic compound in experimental mice.

  16. An assessment of immediate DNA damage to occupationally exposed workers to low dose ionizing radiation by using the comet assay.

    PubMed

    Martínez, Angélica; Coleman, Matthew; Romero-Talamás, Carlos Alejandro; Frias, Sara

    2010-01-01

    Several cytogenetic studies have shown an increased frequency of chromosomal aberrations for workers exposed to low dose ionizing radiation, however the dose, type of radiation and management vary among the areas of work; it is possible that this variation may generate different quantity of DNA damage, detectable within the first hours after exposure of the personnel. In this study we assessed early DNA lesions caused by exposure to low doses of ionizing radiation in 41 workers from the departments of Radiology, Nuclear Medicine and Radiotherapy and a group of 20 healthy unexposed individuals, all from the same Institution. Blood samples were obtained from exposed and unexposed subjects for analysis of DNA damage using the comet assay. The migration of the comet's tail was compared before and after the workday, as well as among the groups; the relationship between DNA migration and the exposure dose of the month was also obtained. A significant increase in damage to DNA was seen after workday for the occupationally exposed group (p < 0.01) as compared with the samples before workday as well as with those from the unexposed group. A positive correlation was found between the monthly dose of radiation and the migration length of DNA before and after the workday (p < 0.01). There were significant differences in the length of the comet tails among workers from different departments: workers from Radiology (28.6 microm) have less DNA damage than those from Nuclear Medicine and Radiotherapy (92.5 microm, 63.4 microm respectively) departments. All the workers occupationally exposed showed an increase in DNA fragmentation after the workday. The amount of radiation in all three services is different, in Nuclear Medicine and Radiotherapy the workers showed a greater monthly dose of exposure and greater DNA damage than the Radiology workers. The longer tails were observed in Nuclear Medicine where radionuclides are used; these radioactive substances are handled and

  17. Evaluation of the Comet Assay for Assessing the Dose-Response Relationship of DNA Damage Induced by Ionizing Radiation

    PubMed Central

    Wang, Yan; Xu, Chang; Du, Li Qing; Cao, Jia; Liu, Jian Xiang; Su, Xu; Zhao, Hui; Fan, Fei-Yue; Wang, Bing; Katsube, Takanori; Fan, Sai Jun; Liu, Qiang

    2013-01-01

    Dose- and time-response curves were combined to assess the potential of the comet assay in radiation biodosimetry. The neutral comet assay was used to detect DNA double-strand breaks in lymphocytes caused by γ-ray irradiation. A clear dose-response relationship with DNA double-strand breaks using the comet assay was found at different times after irradiation (p < 0.001). A time-response relationship was also found within 72 h after irradiation (p < 0.001). The curves for DNA double-strand breaks and DNA repair in vitro of human lymphocytes presented a nice model, and a smooth, three-dimensional plane model was obtained when the two curves were combined. PMID:24240807

  18. In vivo assessment of DNA damage and protective effects of extracts from Miconia species using the comet assay and micronucleus test.

    PubMed

    Serpeloni, Juliana Mara; Bisarro dos Reis, Mariana; Rodrigues, Juliana; Campaner dos Santos, Lourdes; Vilegas, Wagner; Varanda, Eliana A; Dokkedal, Anne L; Cólus, Ilce Mara S

    2008-11-01

    The genus Miconia comprises approximately 1000 species belonging to the Melastomataceae family. Several crude plant extracts from Miconia and their isolated compounds have shown biological activities, such as analgesic and anti-neoplastic action; however, no studies concerning their effects on DNA are available. The present study aimed to evaluate, in vivo, the genotoxic and mutagenic effects of four species of plants from Miconia genus using the comet assay and micronucleus test. Their possible protective effects were also evaluated in experiments associating the plant extracts with cyclophosphamide (CPA). The methanolic extracts of Miconia albicans, Miconia cabucu, Miconia rubiginosa, Miconia stenostachya and the chloroformic extract of M. albicans were investigated. For genotoxic and mutagenic evaluations, three concentrations were tested, 200, 400 and 540 mg/kg body weight (bw), based on the solubility limit of the extract in distilled water. For the protective effects, only the highest dose was evaluated against 40 mg/kg bw of CPA. Blood was removed from mice tails pre- (T0) and post-treatment (T1-30 h) for the micronucleus test and 24 h post-treatment for the comet assay. The Student's t-test was used to compare data obtained at T0 and T1, the analysis of variance-Tukey test was used to compare between groups in the micronucleus test and the Kruskal-Wallis and Dunn's test were used to compare different groups in the comet assay. All the extracts induced alterations in DNA migration (comet assay); however, no mutagenic effect was observed in the micronucleus assay. All extracts showed a protective effect against CPA in both assays. Our study showed that the use of crude extracts could be more advantageous than the use of isolated compounds. The interaction between phytochemicals in the extracts showed efficacy in reducing mutagenicity and improving the protective effects.

  19. 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

  20. 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

  1. Photogenotoxicity of skin phototumorigenic fluoroquinolone antibiotics detected using the comet assay.

    PubMed

    Reavy, H J; Traynor, N J; Gibbs, N K

    1997-09-01

    The fluoroquinolone (FQ) antibiotics photosensitize human skin to solar UV radiation and are reported to photosensitize tumor formation in mouse skin. As tumor initiation will not occur without genotoxic insult, we examined the potential of ciprofloxacin, lomefloxacin, fleroxacin, BAYy3118 (a recently developed monofluorinated quinolone) and a nalidixic acid to photosensitize DNA damage in V79 hamster fibroblasts in vitro. Cells were exposed to 37.5 kJ/m2 UVA (320-400 nm; glass filtered Sylvania psoralen + UVA (PUVA) tubes; calibrated Waldmann radiometer) at 4 degrees C in the presence of FQ and immediately afterwards embedded in agarose, lysed and placed in an electrophoretic field at pH 12. Under these denaturing conditions, the presence of DNA single-strand breaks (SSB), alkali-labile sites (ALS) and double-strand breaks (DSB) can be visualized as DNA migrating away from the nucleus (characteristic "comet" appearance) after staining with a specific fluorochrome. At FQ concentrations that induced minimal loss of cell viability (neutral red uptake assay) the compounds tested induced comets with a rank order of BAYy3118 > norfloxacin > ciprofloxacin > lomefloxacin > fleroxacin > nalidixic acid. If cells were incubated after treatment for 1 h at 37 degrees C, the comet score decreased, suggesting efficient removal of SSB/ALS/DSB. Addition of the DNA polymerase(alpha) inhibitor, aphidicolin, to cells treated with either ciprofloxacin alone or ciprofloxacin + UVA resulted in an accumulation of SSB due to the endo/exonuclease steps of excision repair. We have demonstrated that the FQ are photogenotoxic in mammalian cells but the FQ-photosensitized SSB are efficiently repaired. Preliminary evidence that ciprofloxacin photosensitizes the formation of DNA lesions warranting excision repair may indicate production of more mutagenic lesions.

  2. Application of single-cell gel electrophoresis (comet) assay for assessing levels of DNA damage in canine and feline leukocytes.

    PubMed

    Heaton, Paul R; Ransley, Raymond; Charlton, Chris J; Mann, Sarah J; Stevenson, Joy; Smith, Brigitte H E; Rawlings, John M; Harper, E Jean

    2002-06-01

    Increasing evidence suggests involvement of free-radical species in the development of oxidative DNA damage, the consequences of which have been implicated in a number of degenerative disorders associated with the aging process. Here we report the application of a single-cell gel electrophoresis (comet) assay for assessing levels of DNA damage in canine and feline leukocytes. Leukocytes were collected from 24 healthy adult cats and dogs and subjected to DNA damage ex vivo by exposure to a range of hydrogen peroxide (H(2)O(2)) concentrations (0-250 micromol/L). The optimal concentration of H(2)O(2) to induce a significant increase in DNA damage was 100 micromol/L for both canine and feline leukocyte samples. Levels of DNA damage were assessed and quantified by visual and computer image analysis. The results obtained showed high correlations between visual scoring and computer image analysis for feline samples (percentage DNA in tail, R(2) > 0.99; tail moment, R(2) > 0.95; tail length, R(2) > 0.90) and canine samples (percentage DNA in tail, R(2) > 0.97; tail moment, R(2) > 0.95; tail length, R(2) > 0.91). In conclusion, this method provides a way of assessing levels of DNA damage utilizing visual and/or computer image analysis in the feline and canine systems. With the capacity of the comet assay to be able to measure end products of free-radical reactions, it is a useful tool for determining the optimal effects of dietary antioxidants on a reliable biomarker of oxidative stress such as cellular DNA status in cats and dogs.

  3. Comprehensive Profiling of Radiosensitive Human Cell Lines with DNA Damage Response Assays Identifies the Neutral Comet Assay as a Potential Surrogate for Clonogenic Survival

    PubMed Central

    Nahas, Shareef A.; Davies, Robert; Fike, Francesca; Nakamura, Kotoka; Du, Liutao; Kayali, Refik; Martin, Nathan T.; Concannon, Patrick; Gatti, Richard A.

    2015-01-01

    In an effort to explore the possible causes of human radiosensitivity and identify more rapid assays for cellular radiosensitivity, we interrogated a set of assays that evaluate cellular functions involved in recognition and repair of DNA double-strand breaks: (1) neutral comet assay, (2) radiation-induced γ-H2AX focus formation, (3) the temporal kinetics of structural maintenance of chromosomes 1 phosphorylation, (4) intra-S-phase checkpoint integrity, and (5) mitochondrial respiration. We characterized a unique panel of 19 “radiosensitive” human lymphoblastoid cell lines from individuals with undiagnosed diseases suggestive of a DNA repair disorder. Radiosensitivity was defined by reduced cellular survival using a clonogenic survival assay. Each assay identified cell lines with defects in DNA damage response functions. The highest concordance rate observed, 89% (17/19), was between an abnormal neutral comet assay and reduced survival by the colony survival assay. Our data also suggested that the neutral comet assay would be a more rapid surrogate for analyzing DNA repair/processing disorders. PMID:21962002

  4. Tiagabine treatment and DNA damage in rat astrocytes: an in vitro study by comet assay.

    PubMed

    Cardile, V; Palumbo, M; Renis, M; Pavone, A; Maci, T; Perciavalle, V

    2001-06-22

    We studied in vitro the effects of Tiagabine on genomic DNA of cortical rat astrocytes. To evaluate DNA damage, we used a relatively simple technique called Single Cell Gel Electrophoresis or Comet assay. Tiagabine was dissolved in culture medium and added at concentration of 1, 10, 20 and 50 microg/ml on 12-day old cultured astrocytes. In presence of 1 and 10 microg/ml of Tiagabine, no DNA damage was observed after 48 h of treatment. A moderate DNA damage was instead observed for cells exposed to 20 microg/ml of antiepileptic drug. Finally, DNA fragmentation was more evident after treatment with 50 microg/ml of Tiagabine. We conclude that Tiagabine, at the usual recommended doses, does not appear to influence negatively the cortical rat astrocytes, inducing DNA fragmentation only at very high concentrations.

  5. 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.

  6. Workshop on Analysis of Returned Comet Nucleus Samples

    NASA Technical Reports Server (NTRS)

    1989-01-01

    This volume contains abstracts that were accepted by the Program Committee for presentation at the workshop on the analysis of returned comet nucleus samples held in Milpitas, California, January 16 to 18, 1989. The abstracts deal with the nature of cometary ices, cryogenic handling and sampling equipment, origin and composition of samples, and spectroscopic, thermal and chemical processing methods of cometary nuclei. Laboratory simulation experimental results on dust samples are reported. Some results obtained from Halley's comet are also included. Microanalytic techniques for examining trace elements of cometary particles, synchrotron x ray fluorescence and instrument neutron activation analysis (INAA), are presented.

  7. Double-face activity of resveratrol in voluntary runners: assessment of DNA damage by comet assay.

    PubMed

    Tomasello, Barbara; Grasso, Salvatore; Malfa, Giuseppe; Stella, Stefania; Favetta, Marco; Renis, Marcella

    2012-05-01

    Voluntary runners are subjected to a massive increase in reactive oxygen/nitrogen species production, which can promote different oxidative stress-related diseases such as premature aging, neurodegenerative disorders, and cancer. The aims of this work were to evaluate the following in peripheral blood cells of voluntary runners: (i) DNA status; (ii) susceptibility to the in vitro insult induced by hydrogen peroxide (H(2)O(2)) as a breaking agent; (iii) capabilities of 3,5,4'-trihydroxystilbene (RESV) in counteracting DNA damage. Twenty-five male voluntary runners were compared with 20 sedentary men, as age-matched controls, and DNA status was evaluated with different versions of comet assay: alkaline, neutral, and Fpg enzyme-modified version to measure 8-OH-deoxyguanosine (8-oxo-dG) levels. The H(2)O(2) and/or RESV treatments were performed directly on agarose-embedded cells (atypical comet assay). The results evidenced DNA damage and levels of 8-oxo-dG higher in runners than in sedentary control subjects. The runners' DNA was more prone to the in vitro-induced oxidative insult (200 μM H(2)O(2)) than that of the control group. Resveratrol (100 μM), depending on the individual basal DNA status, was able to switch from antioxidant to pro-oxidant. Our results, on the one hand, validated the proposed in vitro experimental protocol in order to measure individual DNA status. On the other hand, our data point out the importance of monitoring the athletes' redox status before subjecting them to dietary supplementation treatment.

  8. Alkaline single-cell gel (comet) assay and genotoxicity monitoring using two species of tadpoles.

    PubMed

    Ralph, S; Petras, M; Pandrangi, R; Vrzoc, M

    1996-01-01

    Small bodies of water (e.g., creeks, ponds, and drainage ditches) have received very little attention in genotoxicity studies, yet these areas are important because they are often the first to be affected by industrial effluents, sewage contaminants, accidental spills, internal combustion engine emissions, landfill runoffs, and pesticide uses. To address this deficiency, we examined erythrocytes in two species of tadpoles, Rana clamitans and Bufo americanus, using the alkaline single-cell gel (SCG) ("comet") assay. This approach involves detection, under alkaline conditions, of cell DNA fragments, which on electrophoresis migrate from the nuclear core, resulting in a "comet-with-tail" formation. Exposure of R. clamitans todpoles to a range of concentrations of methyl methanesulfonate (MMS) produced a linear increase in DNA length to DNA core width ratios. This is consistent with findings in a number of other species. Time-dose experiments using MMS suggest that the peak level of DNA damage in R. clamitans todpoles occurred 42 hr after exposure. B. americanus tadpoles exposed to 6.25 mg/l of MMS for 12 hours had a significant increase in DNA damage over that seen in the controls. Freshly caught R. clamitans tadpoles from Highgate and B. americanus tadpoles from Duart, both on the north shore of Lake Erie, gave ratios of 2.78 and 2.07, respectively. This region of Ontario is a prime agricultural area and pesticide use is extensive. Tadpoles from Highgate and Duart, maintained in the laboratory for 4 months and 6 weeks, respectively, gave ratios of 1.29 and 1.44. The results of the SCG procedure in tadpoles indicate that this assay is extremely sensitive and suitable for detecting genotoxicity in the environment.

  9. 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 (SO2), nitrogen oxides (NO2), and particulate matter with diameter up to 10 μm (PM10) 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.

  10. 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.

  11. Adaptation of the neutral bacterial comet assay to assess antimicrobial-mediated DNA double-strand breaks in Escherichia coli

    PubMed Central

    SOLANKY, DIPESH; HAYDEL, SHELLEY E.

    2012-01-01

    This study aimed to determine the mechanism of action of a natural antibacterial clay mineral mixture, designated CB, by investigating the induction of DNA double-strand breaks (DSBs) in Escherichia coli. To quantify DNA damage upon exposure to soluble antimicrobial compounds, we modified a bacterial neutral comet assay, which primarily associates the general length of an electrophoresed chromosome, or comet, with the degree of DSB-associated DNA damage. To appropriately account for antimicrobial-mediated strand fragmentation, suitable control reactions consisting of exposures to water, ethanol, kanamycin, and bleomycin were developed and optimized for the assay. Bacterial exposure to the CB clay resulted in significantly longer comet lengths, compared to water and kanamycin exposures, suggesting that the induction of DNA DSBs contributes to the killing activity of this antibacterial clay mineral mixture. The comet assay protocol described herein provides a general technique for evaluating soluble antimicrobial-derived DNA damage and for comparing DNA fragmentation between experimental and control assays. PMID:22940101

  12. 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, ...

  13. 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, ...

  14. Reference control data obtained from an in vivo comet-micronucleus combination assay using Sprague Dawley rats.

    PubMed

    Kasamoto, Sawako; Masumori, Shoji; Tanaka, Jin; Ueda, Maya; Fukumuro, Masahito; Nagai, Miho; Yamate, Jyoji; Hayashi, Makoto

    2017-04-04

    According to the International Conference on Harmonization Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use (ICH S2(R1)), a positive response in any in vitro assay necessitates additional in vivo test(s) (other tissue/endpoint) in addition to the erythrocyte micronucleus test when Option 1 of the test battery is selected. When Option 2 of the test battery is selected, a bacterial gene mutation test and two in vivo tests with different tissues/endpoint are required. The in vivo alkaline comet assay is recommended as the second in vivo test because it can detect a broad spectrum of DNA damage in any tissue and can be combined with the erythrocyte micronucleus test. Considering animal welfare, a combination assay is preferable to an individual assay. Thus, we validated the protocol for the in vivo comet-micronucleus combination assay in rats with three daily administrations and determined the dose of the positive control (ethyl methanesulfonate; EMS, 200mg/kg/day). We also collected the negative control (vehicle) and positive control (EMS) data from the comet (liver, stomach, and kidney) and micronucleus (bone marrow) combination assay using male Sprague Dawley (SD) rats. The negative control data were comparable to our historical control data obtained from stand-alone assays. The positive control data showed clear and consistent positive responses in both endpoints.

  15. Role of recombinant human erythropoietin loading chitosan-tripolyphosphate nanoparticles in busulfan-induced genotoxicity: Analysis of DNA fragmentation via comet assay in cultured HepG2 cells.

    PubMed

    Ghassemi-Barghi, Nasrin; Varshosaz, Jaleh; Etebari, Mahmoud; Jafarian Dehkordi, Abbas

    2016-10-01

    -treatment conditions, significantly decreased the level of DNA damage induced by busulfan, measured with the comet assay, in HepG2 cells compared to the regular rhEPO group.

  16. 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

  17. 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.

  18. 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.

  19. Analysis of IUE Observations of Hydrogen in Comets

    NASA Technical Reports Server (NTRS)

    Combi, Michael R.; Feldman, Paul D.

    1998-01-01

    The 15-years worth of hydrogen Lyman-alpha observations of cometary comae obtained with the International Ultraviolet Explorer (IUE) satellite had gone generally unanalyzed because of two main modeling complications. First, the inner comae of many bright (gas productive) comets are often optically thick to solar Lyman-alpha radiation. Second, even in the case of a small comet (low gas production) the large IUE aperture is quite small as compared with the immense size of the hydrogen coma, so an accurate model which properly accounts for the spatial distribution of the coma is required to invert the infrared brightnesses to column densities and finally to H atom production rates. Our Monte Carlo particle trajectory model (MCPTM), which for the first time provides the realistic full phase space distribution of H atoms throughout the coma has been used as the basis for the analysis of IUE observations of the inner coma. The MCPTM includes the effects of the vectorial ejection of the H atoms upon dissociation of their parent species (H2O and OH) and of their partial collisional thermalization. Both of these effects are crucial to characterize the velocity distribution of the H atoms. This combination of the MCPTM and spherical radiative transfer code had already been shown to be successful in understanding the moderately optically thick coma of comet P/Giacobini-Zinner and the coma of comet Halley that varied from being slightly to very optically thick. Both of these comets were observed during solar minimum conditions. Solar activity affects both the photochemistry of water and the solar Lyman-alpha radiation flux. The overall plan of this program here was to concentrate on comets observed by IUE at other time during the solar cycle, most importantly during the two solar maxima of 1980 and 1990. Described herein are the work performed and the results obtained.

  20. Genotoxicity assessment of deoxynivalenol in the Caco-2 cell line model using the Comet assay.

    PubMed

    Bony, Sylvie; Carcelen, Monique; Olivier, Laurence; Devaux, Alain

    2006-09-30

    The genotoxic risk associated with deoxynivalenol (DON), a prevalent trichothecene mycotoxin which contaminates cereal-based products has not yet been deeply explored. In this work, the alkaline version of the Comet assay was used to evaluate DNA damage stemming from DON exposure in both dividing and differentiated Caco-2 cells, an epithelial intestinal cell line. To avoid false positive results, cytotoxic and apoptotic thresholds were firstly established using the MTS and neutral red assays and the Hoestch staining method, respectively. Dividing cells were found to be more sensitive to DON than differentiated cells and the lowest IC(10) (0.5 microM) obtained for dividing cells exposed for 72 h was used as the highest working concentration in the genotoxicity study. Both differentiated and dividing cells responded with a dose-dependent relationship to DON in terms of DNA damage in the 0.01-0.5 microM range. These results demonstrated the existence of a genotoxic potential for DON at low concentrations compatible with actual exposure situations and calls for additional studies to determine the functional consequences which could be taken into account for the risk assessment of this food contaminant.

  1. 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

  2. Antigenotoxic activity of watercress extract in an in vitro mammalian system using comet assay.

    PubMed

    Casanova, Natalia A; Carballo, Marta A

    2011-12-01

    Watercress (Cruciferae), an integral part of Mediterranean diets, is a nutritive food which is used in the treatment of several diseases. Oxidative DNA damage seems to play a crucial role in chronic, aging-related diseases and it is considered an important and probably carcinogenic factor. The aim of this work was to determine the impact of watercress extract on cell viability and its potential antigenotoxic properties against induced oxidative damage, using a comet assay and peripheral blood cells as an in vitro model. An aqueous extract of the leaves was prepared using a juice processor, centrifuged, filtered and preserved at -20 °C. Two concentrations of the aqueous extract (13.2 and 26.4 mg/mL) were assayed. No differences were found in cell viability between the control and treated groups at any time. Significant antigenotoxic effects were observed for both concentrations, expressed as the damage index (p = 0.005 at 30 min; p < 0.001 at 60 and 90 min), the percentage reductions in damage being similar between them (67.1-75.2% respectively). These results suggest that the consumption watercress in the diet is a powerful tool for improving health and the quality of life. Copyright © 2011 John Wiley & Sons, Ltd.

  3. Methods for the mineralogical and textural analysis of comet nucleus samples

    NASA Technical Reports Server (NTRS)

    Stoeffler, D.; Dueren, H.; Knoelker, J.

    1989-01-01

    The objectives and instrumental requirements of a petrographic analysis of porous comet nucleus material are reviewed. Assumptions about its composition and texture, and the available techniques for the microscopic analysis of comet analogue material are investigated. New techniques required for the petrographic investigation of natural and artificial comet nucleus samples are also considered.

  4. Genotoxic effects of environmental endocrine disruptors on the aquatic insect Chironomus riparius evaluated using the comet assay.

    PubMed

    Martínez-Paz, Pedro; Morales, Mónica; Martínez-Guitarte, José Luis; Morcillo, Gloria

    2013-12-12

    Genotoxicity is one of the most important toxic endpoints in chemical toxicity testing and environmental risk assessment. The aim of this study was to evaluate the genotoxic potential of various environmental pollutants frequently found in aquatic environments and characterized by their endocrine disrupting activity. Monitoring of DNA damage was undertaken after in vivo exposures of the aquatic larvae of the midge Chironomus riparius, a model organism that represents an abundant and ecologically relevant macroinvertebrate, widely used in freshwater toxicology. DNA-induced damage, resulting in DNA fragmentation, was quantified by the comet assay after short (24 h) and long (96 h) exposures to different concentrations of the selected toxicants: bisphenol A (BPA), nonylphenol (NP), pentachlorophenol (PCP), tributyltin (TBT) and triclosan (TCS). All five compounds were found to have genotoxic activity as demonstrated by significant increases in all the comet parameters (%DNA in tail, tail length, tail moment and Olive tail moment) at all tested concentrations. Persistent exposure did not increase the extent of DNA damage, except for TCS at the highest concentration, but generally there was a reduction in DNA damage thought to be associated with the induction of the detoxification processes and repairing mechanisms. Comparative analysis showed differences in the genotoxic potential between the chemicals, as well as significant time and concentration-dependent variations, which most likely reflect differences in the ability to repair DNA damage under the different treatments. The present report demonstrates the sensitivity of the benthic larvae of C. riparius to these environmental genotoxins suggesting its potential as biomonitor organism in freshwater ecosystems. The results obtained about the DNA-damaging potential of these environmental pollutants reinforce the need for additional studies on the genotoxicity of endocrine active substances that, by linking genotoxic

  5. 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

  6. Measurements of the genotoxic potential of (xeno-)oestrogens in the bivalve mollusc Scrobicularia plana, using the Comet assay.

    PubMed

    Petridis, Panos; Jha, Awadhesh N; Langston, William J

    2009-08-13

    There is increasing concern about the fate and effects of (geno)toxic and endocrine disrupting chemicals in sediments, highlighting the need to develop suitable monitoring tools. The deposit-feeding bivalve mollusc Scrobicularia plana has been put forward as a promising bioindicator of sediment contamination in estuaries. The recent demonstration of intersex in S. plana populations has been attributed to the feminisation of male clams following exposure to (xeno-)oestrogens, yet the mode of action of these endocrine disrupting chemicals (EDCs) remains largely unclear. One hypothesis that warrants further investigation is the possible involvement of genotoxicity. The first objective of this study was to assess whether the blood cells of S. plana are suitable for genotoxicity screening, using the alkaline single cell gel electrophoresis (Comet) assay. This was demonstrated successfully by exposing blood cells under in vitro conditions to a range of concentrations of the reference genotoxin hydrogen peroxide (H(2)O(2)): strong correlations between H(2)O(2) concentration and various comet parameters were found. Subsequently, the Comet assay was used to test whether the natural oestrogen 17beta-oestradiol (E2) and the synthetic (xeno)oestrogens ethinyloestradiol (EE2) and nonylphenol (NP) can produce genotoxic effects in S. plana, which might indicate possible involvement of mutagenicity in the mode of action of intersex development. In these short-term tests, clear genotoxic effects (significantly more DNA in the comet tail) were demonstrated by all EDCs, albeit only at high doses: 100 ng/L E2, 1 microg/L EE2 and 100 microg/L NP in vitro; and 1 microg/L E2 and 1mg/L NP after a 6-day in vivo exposure. Nevertheless, this study provides valuable preliminary data on the application and sensitivity of S. plana blood cells and suggests that the Comet assay is a useful tool, to screen for genotoxicity in in faunal clams and to examine further the links with higher order

  7. 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

  8. Measurement of oxidatively-induced clustered DNA lesions using a novel adaptation of single cell gel electrophoresis (comet assay).

    PubMed

    Georgakilas, Alexandros G; Holt, Stewart M; Hair, Jessica M; Loftin, Charles W

    2010-12-01

    The two basic groups of complex DNA damage are double-strand breaks (DSBs) and non-DSB oxidatively-induced clustered DNA lesions (OCDLs). The single-cell gel electrophoresis (SCGE) or comet assay has been widely used for the detection of low levels of various types of DNA lesions including single-strand breaks (SSBs), DSBs, and oxidized bases per individual cell. There are limited data on the use of the comet assay for the detection of non-DSB clustered DNA lesions using different repair enzymes as enzymatic probes. This unit discusses a novel adaptation of the comet assay used to measure these unique types of lesions. Until now OCDL yields have been measured using primarily pulsed-field agarose gel electrophoresis. The advantages offered by the current approach are: (1) measurement of OCDL levels per individual cell; (2) use of a small number of cells (∼10,000) and relatively low doses of ionizing radiation (1 to 2 Gy) or low levels of oxidative stress, which are not compatible with standard agarose gel electrophoresis; and finally, (3) the assay is fast and allows direct comparison with pulsed-field gel electrophoresis results.

  9. 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.

  10. Geosmin induces genomic instability in the mammalian cell microplate-based comet assay.

    PubMed

    Silva, Aline Flor; Lehmann, Mauricio; Dihl, Rafael Rodrigues

    2015-11-01

    Geosmin (GEO) (trans-1,10-dimethyl-trans-9-decalol) is a metabolite that renders earthy and musty taste and odor to water. Data of GEO genotoxicity on mammalian cells are scarce in the literature. Thus, the present study assessed the genotoxicity of GEO on Chinese hamster ovary (CHO) cells in the microplate-based comet assay. The percent of tail DNA (tail intensity (TI)), tail moment (TM), and tail length (TL) were used as parameters for DNA damage assessment. The results demonstrated that concentrations of GEO of 30 and 60 μg/mL were genotoxic to CHO cells after 4- and 24-h exposure periods, in all parameters evaluated, such as TI, TM, and TL. Additionally, GEO 15 μg/mL was genotoxic in the three parameters only in the 24-h exposure time. The same was observed for GEO 7.5 μg/mL, which induced significant DNA damage observed as TI in the 24-h treatment. The results present evidence that exposure to GEO may be associated with genomic instability in mammalian cells.

  11. Genotoxic effects of boric acid and borax in zebrafish, Danio rerio using alkaline comet assay.

    PubMed

    Gülsoy, Nagihan; Yavas, Cüneyd; Mutlu, Özal

    2015-01-01

    The present study is conducted to determine the potential mechanisms of Boron compounds, boric acid (BA) and borax (BX), on genotoxicity of zebrafish Danio rerio for 24, 48, 72 and 96-hours acute exposure (level:1, 4, 16, 64 mg/l BA and BX) in semi-static bioassay experiment. For that purpose, peripheral erythrocytes were drawn from caudal vein and Comet assay was applied to assess genotoxicity. Acute (96 hours) exposure and high concentrations of boric acid and borax increases % tail DNA and Olive tail moment. Genotoxicity was found for BA as concentration-dependent and BX as concentration and time dependent manner. In general, significant effects (P < 0,05) on both concentrations and exposure times were observed in experimental groups. DNA damage was highest at 96 h and 24 h for all BX and BA concentrations, respectively in peripheral blood of D. rerio. For the first time, our study demonstrates the effect of waterborne BA and BX exposure on genotoxicity at the molecular level, which may contribute to understanding the mechanism of boric acid and borax-induced genotoxicity in fish.

  12. Genotoxic effects of boric acid and borax in zebrafish, Danio rerio using alkaline comet assay

    PubMed Central

    Gülsoy, Nagihan; Yavas, Cüneyd; Mutlu, Özal

    2015-01-01

    The present study is conducted to determine the potential mechanisms of Boron compounds, boric acid (BA) and borax (BX), on genotoxicity of zebrafish Danio rerio for 24, 48, 72 and 96-hours acute exposure (level:1, 4, 16, 64 mg/l BA and BX) in semi-static bioassay experiment. For that purpose, peripheral erythrocytes were drawn from caudal vein and Comet assay was applied to assess genotoxicity. Acute (96 hours) exposure and high concentrations of boric acid and borax increases % tail DNA and Olive tail moment. Genotoxicity was found for BA as concentration-dependent and BX as concentration and time dependent manner. In general, significant effects (P < 0,05) on both concentrations and exposure times were observed in experimental groups. DNA damage was highest at 96 h and 24 h for all BX and BA concentrations, respectively in peripheral blood of D. rerio. For the first time, our study demonstrates the effect of waterborne BA and BX exposure on genotoxicity at the molecular level, which may contribute to understanding the mechanism of boric acid and borax-induced genotoxicity in fish. PMID:26862320

  13. Genotoxicity assessment of cobalt chloride in Eisenia hortensis earthworms coelomocytes by comet assay and micronucleus test.

    PubMed

    Ciğerci, İbrahim Hakkı; Ali, Muhammad Muddassir; Kaygısız, Şöhret Yüksek; Liman, Recep

    2016-02-01

    Cobalt and its different compounds are extensively used worldwide and considered as possible environmental pollutant. Earthworms are useful model organism and its different species are used to monitor soil pollution. No study has been found to detect cobalt chloride (CoCl2) genotoxicity in earthworms. So, current study aimed to evaluate CoCl2 induced genotoxicity in Eisenia hortensis earthworms coelomocytes by alkaline comet assay (CA) and micronucleus (MN) test. The earthworms (n = 10 for each group) were exposed to different series of CoCl2 concentrations (100 ppm, 200 ppm, 300 ppm, 400 ppm, 500 ppm, 600 ppm) to find LD50. The LD50 for CoCl2 was found at 226 ppm. Then, doses of LD50/2, LD50 and 2XLD50 for 48 h were used. CA and MN demonstrated the significant increase (P < 0.05) in DNA damage and chromosomal aberrations. Dose dependent relationship was found. Highest DNA damage and chromosomal aberrations were noticed at 2XLD50. The results concluded that CoCl2 induced DNA damage, cytokinesis failure and chromosomal aberrations in E. hortensis earthworms. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. DNA oxidation: investigating its key role in environmental mutagenesis with the comet assay.

    PubMed

    Azqueta, Amaya; Shaposhnikov, Sergey; Collins, Andrew R

    2009-03-31

    DNA oxidation, which can have potentially serious mutagenic consequences, commonly accompanies exposure to environmental mutagens. Oxidised bases can be measured chromatographically, but spurious oxidation during sample preparation leads to serious over-estimation of low levels of damage. A more reliable approach is to employ endonucleases specific for oxidised bases, to introduce breaks in cellular DNA that are then most commonly measured using the comet assay (alkaline single cell gel electrophoresis). The two enzymes in general use are formamidopyrimidine DNA glycosylase, which detects primarily 8-oxo-7,8-dihydroguanine (8-oxoGua), and endonuclease III which recognises oxidised pyrimidines. We give a brief account of the recommended experimental procedures, and then describe applications in various areas of environmental research. Cultured cell lines or white blood cells have been exposed to a range of environmental mutagens, including natural products, industrial chemicals, radiation and nanoparticles. In vivo exposure of animals and humans to pollutants is more challenging but can give particularly valuable information in relation to real life exposure. Possibly the most useful application is in biomonitoring of human population groups suffering actual exposure to environmental or occupational mutagens. Finally, the potential use of this technique to monitor effects of contaminants in the natural environment has yet to be fully exploited.

  15. Can the use of medical muds cause genotoxicity in eukaryotic cells? A trial using comet assay.

    PubMed

    Gerencsér, Gellért; Szendi, Katalin; Berényi, Károly; Varga, Csaba

    2015-02-01

    Despite the lack of knowledge of their exact effects, peloids (natural muds) are widely applied in clinical treatment and prevention of different diseases, especially in rheumatic and gynecological disorders or skin diseases. Primarily we have information on their inorganic components, but only limited data are available on the organic components and nothing on their mechanism of chemical action. The objective of the present study was to detect the DNA-damaging effects (possible genotoxic effect) of peloid samples using the single-cell comet assay on Long Evans rat lymphocytes, human lymphocytes, and Eisenia fetida coelomocytes. Rat and human lymphocytes were exposed to the in toto peloid samples, in vitro. The Eisenia cells were extracted from the coelom of animals kept in the intact peloid sample. An indicator derived from the DNA fluorescence intensity was used in the statistical evaluation. The predominantly organic (Hévíz) sample showed a significant alteration from the negative control in several cases, while the inorganic (Kolop) applied did not. A higher quantity of organic compounds may have an important role in the emergence of DNA damage. The results revealed that medical muds have not only positive health effects but can also contain substances with potential human toxicity risk. Our research provides essential steps towards the creation of a toxicity profile and the possible safe use of peloids as medicinal therapy.

  16. In vitro comet and micronucleus assays do not predict morphological transforming effects of silica particles in Syrian Hamster Embryo cells.

    PubMed

    Darne, Christian; Coulais, Catherine; Terzetti, Francine; Fontana, Caroline; Binet, Stéphane; Gaté, Laurent; Guichard, Yves

    2016-01-15

    Crystalline silica particles and asbestos have both been classified as carcinogenic by the International Agency for Research on Cancer (IARC). However, because of the limited data available, amorphous silica was not classifiable. In vitro, the carcinogenic potential of natural crystalline and amorphous silica particles has been revealed by the Syrian Hamster Embryo (SHE) cell transformation assay. On the other hand, the genotoxic potential of those substances has not been investigated in SHE cells. And yet, genotoxicity assays are commonly used for hazard evaluation and they are often used as in vitro assays of reference to predict a possible carcinogenic potential. The main objective of this study was to compare the genotoxic potential and the carcinogenic potential of different crystalline and amorphous silica particles in SHE cells. Three silica samples of different crystallinity were used: natural amorphous silica, partially crystallized silica and quartz silica particles. Their genotoxicity were tested through the in vitro micronucleus assay and the comet assay in SHE, and their carcinogenic potential through the SHE transformation assay. In addition, silica samples were also tested with the same genotoxicity assays in V79 hamster-lung cells, a common in vitro model for particle exposure. Results obtained in the micronucleus and the comet assays show that none of the silica was capable of inducing genotoxic effects in SHE cells and only the amorphous silica induced genotoxic effects in V79 cells. However in the SHE cell transformation assays, the partially crystallized and quartz silica were able to induce morphological cell transformation. Together, these data suggest that, in vitro, the short-term genotoxic assays alone are not sufficient to predict the hazard and the carcinogenic potential of this type of particles; SHE transformation assay appears a more reliable tool for this purpose and should be included in the "in vitro battery assays" for hazard

  17. Baseline values of micronuclei and comet assay in the lizard Tupinambis merianae (Teiidae, Squamata).

    PubMed

    Schaumburg, Laura G; Poletta, Gisela L; Siroski, Pablo A; Mudry, Marta D

    2012-10-01

    The Micronucleus test (MN) and Comet assay (CA) are currently the most widely used methods that allow the characterization of DNA damage induced by physical and chemical agents in wild species. The continuous expansion of the cultivated areas in Argentina, since the introduction of transgenic crops, mainly soy, in association with the increased use of pesticides, transformed deeply the natural environments where the lizard Tupinambis merianae (tegu lizard) occurs. Despite the fact that reptiles have shown to be excellent bioindicators of environmental contaminants, there is no record of genotoxicity studies in T. merianae. The aim of the present study was to adjust the MN test and CA protocols to be applied in erythrocytes of T. merianae, and determine the baseline values of DNA damage in this species. We used 20 adult lizards (10 males: 10 females) from Estación Zoológica Experimental "Granja La Esmeralda" (Santa Fe, Argentina). Peripheral blood samples were collected from all animals and the MN test and CA applied according to the protocols established for other reptilian species. We test critical parameters of CA protocol (cell density, unwinding and electrophoresis times) using increasing concentrations of H2O2 (10, 25 and 50 μM) as a known genotoxic agent to induce DNA damage. Based on this, we determined the most suitable conditions for the CA in this species: a cell density of 4×10(3) erythrocytes per slide, 10 min of unwinding and 15 min of electrophoresis at 0.90 V/cm approximately. The baseline frequency of micronuclei (BFMN=MN/1000 erythrocytes counted) determined for this species was 0.95±0.27 and the basal damage index (BDI: calculated from 100 comet images classified in arbitrary units)=103.85±0.97. No differences were observed between sexes in the BFMN or BDI (p>0.05), and no relation was found between baseline values and length or weight of the analyzed animals (p>0.05). These results demonstrated the sensitivity of both biomarkers of

  18. 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.

  19. 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

    2017-07-10

    Sponges are a potential alternative model species to bivalves in pollution biomonitoring and environmental risk assessment in the aquatic ecosystem. Here, 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 non-cytotoxic 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 thebest of our knowledge we have utilised for the first time the alkaline comet assay to detect DNA-strand breaks in marine sponge cells, and demonstrated that exposure to non-cytotoxic 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. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  20. Assessment of the in vivo genotoxicity of isomers of dinitrotoluene using the alkaline Comet and peripheral blood micronucleus assays.

    PubMed

    Lent, Emily May; Crouse, Lee C B; Quinn, Michael J; Wallace, Shannon M

    2012-02-18

    Dinitrotoluene (DNT) is a nitroaromatic explosive that exists as six isomers; two major isomers (2,4- and 2,6-DNT) and four minor isomers (2,3-, 2,5-, 3,4-, and 3,5-DNT). DNT has been found in soil, surface water, and groundwater near ammunition production plants. The major isomers of DNT are classified as "likely to cause cancer in humans."In vitro studies have provided conflicting data regarding the genotoxicity of the minor isomers. Studies indicate that metabolism in the gut and liver are necessary to convert DNT to genotoxic compounds. As such, in the present study the genotoxicity of isomers of DNT was assessed using two in vivo genotoxicity assays. The Comet assay was used to detect DNA damage in liver cells from male Sprague-Dawley rats following oral exposure (14-day) to individual isomers of DNT. The micronucleus assay was conducted using flow cytometric analysis to detect chromosomal damage in peripheral blood. Treatment with 2,3-, 3,4-, 2,4-, 2,5- and 3,5-DNT did not induce DNA damage in liver cells or increase the frequency of micronucleated reticulocytes (MN-RET) in peripheral blood at the doses tested. Treatment with 2,6-DNT induced DNA damage in liver tissue at all doses tested, but did not increase the frequency of micronucleated reticulocytes (MN-RET) in peripheral blood. Thus, 2,4-DNT and the minor isomers were not genotoxic under these test conditions, while 2,6-DNT was genotoxic in the target tissue, the liver. These results support previous research which indicated that the hepatocarcinogenicity of technical grade DNT (TG-DNT) could be attributed to the 2,6-DNT isomer. Published by Elsevier B.V.

  1. 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.

  2. 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.

  3. Genotoxicity of Thermopsis turcica on Allium cepa L. roots revealed by alkaline comet and random amplified polymorphic DNA assays.

    PubMed

    Ciğerci, İbrahim Hakkı; Cenkci, Süleyman; Kargıoğlu, Mustafa; Konuk, Muhsin

    2016-08-01

    This study was undertaken to evaluate genotoxic potential of Thermopsis turcica aqueous extracts on the roots of onion bulb (Allium cepa L.) by comet assay and random amplified polymorphic DNA technique. The Allium root growth inhibition test indicated that the EC50 and 2×EC50 values were 8 and 16 mg/ml concentrations of T. turcica aqueous extracts, respectively. The negative control (distilled water), positive control (methyl methane sulfonate, 10 mg/l) and 8 and 16 mg/ml concentrations of T. turcica extracts were introduced to the roots of onion bulbs for 24 and 96 h. The root growth, DNA damage in root cells and randomly amplified polymorphic DNA (RAPD) profiles of root tissue were used as endpoints of the genotoxicity. The comet assay clearly indicated that dose-dependent single strand DNA breaks in the root nuclei of onions were determined for the treatment concentrations of T. turcica extracts. In comparison to RAPD profile of negative control group, RAPD polymorphisms became evident as disappearance and/or appearance of RAPD bands in treated roots. The diagnostic and phenetic numerical analyses of RAPD profiles obviously indicated dose-dependent genotoxicity induced by Thermopsis extracts. In conclusion, the results clearly indicated that water extract of T. turcica has genotoxic potential on the roots of onion bulbs as shown by comet assay and RAPD technique.

  4. Measurement of DNA damage after acute exposure to pulsed-wave 2450 MHz microwaves in rat brain cells by two alkaline comet assay methods.

    PubMed

    Lagroye, I; Anane, R; Wettring, B A; Moros, E G; Straube, W L; Laregina, M; Niehoff, M; Pickard, W F; Baty, J; Roti Roti, J L

    2004-01-01

    To investigate the effect of 2450 MHz pulsed-wave microwaves on the induction of DNA damage in brain cells of exposed rats and to discover whether proteinase K is needed to detect DNA damage in the brain cells of rats exposed to 2450 MHz microwaves. Sprague-Dawley rats were exposed to 2450 MHz pulsed-wave microwaves and sacrificed 4 h after a 2-h exposure. Rats irradiated whole-body with 1 Gy (137)Cs were included as positive controls. DNA damage was assayed by two variants of the alkaline comet assay on separate aliquots of the same cell preparation. Significant DNA damage was observed in the rat brain cells of rats exposed to gamma-rays using both versions of the alkaline comet assay independent of the presence or absence of proteinase K. However, neither version of the assay could detect any difference in comet length and/or normalized comet moment between sham- and 2450 MHz pulsed-wave microwave-exposed rats, regardless of the inclusion or omission of proteinase K in the comet assay. No DNA damage in brain cells was detected following exposure of rats to 2450 MHz microwaves pulsed-wave at a specific absorption rate of 1.2 W kg(-1) regardless of whether or not proteinase K was included in the assay. Thus, the results support the conclusion that low-level 2450 MHz pulsed-wave microwave exposures do not induce DNA damage detectable by the alkaline comet assay.

  5. 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.

  6. Visual estimation of the percentage of DNA in the tail in the comet assay: evaluation of different approaches in an intercomparison exercise.

    PubMed

    García, Omar; Romero, Ivonne; González, Jorge Ernesto; Moreno, Damaris L; Cuétara, Elizabeth; Rivero, Yesenia; Gutiérrez, Ariadne; Pérez, Carlos L; Alvarez, Aimée; Carnesolta, Deyanira; Guevara, Irania

    2011-02-28

    One of the difficulties in the comparison of results between laboratories working with the comet assay is the great diversity of parameters used to express DNA damage and the lack of conversion factors between the majority of them. Here we report a scorer-independent conversion curve to transform the values of DNA damage reported in arbitrary units (AU) into estimated percentage of DNA in the tail (E%T), and the results obtained in an intercomparison exercise where the effectiveness of this curve and two others proposed in the literature (E%T=AU/4 and E%T=(AU/5)+10) were tested. To obtain the conversion curve, human lymphocytes were first treated with radiation or H(2)O(2). Percentage of DNA in tail (%T) was then measured in 2100 comets (300 comets per treatment) using Casp image analysis software. Subsequently, using these values of %T, categories of 0, 1, 2, 3, and 4 were assigned to comets with %T [0-1), [1-25), [25-45), [45-70), and >70, and DNA damage was calculated in AU, as usual. DNA damage was induced in the interval 24-315AU (1.54-65.23%T). The best-fit conversion curve obtained by regression analysis was E%T=(AU-25.87)/4.46. In the intercomparison exercise, ten scorers from nine laboratories analyzed the same comet images (recorded on a compact disc) visually. The values reported in comet categories were transformed into AU and subsequently into E%T, using the three approaches mentioned above. The best agreement between E%T and %T measured by the software (S%T) was obtained with the conversion curve reported here, where the slope of E%T versus S%T from the ten scorers was not different from 1. Using this conversion curve, the overall mean difference between E%T and S%T was 1.4±2.62 and 57 (81%) of E%T values differ from S%T by less than 5 units. These findings show the strength of the scorer-independent conversion curve as a tool to compare results reported in AU or %T by different laboratories. Copyright © 2010 Elsevier B.V. All rights reserved.

  7. Determination of genotoxic effects of Imazethapyr herbicide in Allium cepa root cells by mitotic activity, chromosome aberration, and comet assay.

    PubMed

    Liman, Recep; Ciğerci, İbrahim Hakkı; Öztürk, Nur Serap

    2015-02-01

    Imazethapyr (IM) is an imidazolinone herbicide that is currently used for broad-spectrum weed control in soybean and other legume crops. In this study, cytotoxic and genotoxic effects of IM were investigated by using mitotic index (MI), mitotic phases, chromosomal abnormalities (CAs) and DNA damage on the root meristem cells of Allium cepa. In Allium root growth inhibition test, EC50 value was determined as 20 ppm, and 0.5xEC50, EC50 and 2xEC50 concentrations of IM herbicide were introduced to onion tuber roots. Distilled water and methyl methane sulfonate (MMS, 10 mg/L) were used as a negative and positive control, respectively. As A. cepa cell cycle is 24 hours, so, application process was carried out for 24, 48, 72 and 96 hours. All the applied doses decreased MIs compared to control group and these declines were found to be statistically meaningful. Analysis of the chromosomes showed that 10 ppm IM except for 48 h induced CAs but 40 ppm IM except for 72 h decreased CAs. DNA damage was found significantly higher in 20 and 40 ppm of IM compared to the control in comet assay. These results indicated that IM herbicide exhibits cytotoxic activity but not genotoxic activity (except 10 ppm) and induced DNA damage in a dose dependent manner in A. cepa root meristematic cells.

  8. Evaluation of the migration of mutagens/carcinogens from PET bottles into mineral water by Tradescantia/micronuclei test, Comet assay on leukocytes and GC/MS.

    PubMed

    Biscardi, D; Monarca, S; De Fusco, R; Senatore, F; Poli, P; Buschini, A; Rossi, C; Zani, C

    2003-01-20

    This study monitored the release of mutagenic/carcinogenic compounds into mineral water (natural and carbonated) from polyethylene terephthalate (PET) bottles, using a plant mutagenicity test which reveals micronuclei formation in Tradescantia pollen cells (Trad/MCN test), a DNA damage assay (Comet assay) on human leukocytes and gas chromatography/mass spectrometry (GC/MS) for the characterisation of migrants. The water samples were collected at a bottling plant and stored in PET bottles for a period ranging from 1 to 12 months. Every month some samples were randomly collected and lyophilised, the residual powders were extracted with organic solvents and then analysed by GC/MS and tested for DNA damage in human leukocytes, or reconstituted with distilled water to obtain concentrates for the exposure of Tradescantia inflorescences. Micronuclei increase in pollen was found only in natural mineral water stored for 2 months. DNA-damaging activity was found in many of the natural and carbonated water samples. Spring water was negative in the plant micronuclei test and the Comet assay, whereas distributed spring water showed DNA-damaging effects, suggesting a possible introduction of genotoxins through the distribution pipelines. GC/MS analysis showed the presence in mineral water of di(2-ethylhexyl)phthalate, a nongenotoxic hepatocarcinogenic plasticizer, after 9 months of storage in PET bottles. Copyright 2002 Elsevier Science B.V.

  9. 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.

  10. 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

  11. Genotoxicity of airborne PM2.5 assessed by salmonella and comet assays in five cities of the Emilia-Romagna (Italy) mutagenicity monitoring network.

    PubMed

    Bocchi, Clara; Bazzini, Cristina; Fontana, Federica; Pinto, Giancarlo; Cassoni, Francesca

    2017-10-11

    Airborne particulate matter (PM) has long been recognized as a potential health hazard and in 2013 was classified as carcinogenic to humans by the International Agency for Research on Cancer. In this study we evaluate and compare mutagenic and genotoxic potencies of PM2.5 collected in three seasons, from 2012 to 2015, in five Italian cities. Mutagenicity was evaluated through the Ames test on TA98 and TA100 strains and, for the measurement of PM clastogenicity, Comet assay was carried out on cultured human lung cells (A549). Organic matter, extracted from urban particulate matter, was also characterized for polycyclic aromatic hydrocarbons (PAHs) and their derivatives content. Samples collected in the colder seasons show the presence of both base pair substitution and frameshift mutagens, with enhanced mutagenic response in the absence of enzyme activation. The highest DNA damage detected with the Comet assay was induced by winter extracts, but different from Salmonella, the relative increase per cubic meter in comet tail for November samples was comparable to July ones. Comparing mutagenicity and genotoxicity with chemical concentrations we found that data from the Salmonella assay correlate with mass concentration and, to a lesser extent, with PAHs, but no association was found with their derivatives, whereas DNA damage correlate only with PAHs measured at one site. These findings demonstrate that to assess the mutagenicity and genotoxicity of complex mixtures it's necessary to use bioassays and that the chemical analysis of pollutants does not take into account the possible inhibitory or synergic effects of exposure. Environ. Mol. Mutagen., 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  12. Analysis of IUE observations of hydrogen in comets

    NASA Technical Reports Server (NTRS)

    Combi, Michael R.; Feldman, Paul D.

    1993-01-01

    The large body of hydrogen Lyman-alpha observations of cometary comae obtained with the International Ultraviolet Explorer satellite has gone generally unanalyzed because of two main modeling complications. First, the inner comae of many bright (gas productive) comets are often optically thick to solar Lyman-alpha radiation. Second, even in the case of a small comet (low gas production) the large IUE aperture is quite small as compared with the immense size of the hydrogen coma, so an accurate model which properly accounts for the spatial distribution of the coma is required to invert the inferred brightnesses to column densities and finally to H atom production rates. Our Monte Carlo particle trajectory model (MPTM), which for the first time provides the realistic full phase space distribution of H atoms throughout the coma was used as the basis for the analysis of IUE observations of the inner coma. The MCPTM includes the effects of the vectorial ejection of the H atoms upon dissociation of their parent species (H2O and OH) and of their partial collisional thermalization. Both of these effects are crucial to characterize the velocity distribution of the H atoms. A new spherical radiative transfer calculation based on our MCPTM was developed to analyze IUE observations of optically thick H comae. The models were applied to observations of comets P/Giacobini-Zinner and P/Halley.

  13. Comet Tempel 2: Orbit, ephemerides and error analysis

    NASA Technical Reports Server (NTRS)

    Yeomans, D. K.

    1978-01-01

    The dynamical behavior of comet Tempel 2 is investigated and the comet is found to be very well behaved and easily predictable. The nongravitational forces affecting the motion of this comet are the smallest of any comet that is affected by nongravitational forces. The sign and time history of these nongravitational forces imply (1) a direct rotation of the comet's nucleus and (2) the comet's ability to outgas has not changed substantially over its entire observational history. The well behaved dynamical motion of the comet, the well observed past apparitions, the small nongravitational forces and the excellent 1988 ground based observing conditions all contribute to relatively small position and velocity errors in 1988 -- the year of a proposed rendezvous space mission to this comet. To assist in planned ground based and earth orbital observations of this comet, ephemerides are given for the 1978-79, 1983-84 and 1988 apparitions.

  14. The potential value of the neutral comet assay and the expression of genes associated with DNA damage in assessing the radiosensitivity of tumor cells.

    PubMed

    Jayakumar, Sundarraj; Bhilwade, Hari N; Pandey, Badri N; Sandur, Santosh K; Chaubey, Ramesh C

    2012-10-09

    The assessment of tumor radiosensitivity would be particularly useful in optimizing the radiation dose during radiotherapy. Therefore, the degree of correlation between radiation-induced DNA damage, as measured by the alkaline and the neutral comet assays, and the clonogenic survival of different human tumor cells was studied. Further, tumor radiosensitivity was compared with the expression of genes associated with the cellular response to radiation damage. Five different human tumor cell lines were chosen and the radiosensitivity of these cells was established by clonogenic assay. Alkaline and neutral comet assays were performed in γ-irradiated cells (2-8Gy; either acute or fractionated). Quantitative PCR was performed to evaluate the expression of DNA damage response genes in control and irradiated cells. The relative radiosensitivity of the cell lines assessed by the extent of DNA damage (neutral comet assay) immediately after irradiation (4Gy or 6Gy) was in agreement with radiosensitivity pattern obtained by the clonogenic assay. The survival fraction of irradiated cells showed a better correlation with the magnitude of DNA damage measured by the neutral comet assay (r=-0.9; P<0.05; 6Gy) than evaluated by alkaline comet assay (r=-0.73; P<0.05; 6Gy). Further, a significant correlation between the clonogenic survival and DNA damage was observed in cells exposed to fractionated doses of radiation. Of 15 genes investigated in the gene expression study, HSP70, KU80 and RAD51 all showed significant positive correlations (r=0.9; P<0.05) with tumor radiosensitivity. Our study clearly demonstrated that the neutral comet assay was better than alkaline comet assay for assessment of radiosensitivities of tumor cells after acute or fractionated doses of irradiation.

  15. 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

  16. Analysis of organic compounds in returned comet nucleus samples

    NASA Technical Reports Server (NTRS)

    Cronin, J. R.

    1989-01-01

    Techniques for analysis of organic compounds in returned comet nucleus samples are described. Interstellar, chondritic and transitional organic components are discussed. Appropriate sampling procedures will be essential to the success of these analyses. It will be necessary to return samples that represent all the various regimes found in the nucleus, e.g., a complete core, volatile components (deep interior), and crustal components (surface minerals, rocks, processed organics such as macromolecular carbon and polymers). Furthermore, sampling, storage, return, and distribution of samples must be done under conditions that preclude contamination of the samples by terrestrial matter.

  17. Genotoxicity of cyanobacterial extracts containing microcystins from Polish water reservoirs as determined by SOS chromotest and comet assay.

    PubMed

    Mankiewicz, Joanna; Walter, Zofia; Tarczynska, Malgorzata; Palyvoda, Olena; Wojtysiak-Staniaszczyk, Magdalena; Zalewski, Maciej

    2002-01-01

    Toxicity of cyanobacterial blooms, an increasing problem around the world, is connected to the increase in bloom samples containing microcystins, caused by excessive eutrophication of drinking- and recreational water reservoirs. Microcystins are the most common group of cyanobacterial hepatotoxins. In Poland they are produced mainly by the Microcystis genus. The toxicity of microcystins has been well documented, but investigation into their genotoxicity has been insufficient relative to the study of their overall toxicity. Therefore, the aim of this study was the estimation and comparison of the genotoxicity of cyanobacterial extracts with microcystins (CEMs) using the SOS chromotest (bacterial test) with Escherichia coli PQ37 and the comet assay with human lymphocytes. Cyanobacterial bloom samples were collected in the summer months from two Polish water reservoirs, one at Sulejów and one at Jeziorsko. The SOS chromotest, which used prokaryotic cells (without metabolic activation), and the comet assay, which used eukaryotic cells, both indicated the potential genotoxic effect of CEMs. Cyanobacterial extracts caused DNA damage in human lymphocytes in vitro. The maximum level of DNA damage was observed after 12 h incubation with CEMs. The bacterial test indicated a dependence of the degree of CEM genotoxicity, the composition, and the concentration of microcystins in each bloom sample examined with the time of exposure. Differences between the genotoxicity of cyanobacterial extract and the standard microcystin-LR were noticeable. This was probably caused by the interaction of different microcystin variants. The results showed that CEMs from Polish water reservoirs were genotoxic, which was reflected by the stimulation of the SOS repair system in bacterial cells (SOS chromotest) and by the damage induced in DNA in human lymphocytes (comet assay).

  18. Application of the comet assay for investigation of oxidative DNA damage in equine peripheral blood mononuclear cells.

    PubMed

    Marlin, David J; Johnson, Lucy; Kingston, Demelza A; Smith, Nicola C; Deaton, Chris M; Mann, Sarah; Heaton, Paul; Van Vugt, Fenneke; Saunders, Kelly; Kydd, Julia; Harris, Pat A

    2004-08-01

    Oxidative stress occurs when antioxidant defense mechanisms are overwhelmed by free radicals and may lead to DNA damage, which has been implicated in processes such as aging and diseases such as cancer. The two main techniques presently used to quantify DNA damage are measurement of 8-hydroxydeoxyguanosine and the Comet assay (also known as single-cell gel electrophoresis). The aim of this study was to apply the comet assay to equine peripheral blood mononuclear cells (PBMCs) and identify two conditions in which we hypothesized that oxidative DNA damage would be increased in PBMCs: aging and equine recurrent airway obstruction (RAO, a condition similar to human asthma). The images obtained were similar to those previously published for humans, cats, and dogs. The optimum concentration of H(2)O(2) to estimate susceptibility to exogenous damage was 50 microM. Mean intraassay coefficients of variation were 4.7 and 9.7% for endogenous and exogenous tail-DNA quantities, respectively, and 7.3 and 8.3%, respectively, for interassay coefficients. There was no significant difference in either endogenous or exogenous percentages of tail DNA for samples collected from six ponies on three consecutive days. There was no significant difference in endogenous, exogenous, or exogenous (corrected for endogenous) oxidative DNA damage between mature and aged ponies. However, young pony foals had significantly less endogenous DNA damage than mature or aged ponies (P < 0.05). RAO-affected horses without airway inflammation (i.e., in clinical remission) had significantly greater endogenous damage compared with non-RAO-affected control animals (P = 0.009). There was a significant correlation between endogenous percentage of tail DNA in PBMCs and red blood cell hemolysate glutathione concentration (r = 0.720; P < 0.001). In conclusion, the comet assay appears to be suitable for investigating DNA damage in equine PBMCs.

  19. Measurement of X-ray-induced DNA double-strand breaks at various stages of the cell cycle using the total fluorescence as a comet assay parameter

    NASA Astrophysics Data System (ADS)

    Attia, Atef M. M.; Nabil, Ghada M.; Frankenberg, Dieter; Frankenberg-Schwager, M.

    2011-11-01

    The aim of the study was to develop a protocol for both estimating cell cycle position and the level of ionizing radiation-induced DNA dsb using the neutral comet assay. Using DNA histograms, cell cycle positions were determined for human dermal fibroblasts. The tail intensity was used to estimate the level of DNA damage induced by X-rays, at different positions of the cell cycle. The results of tail intensity versus DNA content bivariate analysis of exponentially growing cells showed a remarkable decrease in tail intensity with transition of cells from G1 to S-phase and increases slightly with transition to G2/M phase. This effect is observed at all doses including unirradiated cells, indicating that the effect is not caused by X-rays and the comet assay based on the current tail parameters is not relevant to measure DNA damage at various stages of the cell cycle. The results of dose response curves showed a linear decrease in the comet fluorescence with the X-ray dose. This observation provides a basis for estimating the fraction of damaged DNA, based on the fluorescence decrement induced by ionizing radiation. The results of this new approach showed a linear increase in DNA damage with dose, at various stages of the cell cycle, with rates, which vary in the following order G0>G2/M>S/G1 cells.These results suggest that G0 and G2/M cells are the most sensitive to X-rays among all phases of the cell cycle and suggest synchronization of cells at these phases to increase the cellular radiosensitivity during radiotherapy.

  20. Preliminary study of genotoxicity evaluation of orthodontic miniscrews on mucosa oral cells by the alkaline comet assay.

    PubMed

    Martín-Cameán, Ana; Puerto, María; Jos, Ángeles; Azqueta, Amaya; Iglesias-Linares, Alejandro; Solano, Enrique; Cameán, Ana M

    2015-01-01

    Miniscrew implants are widely used nowadays in orthodontic treatments due to their good results in clinical practice. However, data regarding the biocompatibility of commercially available orthodontic miniscrews and temporary devices are very scarce, and their role as genotoxicity inducers has been not previously evaluated with the alkaline comet assay. The aim of this study was to investigate the DNA damage in buccal cells of patients subjected to orthodontic treatments. The alkaline comet assay has been applied in oral mucosa cells from patients treated with conventional orthodontic treatment in comparison to patients treated additionally with miniscrews, non-treated volunteers (control) and smoking volunteers (positive control). The application of orthodontic appliances and miniscrews induced significant and similar (2-fold) increases of %DNA in tail in comparison to control group. Females experienced a significant increase in %DNA in all the treatments in comparison to the control group, whereas males showed significant damage only with the combined orthodontic and miniscrew treatment. In conclusion, conventional orthodontic appliances induced genotoxicity, and the incorporation of miniscrews assayed did not imply any additional increase of DNA damage.

  1. Genotoxicity evaluation of locally produced dental porcelain--an in vitro study using the Ames and Comet assays.

    PubMed

    Noushad, Mohammed; Kannan, Thirumulu Ponnuraj; Husein, Adam; Abdullah, Haswati; Ismail, Abdul Rashid

    2009-09-01

    The aim of this study was to determine the genotoxicity of a locally produced dental porcelain (Universiti Sains Malaysia, Malaysia) using the Ames and Comet assays. In the Ames assay, four genotypic variants of the Salmonella strains (TA98, TA100, TA1537 and TA1535) carrying mutations in several genes were used. The dental porcelain was incubated with these four strains in five different doses both in the presence and absence of metabolic activation (S9) and the result was assessed based on the number of revertant colonies. Concurrently, appropriate positive controls were used so as to validate the test. The average number of revertant colonies per plate treated with locally produced dental porcelain was less than double as compared to that of negative control. In the Comet assay, L929 (CCL-1 ATCC, USA) mouse fibroblast cells were treated with the dental porcelain in three different concentrations along with concurrent negative and positive controls. The tail moment which was used as a measurement of DNA damage was almost equal to that of the negative control, suggesting that the locally produced dental porcelain did not induce any DNA damage. The results indicated that the locally produced dental porcelain is non-genotoxic under the present test conditions.

  2. 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.

  3. 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.

  4. Assessment of occupational exposure to welding fumes by inductively coupled plasma-mass spectroscopy and by the alkaline Comet assay.

    PubMed

    Botta, Céline; Iarmarcovai, Gwenaëlle; Chaspoul, Florence; Sari-Minodier, Irène; Pompili, Jocelyne; Orsière, Thierry; Bergé-Lefranc, Jean-Louis; Botta, Alain; Gallice, Philippe; De Méo, Michel

    2006-05-01

    Welding fumes are classified as possibly carcinogenic to humans (Group 2B) by the International Agency for Research on Cancer. In the current study, blood and urine concentrations of aluminum (Al), cadmium (Cd), cobalt (Co), chromium (Cr), manganese (Mn), nickel (Ni), lead (Pb), and zinc (Zn) were monitored by inductively coupled plasma-mass spectrometry (ICP-MS) in 30 welders and in 22 controls. In addition, DNA damage was examined in the lymphocytes of these subjects by the alkaline Comet assay. Two biological samples were taken from the welders at the beginning (BW) and at the end (EW) of a work week. In controls, collection of samples was limited to BW. Blood concentrations of Cd, Co, Cr, Ni, and Pb were higher in the welders than in the control group while higher concentrations of Al, Cd, Co, Cr, Ni, and Pb were detected in welder urines. There was no significant difference in the metal concentrations for the BW and EW welder samples. Increased levels of DNA damage were found in lymphocytes from welders as compared to the controls, and 20/30 welders had higher levels of DNA lesions in the EW than in the BW samples. Age had a significant effect on DNA damage in the control group. Spearman's rank correlation analysis indicated that there were positive correlations between blood concentrations of Al, Co, Ni, and Pb and the levels of DNA damage. A negative correlation was found between DNA damage and Mn in blood, while there was a positive correlation between urinary Mn concentration and DNA damage. These data indicate that occupational exposure to welding fumes increases DNA damage in lymphocytes. Copyright (c) 2006 Wiley-Liss, Inc.

  5. The effects on DNA migration of altering parameters in the comet assay protocol such as agarose density, electrophoresis conditions and durations of the enzyme or the alkaline treatments.

    PubMed

    Ersson, Clara; Möller, Lennart

    2011-11-01

    The single cell gel electrophoresis (comet assay) is a popular method for measuring DNA migration as an estimate of DNA damage. No standardised comet assay protocol exists, which make comparisons between studies complicated. In a previous inter-laboratory validation study of the comet assay, we identified important parameters in the protocol that might affect DNA migration. The aim of this study was to assess how different comet assay protocols affect DNA migration. The results in this study suggest that (i) there is a significant linear dose-response relationship between the agarose gel's density and DNA migration and that damaged cells are more sensitive to the agarose gel's density; (ii) incubation with formamidopyrimidine DNA glycosylase for 10 min is inadequate, whereas 30 min is sufficient; (iii) the typically used 20 min of alkaline treatment might be to short when analysing samples that contain particular alkali-labile sites (ALS) and (iv) the duration of electrophoresis as well as the strength of the electric field applied affects the DNA migration. By using protocol-specific calibration curves, it is possible to reduce the variation in DNA migration caused by differences in comet assay protocols. This does, however, not completely remove the impact of the durations of alkaline treatment and electrophoresis when analysing cells containing ALS that are relatively resistant to high alkaline treatment.

  6. Assessment of DNA damage by comet assay and fast halo assay in buccal epithelial cells of Indian women chronically exposed to biomass smoke.

    PubMed

    Mondal, Nandan Kumar; Bhattacharya, Purba; Ray, Manas Ranjan

    2011-07-01

    Genotoxicity of indoor air pollution from biomass burning was evaluated in buccal epithelial cells (BECs) of 85 pre-menopausal Indian women who were engaged in cooking with biomass (wood, dung, crop residues) and 76 age-matched control women who were cooking with cleaner fuel liquefied petroleum gas (LPG). DNA damage was evaluated by comet assay and fast halo assay (FHA). The concentrations of particulate matter with aerodynamic diameters of less than 10 and 2.5 μm (PM(10) and PM(2.5), respectively) in indoor air were measured by real-time aerosol monitor. Generation of reactive oxygen species (ROS) was measured by flow cytometry and the level of superoxide dismutase (SOD) by spectrophotometry. Compared with control, BEC of biomass users illustrated 2.6-times higher comet tail % DNA (32.2 vs. 12.4, p < 0.001), 2.7-times greater comet tail length (37.8 μm vs. 14.2 μm, p < 0.001) and 2.2-times more olive tail moment (7.1 vs. 3.2, p < 0.001), suggesting marked increase in DNA damage. FHA also showed 5-times more mean nuclear diffusion factor (9.2 vs. 1.8, p < 0.0001) in BEC of biomass users, confirming sharp rise in DNA single strand breaks. Airway cells of biomass-using women showed 51% rise in ROS generation but 28% reduction in SOD, suggesting oxidative stress in the airways. Indoor air of biomass-using households had 3-times more PM(10) and PM(2.5) than LPG-using families, and DNA damage showed positive association with PM(10) and PM(2.5) levels controlling education, kitchen location and family income as potential confounders. In summary, chronic inhalation of biomass smoke elicits oxidative stress and extensive DNA damage in BEC.

  7. Double-stranded DNA breaks hidden in the neutral Comet assay suggest a role of the sperm nuclear matrix in DNA integrity maintenance.

    PubMed

    Ribas-Maynou, J; Gawecka, J E; Benet, J; Ward, W S

    2014-04-01

    We used a mouse model in which sperm DNA damage was induced to understand the relationship of double-stranded DNA (dsDNA) breaks to sperm chromatin structure and to the Comet assay. Sperm chromatin fragmentation (SCF) produces dsDNA breaks located on the matrix attachment regions, between protamine toroids. In this model, epididymal sperm induced to undergo SCF can religate dsDNA breaks while vas deferens sperm cannot. Here, we demonstrated that the conventional neutral Comet assay underestimates the epididymal SCF breaks because the broken DNA ends remain attached to the nuclear matrix, causing the DNA to remain associated with the dispersion halo, and the Comet tails to be weak. Therefore, we term these hidden dsDNA breaks. When the Comet assay was modified to include an additional incubation with sodium dodecyl sulfate (SDS) and dithiothreitol (DTT) after the conventional lysis, thereby solubilizing the nuclear matrix, the broken DNA was released from the matrix, which resulted in a reduction of the sperm head halo and an increase in the Comet tail length, exposing the hidden dsDNA breaks. Conversely, SCF-induced vas deferens sperm had small halos and long tails with the conventional neutral Comet assay, suggesting that the broken DNA ends were not tethered to the nuclear matrix. These results suggest that the attachment to the nuclear matrix is crucial for the religation of SCF-induced DNA breaks in sperm. Our data suggest that the neutral Comet assay identifies only dsDNA breaks that are released from the nuclear matrix and that the addition of an SDS treatment can reveal these hidden dsDNA breaks.

  8. Double-stranded DNA breaks hidden in the neutral Comet assay suggest a role of the sperm nuclear matrix in DNA integrity maintenance

    PubMed Central

    Ribas-Maynou, J.; Gawecka, J.E.; Benet, J.; Ward, W.S.

    2014-01-01

    We used a mouse model in which sperm DNA damage was induced to understand the relationship of double-stranded DNA (dsDNA) breaks to sperm chromatin structure and to the Comet assay. Sperm chromatin fragmentation (SCF) produces dsDNA breaks located on the matrix attachment regions, between protamine toroids. In this model, epididymal sperm induced to undergo SCF can religate dsDNA breaks while vas deferens sperm cannot. Here, we demonstrated that the conventional neutral Comet assay underestimates the epididymal SCF breaks because the broken DNA ends remain attached to the nuclear matrix, causing the DNA to remain associated with the dispersion halo, and the Comet tails to be weak. Therefore, we term these hidden dsDNA breaks. When the Comet assay was modified to include an additional incubation with sodium dodecyl sulfate (SDS) and dithiothreitol (DTT) after the conventional lysis, thereby solubilizing the nuclear matrix, the broken DNA was released from the matrix, which resulted in a reduction of the sperm head halo and an increase in the Comet tail length, exposing the hidden dsDNA breaks. Conversely, SCF-induced vas deferens sperm had small halos and long tails with the conventional neutral Comet assay, suggesting that the broken DNA ends were not tethered to the nuclear matrix. These results suggest that the attachment to the nuclear matrix is crucial for the religation of SCF-induced DNA breaks in sperm. Our data suggest that the neutral Comet assay identifies only dsDNA breaks that are released from the nuclear matrix and that the addition of an SDS treatment can reveal these hidden dsDNA breaks. PMID:24282283

  9. 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 (EC50) of DNA fragmentation from the dose-response relationships indicated toluene has the highest EC50 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.

  10. 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.

  11. Construction and validation of a dose-response curve using the comet assay to determine human radiosensitivity to ionizing radiation.

    PubMed

    Güerci, A; Zúñiga, L; Marcos, R

    2011-01-01

    Individual radiosensitivity is an individual characteristic associated with an increased reaction to ionizing radiation. The purpose of our work is to establish a dose-response curve useful to classify individuals as radiosensitive or radioresistant. Thus, a dose-response curve was constructed by measuring in vitro responses to increasing doses (0 to 8 Gy) of gamma radiation in the comet assay. The obtained curve fit well with a linear equation in the range of 0 to 8 Gy. The overall dose-response curve was constructed for percent DNA in tail, as a measure of the genetic damage induced by irradiation. To probe the goodness of the constructed curve, a validation study was carried out with whole blood from two donors in a blind study. Results show that, for the two applied doses (2 and 6 Gy), the obtained values fit well inside the interval of confidence of the curve. In conclusion, our results demonstrate the usefulness of the comet assay in determining individual responses to defined doses of gamma radiation. The standard dose-response curve constructed may be used to detect individuals departing from reference values.

  12. Evaluation of DNA damage induced by environmental exposure to mercury in Liza aurata using the comet assay.

    PubMed

    Pereira, Carla Sofia Alves; Guilherme, Sofia Isabel Antunes Gomes; Barroso, Carlos Miguel Miguez; Verschaeve, Luc; Pacheco, Mário Guilherme Garcês; Mendo, Sónia Alexandra Leite Velho

    2010-01-01

    Mercury (Hg) is one of the major aquatic contaminants even though emissions have been reduced over the years. Despite the relative abundance of investigations carried out on Hg toxicity, there is a scarcity of studies on its DNA damaging effects in fish under realistic exposure conditions. This study assessed the Hg genotoxicity in Golden grey mullets (Liza aurata) at Laranjo basin, a particularly contaminated area of Ria de Aveiro (Portugal) well known for its Hg contamination gradient. (1) Fish were seasonally caught at Laranjo basin and at a reference site (S. Jacinto), and (2) animals from the reference site were transplanted and caged (at bottom and surface), for 3 days, in two different locations within Laranjo basin. Using the comet assay, blood was analyzed for genetic damage and apoptotic cell frequency. The seasonal survey showed greater DNA damage in the Hg-contaminated area for all sampling seasons excluding winter. The temporal variation pattern of DNA lesions was: summer approximately autumn > winter > spring. Fish caged at Laranjo also exhibited greater DNA damage than those caged at the reference site, highlighting the importance of gill uptake on the toxicity of this metal. No increased susceptibility to apoptosis was detected in either wild or caged fish, indicating that mercury damages DNA of blood cells by a nonapoptotic mechanism. Both L. aurata and the comet assay proved to be sensitive and suitable for genotoxicity biomonitoring in mercury-contaminated coastal systems.

  13. 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.

  14. 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.

  15. 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

  16. Use of the Comet test and micronucleus assay on human white blood cells for in vitro assessment of genotoxicity induced by different drinking water disinfection protocols.

    PubMed

    Maffei, Francesca; Buschini, Annamaria; Rossi, Carlo; Poli, Paola; Forti, Giorgio Cantelli; Hrelia, Patrizia

    2005-08-01

    Surface water disinfection can lead to the formation of mutagenic/carcinogenic by-products derived from reactions with naturally occurring inorganic compounds. We investigated the feasibility and potential usefulness of an integrated approach to genotoxicity analysis of drinking water. The approach employed the Comet and micronucleus (MN) assays to evaluate the DNA and chromosomal damage produced by water extracts in human blood cells. Surface water samples from Lago Trasimeno (Italy) were collected in different seasons (July 2000, October 2000, February 2001, and June 2001), and samples were disinfected with sodium hypochloride (NaClO), chlorine dioxide (ClO(2)), or peracetic acid (PAA). Extracts of untreated and treated water were incubated with primary human leukocytes. The Comet assay revealed both strong seasonal variations and differences between samples processed by the three disinfection protocols. The three disinfectants increased the genotoxicity of the water collected in July 2000 and October 2000, with PAA producing the greatest amount of DNA damage. Extracts of raw water collected in February 2001 produced so much DNA damage that the relative genotoxic potentials of the three disinfectants could not be evaluated. No increase in MN frequency was detected in any of the samples. The multi-endpoint MN assay indicated, however, that our study samples (especially the sample collected in the February 2001) were cytotoxic. We conclude that this integrated approach to genotoxicity assessment may be useful both for the quality control of raw drinking water and to help compare the potential health risks associated with alternative disinfection processes. Copyright 2005 Wiley-Liss, Inc.

  17. Evaluation of DNA damage in agricultural workers exposed to pesticides using single cell gel electrophoresis (comet) assay.

    PubMed

    Kaur, Raminderjeet; Kaur, Satbir; Lata, Mukesh

    2011-09-01

    Pesticides are used in agriculture to protect crops, but they pose a potential risk to farmers and environment. The aim of the present study is to investigate the relation between the occupational exposure to various pesticides and the presence of DNA damage. Blood samples of 210 exposed workers (after a day of intense spraying) and 50 control subjects belonging to various districts of Punjab (India) were evaluated using Comet assay. Sixty workers who showed DNA damage were selected for follow up at 5-6 months after the first sampling during a low or null spraying period. Significant differences were found in DNA damage between freshly exposed workers and controls and freshly exposed and followed up cases. There was significant increase in the comet parameters viz. mean comet tail length and frequency of cells showing migration in exposed workers as compared to controls (72.22 ± 20.76 vs. 46.92 ± 8.17, P<0.001; 31.79 vs. 5.77, P<0.001). In the second samples, followed up cases showed significant decrease in frequency of damaged cells as compared to freshly exposed workers of first sampling (P<0.05). The confounding factors such as variable duration of pesticide exposure, age, smoking, drinking and dietary habits etc which were expected to modulate the damage, were instead found to have no significant effect on DNA fragmentation. The evidence of a genetic hazard related to exposure resulting from the intensive use of pesticides stresses the need for educational programs for agricultural workers to reduce the use of chemicals in agriculture.

  18. Evaluation of Cytogenetic and Genotoxic Effects of Oxalic Acid by the Alkaline Comet Assay and QRT PCR in Human Buccal Epithelial Cells.

    PubMed

    Unlu, Sibel; Saglar, Emel

    2015-12-01

    To evaluate the cytogenetic and genotoxic effects of oxalic acid. Buccal epithelial cells were obtained and cells were suspended in phosphate buffered saline. Increasing amounts of oxalic acids were added to cell suspensions and incubated in 37 degrees C, 5% CO2 for 30 minutes. Comets were scored using a computer-based image analysis system, and tail moments were taken as a measure of DNA damage. The transcriptional changes of HIPK2, GADD45A, DDB2, p53, PCNA, NBS1, and MDM2 genes were also investigated by qRT-PCR using B2M and GAPDH genes as references. It was found that DNA damage occurred in parallel with increasing amounts of oxalic acid according to tail moment measurements, and also expressions of the selected genes have increased. It can be concluded that along with increasing amounts of oxalic acid, cell damage was detected, and both comet assay and expression of the selected genes can be used in the assessment of the damage caused by oxalic acid.

  19. Workshop on Analysis of Returned Comet Nucleus Samples

    NASA Astrophysics Data System (ADS)

    Chang, Sherwood

    This volume contains abstracts that have been accepted by the Program Committee for presentation at the Workshop on Analysis of Returned Comet Nucleus Samples, held in Milpitas, California, January 16-18, 1989. Conveners are Sherwood Chang (NASA Ames Research Center) and Larry Nyquist (NASA Johnson Space Center). Program Committee members are Thomas Ahrens (ex-officio; California Institute of Technology), Lou Allamandola (NASA Ames Research Center), David Blake (NASA Ames Research Center), Donald Brownlee (University of Washington, Seattle), Theodore E. Bunch (NASA Ames Research Center), Humberto Campins (Planetary Science Institute), Jeff Cuzzi (NASA Ames Research Center), Eberhard Griin (Max-Plank-Institut fiir Kemphysik), Martha Hanner (Jet Propulsion Laboratory), Alan Harris (Jet Propulsion Laboratory), John Kerrid-e (University of Califomia, Los Angeles), Yves Langevin (University of Paris), Gerhard Schwehm (ESTEC), and Paul Weissman (Jet Propulsion Laboratory). Logistics and administrative support for the workshop were provided by the Lunar and Planetary Institute Projects Office.

  20. 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.

  1. An investigation of the DNA-damaging ability of benzene and its metabolites in human lymphocytes, using the Comet assay

    SciTech Connect

    Anderson, D.; Yu, T.W.; Schmezer, P. |

    1995-12-31

    Benzene and five of its known metabolites-muconic acid, hydroquinone, catechol, p-benzoquinone, and benzentriol-were examined for DNA damage in human lymphocytes using the alkaline Comet assay, and conditions were optimised to determine responses. When comets were measured by eye after treatment with hydrogen peroxide (H{sup 2}O{sup 2}), the positive control, and each compound for 0.5 hr, only H{sup 2}O{sup 2} and benzenetrial induced pronounced DNA damage without metabolic activation. The effect of catechol was moderate compared, with that of benzenetriol. There was a very weak effect of benzene in the absence of rat liver S-9 mix. In the presence of S-9 mix, benzene was not activated. The effect of benzenetriol was greatly reduced by the external metabolishing system, but p-benzoquinone became activated o some extent. Catalase abolished the effect of benzenetriol, suggesting that H{sup 2}O{sup 2} formed during autoxidation may be responsible for the DNA-damaging ability of this metabolite. Mitogen-stimulated cycling cells were less sensitive to H{sup 2}O{sup 2} and benzenetrial than unstimulated G{sub O} lymphocytes. Effects tended to become more pronounced at high doses and after longer exposures, although this was not always consistent from experiment to experiment. In conclusion, benzene and all metabolites investigated gave positive responses. Where altered responses were observed, they were significantly different from the corresponding controls. 46 refs., 7 tabs.

  2. Protective activity of cedron (Aloysia triphylla) infusion over genetic damage induced by cisplatin evaluated by the comet assay technique.

    PubMed

    Zamorano-Ponce, Enrique; Fernández, Julia; Vargas, Gilda; Rivera, Pilar; Carballo, Marta A

    2004-08-30

    Using the comet assay technique, this paper examines the protection from the cisplatin-induced genetic damage in mouse bone marrow cells provided by cedron-leaf infusion. Animals were separated into six groups: (I) untreated, (II) negative control, (III) treated with cedron-leaf infusion (5%), (IV) treated with cisplatin (6 mg/kg b.w.), (V) pretreated with infusion and treated with cisplatin and (VI) positive control (cyclophosphamide, 20 mg/kg b.w.). Based on the tail moment values found, four types of comets were distinguished. No statistical differences (P<0.01) were found between untreated animals, negative control and infusion treated mice. As expected, treatment of mice with a single dose of cis-DDP-induced genetic damage and the pretreatment with infusion prior to cis-DDP injection inhibited the capacity of cisplatin to induce genetic damage. Cell viability was up to 90% in all cases. The results suggest that infusion could exert its in vivo antigenotoxic action by enhancing the antioxidant status of bone marrow cells. The found could be attributed to its scavenging potency towards free radicals.

  3. Genotoxicity of select herbicides in Rana catesbeiana tadpoles using the alkaline single-cell gel DNA electrophoresis (comet) assay.

    PubMed

    Clements, C; Ralph, S; Petras, M

    1997-01-01

    Pesticides are broadly used for pest control in agriculture despite possible negative impacts they may pose to the environment. Thus, we examined the DNA damage caused by five herbicides commonly used in southern Ontario (Canada). Erythrocytes from Rana catesbeiana (bullfrog) tadpoles were evaluated for DNA damage following exposure to selected herbicides, using the alkaline single-cell gel DNA electrophoresis (SCG) or "comet" assay [Singh et al. (1988): Exp Cell Res 175:184-191; Ralph et al. (1996): Eviron Mol Mutagen 28:112-120]. This approach involves detection, under alkaline conditions, of DNA fragments that upon electrophoresis migrate from the nuclear care, resulting in a comet formation. The herbicides tested, along with their active ingredients, were AAtrex Nine-O (atrazine), Dual-960E (metalochlor), Roundup (glyphosate), Sencor-500F (metribuzin), and Amsol (2,4-D amine). Tadpoles were exposed in the laboratory for a 24-hr period to several concentrations of the herbicides dissolved in dechlorinated water. Methyl methanesulphonate was used as a positive control. The herbicides AAtrex Nine-O-, Dual-960E-, Roundup-, and Sencor-500F-treated tadpoles showed significant DNA damage when compared with unexposed control animals, whereas, Amsol-treated tadpoles did not. Unlike the other responding herbicides, Sencor-500F did not show a relationship between dosage and DNA damage. In summary, the results indicate that at least some of the herbicides currently used in southern Ontario are capable of inducing DNA damage in tadpoles.

  4. Assessment of genotoxic damage by the comet assay in white storks (Ciconia ciconia) after the Doñana Ecological Disaster.

    PubMed

    Pastor, N; López-Lázaro, M; Tella, J L; Baos, R; Hiraldo, F; Cortés, F

    2001-05-01

    Single cell gel electrophoresis, the so-called "Comet" assay, was performed as a genotoxicity test in white storks sampled in an area heavily contaminated after the ecological disaster in south western Spain. This disaster occurred as a consequence of a massive toxic spillage of acid waste rich in heavy metals that impacted on the Doñana National Park. The importance of this protected area as a breeding and wintering site for many endangered bird species makes this analysis of DNA damage of special interest. Our results clearly show that white storks born in the contaminated area 1 year after the toxic spill bear a high burden of genetic damage as compared with control individuals. The possible implications for future survival as well as reproductive rate are discussed.

  5. The comet assay as a rapid test in biomonitoring occupational exposure to DNA-damaging agents and effect of confounding factors.

    PubMed

    Møller, P; Knudsen, L E; Loft, S; Wallin, H

    2000-10-01

    Within the last decade, the comet assay has been used with increasing popularity to investigate the level of DNA damage in terms of strand breaks and alkaline labile sites in biomonitoring studies. The assay is easily performed on WBCs and has been included in a wide range of biomonitoring studies of occupational exposures encompassing styrene, vinyl chloride, 1,3-butadiene, pesticides, hair dyes, antineoplastic agents, organic solvents, sewage and waste materials, wood dust, and ionizing radiation. Eleven of the occupational studies were positive, whereas seven were negative. Notably, the negative studies appeared to have less power than the positive studies. Also, there were poor dose-response relationships in many of the biomonitoring studies. Many factors have been reported to produce effects by the comet assay, e.g., age, air pollution exposure, diet, exercise, gender, infection, residential radon exposure, smoking, and season. Until now, the use of the comet assay has been hampered by the uncertainty of the influence of confounding factors. We argue that none of the confounding factors are unequivocally positive in the majority of the studies. We recommend that age, gender, and smoking status be used as criteria for the selection of populations and that data on exercise, diet, and recent infections be registered before blood sampling. Samples from exposed and unexposed populations should be collected at the same time to avoid seasonal variation. In general, the comet assay is considered a suitable and fast test for DNA-damaging potential in biomonitoring studies.

  6. Genotoxicity of field-collected inter-tidal sediments from Cork Harbor, Ireland, to juvenile turbot (Scophthalmus maximus L.) as measured by the Comet assay.

    PubMed

    Kilemade, M F; Hartl, M G J; Sheehan, D; Mothersill, C; Van Pelt, F N A M; O'Halloran, J; O'Brien, N M

    2004-01-01

    The alkaline single cell gel electrophoresis (SCGE) or Comet assay was employed to test the potential of surficial sediment collected from Cork Harbor, Ireland, to induce DNA damage in turbot (Scophthalmus maximus L.) in a laboratory exposure experiment. Turbot were exposed for 21 days to field-collected sediment from Cork Harbor and from a relatively clean reference site at Ballymacoda and sampled at 0, 7, 14, and 21 days. As a positive control for the sediment exposure experiment, a subsample of the turbot was exposed to cadmium chloride-spiked seawater. DNA damage analysis was performed on epidermal, gill, spleen, liver, and whole blood cell preparations. Liver, gill, and blood were the most sensitive tissues while a lower level of damage was detected in the epidermis and spleen. The blood was determined to be a suitable predictor of DNA damage in the whole organism. Chemical analysis of the sediment indicated that polycyclic aromatic hydrocarbons formed the bulk of the contaminants, with the harbor sites having almost double the levels of those from the reference site. The data indicated that turbot exposed to sediments from Cork Harbor elicited a significant increase in DNA damage in comparison with those exposed to sediment from the reference site and that exposure to the contaminated sediments caused a multi-organ genotoxic response. Results from the study indicate a relationship between the presence of genotoxicants in sediment and DNA damage. This finding was encouraging with regard to the potential use of the Comet assay as part of a marine biomonitoring strategy.

  7. Integrity of human sperm DNA assessed by the neutral comet assay and its relationship to semen parameters and clinical outcomes for the IVF-ET program

    PubMed Central

    Chung, Da-Yeon; Choi, Soon-Young; Kim, Jong-Hyun; Kim, Gi-Young; Lee, Jae-Seok; Lee, Hee-Sun; Kim, Myung-Hee; Roh, Sung-Il

    2011-01-01

    Objective To explore potential relationships between sperm DNA integrity and both semen parameters and clinical outcomes. Methods Semen analysis of 498 samples was performed according to the 2010 criteria of the World Health Organization. The sperm DNA fragmentation Index (DFI) of the semen samples was assessed using a neutral comet assay. Results Sperm DFI showed a significant correlation with semen parameters, including the patient's age, sperm viability, motility, morphology, and number of leukocytes (p<0.05). The sperm DFI values for asthenozoospermic (15.2%), oligoteratozoospermic (18.3%), asthenoteratozoospermic (17.5%), and oligoasthenoteratozoospermic semen samples (21.3%) were significantly higher than that observed in normozoospermic semen samples (10.5%, p<0.05). A sperm DFI value of 14% was used as a threshold of sperm DFI in assessing whether DNA was highly damaged. In 114 IVF-ET cycles, the fertilization rate of the sperm DFI <14% group (70 cycles, 61.7%) was significantly higher than that observed for the ≥14% group (44 cycles, 55.3%), but there was no difference in the other clinical outcomes between the two groups. In the ≥14% group, the pregnancy rates of the ICSI cycles (40.0%) and half-ICSI (44.0%) were higher than conventional IVF cycles (30.7%), but the difference was not statistically significant. Conclusion Along with the conventional semen analysis, the sperm DFI assessed using the comet assay was shown to improve the quality of the semen evaluation. To evaluate the precise effect of ICSI on pregnancy rates in the patients who demonstrate high sperm DFI values, further study is necessary. PMID:22384412

  8. The sensitivity of the alkaline comet assay in detecting DNA lesions induced by X rays, gamma rays and alpha particles.

    PubMed

    Rössler, U; Hornhardt, S; Seidl, C; Müller-Laue, E; Walsh, L; Panzer, W; Schmid, E; Senekowitsch-Schmidtke, R; Gomolka, M

    2006-01-01

    Experiments were designed and performed in order to investigate whether or not the different cellular energy deposition patterns of photon radiation with different energies (29 kV, 220 kV X rays; Co-60, Cs-137-gamma-rays) and alpha-radiation from an Am-241 source differ in DNA damage induction capacity in human cells. For this purpose, the alkaline comet assay (single cell gel electrophoresis) was applied to measure the amount of DNA damage in relation to the dose received. The comet assay data for the parameters '% DNA in the tail' and 'tail moment' for human peripheral lymphocytes did not indicate any difference in the initial radiation damage produced by 29 kV X rays relative to the reference radiations, 220 kV X rays and the gamma rays, whether for the total mean dose range of 0-3 Gy nor in the low-dose range. In contrast, when the 'tail length' data were analysed saturation of the fitted dose response curve appeared for X rays at about 1.5 Gy but was not apparent for gamma rays up to 3 Gy. Preliminary data for alpha exposures of HSC45-M2 cells showed a significant increase in DNA damage only at high doses (>2 Gy Am-241), but the damage at 2 Gy exceeded the damage induced at 2 Gy by Cs-137-gamma-rays by a factor of 2.5. In contrast, other experiments involving different cell systems and DNA damage indicators such as chromosomal aberrations have detected a significant increase in DNA damage at much lower doses, that is at 0.02 Gy for Am-241 and depicte a higher biological effectiveness. These results indicate that differences in biological effects arise through downstream processing of complex DNA damage.

  9. Genotoxicity of chlorpyrifos in freshwater fish Labeo rohita using Alkaline Single-cell Gel Electrophoresis (Comet) assay.

    PubMed

    Ismail, Muhammad; Khan, Qaiser Mahmood; Ali, Rahat; Ali, Tayyaba; Mobeen, Ameena

    2014-10-01

    Chlorpyrifos is a widely used insecticide of organophosphate group, which causes severe toxicological effects in non target aquatic organisms especially in fish. In the present study the genotoxic effects of sublethal concentrations of chlorpyrifos were observed in the erythrocytes and gill cells of Labeo rohita (commonly known as rohu) using the Alkaline Single-Cell Gel Electrophoresis (Comet) assay. Effects of chlorpyrifos on the behavior of the fish were also investigated. The 96 h LC50 value of chlorpyrifos, estimated by Trimmed Spearman-Karber (TSK) in static bioassay, was found to be 442.8 µg/L. On the basis of LC50 value, the fish were exposed to three sublethal concentrations of chlorpyrifos (SL-I ∼221.4 µg/L, SL- II ∼110.7 µg/L and SL-III ∼73.8 µg/L) for 96 h. Blood and gill samples were collected at every 24 h and were subjected to the Comet assay. The observed DNA damage was concentration dependent and time dependent and those levels of DNA damage in between the tested concentrations and times were significantly different (p < 0.01). It was also found that the gill cells are more sensitive to chlorpyrifos, though; it revealed more DNA damage as compared to the erythrocytes of fish. Fish exposed to different concentrations of chlorpyrifos showed different neurotoxic behavioral responses. It was concluded that chlorpyrifos is a genotoxic and neurotoxic insecticide causing DNA damage and neurotoxic effects in Labeo rohita.

  10. Effect of electromagnetic radiofrequency radiation on the rats' brain, liver and kidney cells measured by comet assay.

    PubMed

    Trosić, Ivancica; Pavicić, Ivan; Milković-Kraus, Sanja; Mladinić, Marin; Zeljezić, Davor

    2011-12-01

    The goal of study was to evaluate DNA damage in rat's renal, liver and brain cells after in vivo exposure to radiofrequency/microwave (Rf/Mw) radiation of cellular phone frequencies range. To determine DNA damage, a single cell gel electrophoresis/comet assay was used. Wistar rats (male, 12 week old, approximate body weight 350 g) (N = 9) were exposed to the carrier frequency of 915 MHz with Global System Mobile signal modulation (GSM), power density of 2.4 W/m2, whole body average specific absorption rate SAR of 0.6 W/kg. The animals were irradiated for one hour/day, seven days/week during two weeks period. The exposure set-up was Gigahertz Transversal Electromagnetic Mode Cell (GTEM--cell). Sham irradiated controls (N = 9) were apart of the study. The body temperature was measured before and after exposure. There were no differences in temperature in between control and treated animals. Comet assay parameters such as the tail length and tail intensity were evaluated. In comparison with tail length in controls (13.5 +/- 0.7 microm), the tail was slightly elongated in brain cells of irradiated animals (14.0 +/- 0.3 microm). The tail length obtained for liver (14.5 +/- 0.3 microm) and kidney (13.9 +/- 0.5 microm) homogenates notably differs in comparison with matched sham controls (13.6 +/- 0.3 microm) and (12.9 +/- 0.9 microm). Differences in tail intensity between control and exposed animals were not significant. The results of this study suggest that, under the experimental conditions applied, repeated 915 MHz irradiation could be a cause of DNA breaks in renal and liver cells, but not affect the cell genome at the higher extent compared to the basal damage.

  11. Effects of freezing-thawing on DNA integrity of boar spermatozoa assessed by the neutral comet assay.

    PubMed

    Fraser, L; Strzezek, J

    2005-12-01

    A modified version of the neutral comet assay was employed to evaluate the effect of the freezing-thawing process on boar-sperm DNA integrity. The sperm-rich fractions were collected from four mature boars and frozen into aluminium tubes and straws after extension in lactose-hen egg yolk-glycerol extender (lactose-HEY-G) or an extender containing lactose, lyophilized lipoprotein fractions extracted from ostrich egg yolk and glycerol (lactose-LPFo-G). The semen samples were also frozen in a standard boar semen extender (Kortowo-3), without the addition of cryoprotective substances. Post-thaw sperm motility and plasma membrane integrity, assessed by SYBR-14/PI and Hoechst 33258 stains, declined (p < or = 0.05) with a corresponding increase (p < or = 0.05) in sperm DNA damage, regardless of the extender type and packaging material. Spermatozoa frozen in lactose-HEY-G or lactose-LPFo-G extender showed lower (p < or = 0.05) DNA damage than those frozen in the absence of cryoprotective substances. The addition of HEY or LPFo to the freezing extender helped reduce the rate of cryo-damage to sperm DNA, which varied among the boars. Inter-boar variations in post-thaw DNA damage were more pronounced in sperm samples frozen in lactose-HEY-G or lactose-LPFo-G extender. The results of this study show that the freezing-thawing process affects the DNA integrity of boar spermatozoa, irrespective of the extender type and packaging material. Furthermore, the use of whole hen egg yolk and ostrich lyophilized lipoprotein fractions in the freezing extender gave similar results regarding sperm DNA integrity. It can be concluded that the neutral comet assay can be used in conjunction with routine sperm parameters for assessment of post-thaw quality of boar semen.

  12. 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...

  13. Leucocytes isolated from simply frozen whole blood can be used in human biomonitoring for DNA damage measurement with the comet assay.

    PubMed

    Akor-Dewu, Maryam B; El Yamani, Naouale; Bilyk, Olena; Holtung, Linda; Tjelle, Torunn E; Blomhoff, Rune; Collins, Andrew R

    2014-04-01

    Preservation of human blood cells for DNA damage analysis with the comet assay conventionally involves the isolation of mononuclear cells by centrifugation, suspension in freezing medium and slow freezing to -80 °C-a laborious process. A recent publication (Al-Salmani et al. Free Rad Biol Med 2011; 51: 719-725) describes a simple method in which small volumes of whole blood are frozen to -20 or -80 °C; on subsequent thawing, the comet assay is performed, with no indication of elevated DNA strand breakage resulting from the rapid freezing. However, leucocytes in whole blood (whether fresh or frozen) are abnormally resistant to damage by H2 O2 , and so a common test of antioxidant status (resistance to strand breakage by H2 O2 ) cannot be used. We have refined this method by separating the leucocytes from the thawed blood; we find that, after three washes, the cells respond normally to H2 O2 . In addition, we have measured specific endogenous base damage (oxidized purines) in the isolated leucocytes, using the enzyme formamidopyrimidine DNA glycosylase. In a study of blood samples from 10 subjects, H2 O2 sensitivity and endogenous damage-both reflecting the antioxidant status of the cells-correlated significantly. This modified approach to sample collection and storage is particularly applicable when the available volume of blood is limited and has great potential in biomonitoring and ecogenotoxicology studies where samples are obtained in the field or at sites remote from the testing laboratory.

  14. Ephemeris data and error analysis in support of a Comet Encke intercept mission

    NASA Technical Reports Server (NTRS)

    Yeomans, D. K.

    1974-01-01

    Utilizing an orbit determination based upon 65 observations over the 1961 - 1973 interval, ephemeris data were generated for the 1976-77, 1980-81 and 1983-84 apparitions of short period comet Encke. For the 1980-81 apparition, results from a statistical error analysis are outlined. All ephemeris and error analysis computations include the effects of planetary perturbations as well as the nongravitational accelerations introduced by the outgassing cometary nucleus. In 1980, excellent observing conditions and a close approach of comet Encke to the earth permit relatively small uncertainties in the cometary position errors and provide an excellent opportunity for a close flyby of a physically interesting comet.

  15. 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

  16. Differences in quantification of DNA double-strand breaks assessed by 53BP1/γH2AX focus formation assays and the comet assay in mammalian cells treated with irradiation and N-acetyl-L-cysteine.

    PubMed

    Kurashige, Tomomi; Shimamura, Mika; Nagayama, Yuji

    2016-06-01

    The biological effect of ionizing radiation (IR) on genomic DNA is thought to be either direct or indirect; the latter is mediated by IR induction of free radicals and reactive oxygen species (ROS). This study was designed to evaluate the effect of N-acetyl-L-cysteine (NAC), a well-known ROS-scavenging antioxidant, on IR induction of genotoxicity, cytotoxicity and ROS production in mammalian cells, and aimed to clarify the conflicting data in previous publications. Although we clearly demonstrate the beneficial effect of NAC on IR-induced genotoxicity and cytotoxicity (determined using the micronucleus assay and cell viability/clonogenic assays), the data on NAC's effect on DNA double-strand break (DSB) formation were inconsistent in different assays. Specifically, mitigation of IR-induced DSBs by NAC was readily detected by the neutral comet assay, but not by the γH2AX or 53BP1 focus assays. NAC is a glutathione precursor and exerts its effect after conversion to glutathione, and presumably it has its own biological activity. Assuming that the focus assay reflects the biological responses to DSBs (detection and repair), while the comet assay reflects the physical status of genomic DNA, our results indicate that the comet assay could readily detect the antioxidant effect of NAC on DSB formation. However, NAC's biological effect might affect the detection of DSB repair by the focus assays. Our data illustrate that multiple parameters should be carefully used to analyze DNA damage when studying potential candidates for radioprotective compounds.

  17. An analysis of the BVRI colors of 22 active comets

    NASA Astrophysics Data System (ADS)

    Betzler, A. S.; Almeida, R. S.; Cerqueira, W. J.; Araujo, L. A.; Prazeres, C. J. M.; Jesus, J. N.; Bispo, P. A. S.; Andrade, V. B.; Freitas, Y. A. S.; Betzler, L. B. S.

    2017-08-01

    Our aim was to analyze the variation of Johnson-Kron-Cousins BVRI color indexes of a sample with 22 active comets of various dynamic groups with the time, geometrical, observational and dynamical parameters. We performed photometric observations of 16 comets between 2010 and 2014, using robotic telescopes in three continents. In addition to the sample, we used data of six comets available in the literature. A statistical comparison between the distributions of color indexes was performed using the Kruskal-Wallis H-test. The color indexes of active comets can vary a few tenths up to a magnitude on time scales that range from hours to weeks. Using the B-V colors of the observed comets, we generated a relationship that correlates the cometary visual and CCD magnitudes. We did not identify any relationship between B-V and V-R colors with heliocentric distance and phase angle. The color B-V is correlated with the photometric aperture that can be described by a logarithmic function. We did not identify any differences in the distribution of B-V color among the comets analyzed at a confidence level equal to or greater than 95%. The mean color of active comets are B-R = 1.20 ± 0.24 , B-V = 0.76 ± 0.16 and V-R = 0.42 ± 0.16 . Active comets with V-R colors outside the three standard deviation interval can be considered objects with unusual physical characteristics.

  18. Measurement of DNA base and nucleotide excision repair activities in mammalian cells and tissues using the comet assay--a methodological overview.

    PubMed

    Azqueta, Amaya; Langie, Sabine A S; Slyskova, Jana; Collins, Andrew R

    2013-11-01

    There is an increasing demand for phenotyping assays in the field of human functional genetics. DNA repair activity is representative of this functional approach, being seen as a valuable biomarker related to cancer risk. Repair activity is evaluated by incubating a cell extract with a DNA substrate containing lesions specific for the DNA repair pathway of interest. Enzymic incision at the lesion sites can be measured by means of the comet assay (single cell gel electrophoresis). The assay is particularly applicable for evaluation of base and nucleotide excision repair pathways (BER and NER). Substrate DNA containing oxidised purines gives a measure of BER, while UV-induced photolesions are the substrate for NER. While applications of comet-based DNA repair assays continue to increase, there are no commonly accepted standard protocols, which complicates inter-laboratory comparisons of results. Here we provide a comprehensive summary of protocols for the comet-based BER- and NER-specific in vitro DNA repair assays that can be applied to a wide spectrum of biological material--cultured cell lines, blood cells, animal tissue samples and human biopsies. Our intention is to provide a detailed and user-friendly account of the assays, including practical tips and recommendations to help in setting them up. By proposing standard protocols, we hope to facilitate comparison of results obtained in different laboratories.

  19. Terminal navigation analysis for the 1980 comet Encke slow flyby mission

    NASA Technical Reports Server (NTRS)

    Jacobson, R. A.; Mcdanell, J. P.; Rinker, G. C.

    1973-01-01

    The initial results of a terminal navigation analysis for the proposed 1980 solar electric slow flyby mission to the comet Encke are presented. The navigation technique employs onboard optical measurements with the scientific television camera, groundbased observations of the spacecraft and comet, and groundbased orbit determination and thrust vector update computation. The knowledge and delivery accuracies of the spacecraft are evaluated as a function of the important parameters affecting the terminal navigation. These include optical measurement accuracy, thruster noise level, duration of the planned terminal coast period, comet ephemeris uncertainty, guidance initiation time, guidance update frequency, and optical data rate.

  20. Application of the micronucleus test and comet assay in Trachemys callirostris erythrocytes as a model for in situ genotoxic monitoring.

    PubMed

    Zapata, Lina M; Bock, Brian C; Orozco, Luz Yaneth; Palacio, Jaime A

    2016-05-01

    Trachemys callirostris is a turtle species endemic to northern South America. In northern Colombia it occurs in the middle and lower Magdalena River drainage and its principal tributaries (lower Cauca and San Jorge rivers) and in other minor drainages such as the lower Sinú River. In recent years, industrial, agricultural, and mining activities have altered natural habitats in Colombia where this species occurs, and many of the pollutants released there are known to induce genetic alterations in wildlife species. The micronucleus test and comet assay are two of the most widely used methods to characterize DNA damage induced by physical and chemical agents in wildlife species, but have not been employed previously for genotoxic evaluations in T. callirostris. The goal of this study was to optimize these genotoxic biomarkers for T. callirostris erythrocytes in order to establish levels of DNA damage in this species and thereby evaluate its potential as a sentinel species for monitoring genotoxic effects in freshwater environments in northern Colombia. Both genotoxic techniques were applied on peripheral blood erythrocytes from 20 captive-reared T. callirostris individuals as a negative control, as well as from samples obtained from 49 individuals collected in Magangué (Magdalena River drainage) and 24 individuals collected in Lorica (Sinú River drainage) in northern Colombia. Negative control individuals exhibited a baseline frequency of micronuclei of 0.78±0.58 and baseline values for comet tail length and tail moment of 3.34±0.24µm and 10.70±5.5, respectively. In contrast, samples from both field sites exhibited significantly greater evidence of genotoxic effects for both tests. The mean MN frequencies in the samples from Magangué and Lorica were 8.04±7.08 and 12.19±12.94, respectively. The mean tail length for samples from Magangué and Lorica were 5.78±3.18 and 15.46±7.39, respectively. Finally, the mean tail moment for samples from Magangué and

  1. 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.

  2. New osculating orbits for 110 comets and analysis of original orbits for 200 comets

    NASA Technical Reports Server (NTRS)

    Marsden, B. G.; Sekanina, Z.; Everhart, E.

    1978-01-01

    Osculating orbits are presented for 110 nearly parabolic comets. Combining these with selected orbit determinations from other sources, a total of 200 orbits are considered where the available observations yield a result of very good (first-class) or good (second-class) quality. For each of these, the original and future orbits (referred to the barycenter of the solar system) are calculated. The Oort effect (a tendency for original reciprocal semimajor axis values to range from zero to +100 millionths per AU) is clearly seen among the first-class orbits but not among the second-class orbits. Modifications in original reciprocal semimajor axis values due to the effects of nongravitational forces are considered.

  3. 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.

  4. Assessing the genotoxic potentials of arsenic in tilapia (Oreochromis mossambicus) using alkaline comet assay and micronucleus test.

    PubMed

    Ahmed, Md Kawser; Habibullah-Al-Mamun, Md; Hossain, M Anwar; Arif, Mohammad; Parvin, Elora; Akter, Mosammat Salma; Khan, Mohammad Shahneawz; Islam, Md Monirul

    2011-06-01

    This experiment was conducted to study the genotoxic potentials of sodium arsenite (NaAsO(2)) in freshwater fish Oreochromis mossambicus by using alkaline comet assay and micronucleus (MN) test. Fish were exposed to three different concentrations (3 ppm, 28 ppm and 56 ppm) of arsenic and gill, liver and blood tissue samples were collected after 48 h, 96 h and 192 h of exposure. Arsenic exposure induced DNA damage in all tissues examined in a concentration dependent manner. A significant (p<0.05) increase in the comet tail DNA (%) of the exposed fish liver, gill, and blood was observed after 48 h and 96 h of exposure, but a decline in DNA damage was recorded in all the tissues at all the three concentrations studied after 192 h of exposure. Liver tissue exhibited significantly (p<0.05) higher DNA damage at all the concentrations examined, followed by gill and blood. Higher liver tail DNA (51.38 ± 0.21%) refers that it is more prone to injury to arsenic toxicity than the gill and blood. In blood samples arsenic induced micronucleus formation in a concentration dependent manner and highest (5.8 ± 0.46%) value was recorded in 56 ppm after 96 h of exposure, whereas, it was decreased after 192 h of exposure at all the three concentrations of NaAsO(2) examined which refers to the DNA repairing ability of fish to arsenic toxicity. The results of this study depict the genotoxic potentials of arsenic to fish which in turns provide insight on advanced study in aquatic toxicology. Copyright © 2011 Elsevier Ltd. All rights reserved.

  5. Evaluation of drinking water treatment combined filter backwash water recycling technology based on comet and micronucleus assay.

    PubMed

    Chen, Ting; Xu, Yongpeng; Liu, Zhiquan; Zhu, Shijun; Shi, Wenxin; Cui, Fuyi

    2016-04-01

    Based on the fact that recycling of combined filter backwash water (CFBW) directly to drinking water treatment plants (WTP) is considered to be a feasible method to enhance pollutant removal efficiency, we were motivated to evaluate the genotoxicity of water samples from two pilot-scale drinking water treatment systems, one with recycling of combined backwash water, the other one with a conventional process. An integrated approach of the comet and micronucleus (MN) assays was used with zebrafish (Danio rerio) to investigate the water genotoxicity in this study. The total organic carbon (TOC), dissolved organic carbon (DOC), and trihalomethane formation potential (THMFP), of the recycling process were lower than that of the conventional process. All the results showed that there was no statistically significant difference (P>0.05) between the conventional and recycling processes, and indicated that the genotoxicity of water samples from the recycling process did not accumulate in 15 day continuous recycling trial. It was worth noting that there was correlation between the concentrations of TOC, DOC, UV254, and THMFPs in water and the DNA damage score, with corresponding R(2) values of 0.68, 0.63, 0.28, and 0.64. Nevertheless, both DNA strand breaks and MN frequency of all water samples after disinfection were higher than that of water samples from the two treatment units, which meant that the disinfection by-products (DBPs) formed by disinfection could increase the DNA damage. Both the comet and MN tests suggest that the recycling process did not increase the genotoxicity risk, compared to the traditional process. Copyright © 2015. Published by Elsevier B.V.

  6. The Comet Assay and its applications in the field of ecotoxicology: a mature tool that continues to expand its perspectives.

    PubMed

    de Lapuente, Joaquín; Lourenço, Joana; Mendo, Sónia A; Borràs, Miquel; Martins, Marta G; Costa, Pedro M; Pacheco, Mário

    2015-01-01

    Since Singh and colleagues, in 1988, launched to the scientific community the alkaline Single Cell Gel Electrophoresis (SCGE) protocol, or Comet Assay, its uses and applications has been increasing. The thematic areas of its current employment in the evaluation of genetic toxicity are vast, either in vitro or in vivo, both in the laboratory and in the environment, terrestrial or aquatic. It has been applied to a wide range of experimental models: bacteria, fungi, cells culture, arthropods, fishes, amphibians, reptiles, mammals, and humans. This document is intended to be a comprehensive review of what has been published to date on the field of ecotoxicology, aiming at the following main aspects: (i) to show the most relevant experimental models used as bioindicators both in the laboratory and in the field. Fishes are clearly the most adopted group, reflecting their popularity as bioindicator models, as well as a primary concern over the aquatic environment health. Amphibians are among the most sensitive organisms to environmental changes, mainly due to an early aquatic-dependent development stage and a highly permeable skin. Moreover, in the terrestrial approach, earthworms, plants or mammalians are excellent organisms to be used as experimental models for genotoxic evaluation of pollutants, complex mix of pollutants and chemicals, in both laboratory and natural environment. (ii) To review the development and modifications of the protocols used and the cell types (or tissues) used. The most recent developments concern the adoption of the enzyme linked assay (digestion with lesion-specific repair endonucleases) and prediction of the ability to repair of oxidative DNA damage, which is becoming a widespread approach, albeit challenging. For practical/technical reasons, blood is the most common choice but tissues/cells like gills, sperm cells, early larval stages, coelomocytes, liver or kidney have been also used. (iii) To highlight correlations with other biomarkers

  7. The Comet Assay and its applications in the field of ecotoxicology: a mature tool that continues to expand its perspectives

    PubMed Central

    de Lapuente, Joaquín; Lourenço, Joana; Mendo, Sónia A.; Borràs, Miquel; Martins, Marta G.; Costa, Pedro M.; Pacheco, Mário

    2015-01-01

    Since Singh and colleagues, in 1988, launched to the scientific community the alkaline Single Cell Gel Electrophoresis (SCGE) protocol, or Comet Assay, its uses and applications has been increasing. The thematic areas of its current employment in the evaluation of genetic toxicity are vast, either in vitro or in vivo, both in the laboratory and in the environment, terrestrial or aquatic. It has been applied to a wide range of experimental models: bacteria, fungi, cells culture, arthropods, fishes, amphibians, reptiles, mammals, and humans. This document is intended to be a comprehensive review of what has been published to date on the field of ecotoxicology, aiming at the following main aspects: (i) to show the most relevant experimental models used as bioindicators both in the laboratory and in the field. Fishes are clearly the most adopted group, reflecting their popularity as bioindicator models, as well as a primary concern over the aquatic environment health. Amphibians are among the most sensitive organisms to environmental changes, mainly due to an early aquatic-dependent development stage and a highly permeable skin. Moreover, in the terrestrial approach, earthworms, plants or mammalians are excellent organisms to be used as experimental models for genotoxic evaluation of pollutants, complex mix of pollutants and chemicals, in both laboratory and natural environment. (ii) To review the development and modifications of the protocols used and the cell types (or tissues) used. The most recent developments concern the adoption of the enzyme linked assay (digestion with lesion-specific repair endonucleases) and prediction of the ability to repair of oxidative DNA damage, which is becoming a widespread approach, albeit challenging. For practical/technical reasons, blood is the most common choice but tissues/cells like gills, sperm cells, early larval stages, coelomocytes, liver or kidney have been also used. (iii) To highlight correlations with other biomarkers

  8. The genotoxic effects of benzo[a]pyrene and methamidophos on black porgy evaluated by comet assay

    NASA Astrophysics Data System (ADS)

    Liu, Rixian; Hong, Huasheng; Wang, Xinhong; Wang, Kejian; Wang, Chunguang

    2005-12-01

    In this study, two common pollutants (benzo[a]pyrene and methamidophos) in marine environment were tested by comet assay for their inducement of in vivo genotoxic effect to the blood cells of black porgy ( Acanthopagrus schlegeli). The fish was exposed to 2 μg/L of benzo[a]pyrene (BaP) and methamidophos, and their mixture. The assay was performed on whole blood at 2 h, 5 h, 24 h and 96 h exposure intervals. A significant increase in DNA damage was observed in each treatment with the pollutants. Additive effect of BaP and methamidophos was also found in the experiment. However, the decrease ratios of DNA damage for 5 h and 96 h exposure interals compared with 2 h and 24 h exposure ones, respectively, were noticed. This phenomenon may be explained by the function of repairing process via enzyme cytochrome P450 in the animal. Evidence of the genotoxicity of organophosphorus pesticides (OPs) and polynuclear aromatic hydrocarbons (PAHs) on marine fish are discussed in this paper.

  9. 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.

  10. Aerothermodynamic Analysis of Commercial Experiment Transporter (COMET) Reentry Capsule

    NASA Technical Reports Server (NTRS)

    Wood, William A.; Gnoffo, Peter A.; Rault, Didier F. G.

    1996-01-01

    An aerothermodynamic analysis of the Commercial Experiment Transporter (COMET) reentry capsule has been performed using the laminar thin-layer Navier-Stokes solver Langley Aerothermodynamic Upwind Relaxation Algorithm. Flowfield solutions were obtained at Mach numbers 1.5, 2, 5, 10, 15, 20, 25, and 27.5. Axisymmetric and 5, 10, and 20 degree angles of attack were considered across the Mach-number range, with the Mach 25 conditions taken to 90 degrees angle of attack and the Mach 27.5 cases taken to 60 degrees angle of attack. Detailed surface heat-transfer rates were computed at Mach 20 and 25, revealing that heating rates on the heat-shield shoulder ,can exceed the stagnation-point heating by 230 percent. Finite-rate chemistry solutions were performed above Mach 10, otherwise perfect gas computations were made. Drag, lift, and pitching moment coefficients are computed and details of a wake flow are presented. The effect of including the wake in the solution domain was investigated and base pressure corrections to forebody drag coefficients were numerically determined for the lower Mach numbers. Pitching moment comparisons are made with direct simulation Monte Carlo results in the more rarefied flow at the highest Mach numbers, showing agreement within two-percent. Thin-layer Navier-Stokes computations of the axial force are found to be 15 percent higher across the speed range than the empirical/Newtonian based results used during the initial trajectory analyses.

  11. Genotoxic effects of the alkaloids harman and harmine assessed by comet assay and chromosome aberration test in mammalian cells in vitro.

    PubMed

    Boeira, J M; da Silva, J; Erdtmann, B; Henriques, J A

    2001-12-01

    Harman and harmine are beta-carboline alkaloids which are present in plants widely used in medical practice, in beverages used for religious purposes in Brazil, as well as in tobacco smoke and over cooked food. In view of the controversial results observed in the literature about the mutagenic effects of these alkaloids, we studied their cytotoxic and genotoxic effects in V79 Chinese hamster lung fibroblasts in vitro using single-cell gel assay, Comet assay, either in the presence or in absence of an exogenous metabolic activation system (S9-mix), and by the chromosome aberration test without S9-mix. Harmine was more cytotoxic than harman. Both harman and harmine increased aberrant cell frequency and induced DNA damage by the Comet assay. These results suggest that harman and harmine are genotoxic in V79 cells, probably as a consequence of their ability to induce DNA strand breaks.

  12. Terrestrial analysis of the organic component of comet dust.

    PubMed

    Sandford, Scott A

    2008-01-01

    The nature of cometary organics is of great interest, both because these materials are thought to represent a reservoir of the original carbon-containing materials from which everything else in our solar system was made and because these materials may have played key roles in the origin of life on Earth. Because these organic materials are the products of a series of universal chemical processes expected to operate in the interstellar media and star-formation regions of all galaxies, the nature of cometary organics also provides information on the composition of organics in other planetary systems and, by extension, provides insights into the possible abundance of life elsewhere in the universe. Our current understanding of cometary organics represents a synthesis of information from telescopic and spacecraft observations of individual comets, the study of meteoritic materials, laboratory simulations, and, now, the study of samples collected directly from a comet, Comet P81/Wild 2.

  13. Genotoxicity of a thiosulfonate compound derived from Allium sp. intended to be used in active food packaging: In vivo comet assay and micronucleus test.

    PubMed

    Mellado-García, Pilar; Puerto, María; Prieto, Ana I; Pichardo, Silvia; Martín-Cameán, Ana; Moyano, Rosario; Blanco, Alfonso; Cameán, Ana M

    2016-04-01

    Components of Allium species have antimicrobial and antioxidant properties. A commercial Allium sp. extract (Proallium AP(®)), of which the main constituent is propyl thiosulphinate oxide (PTSO), is being used in the development of active food packaging. In previous in vitro genotoxicity studies, PTSO, in the presence of metabolic activation, increased the appearance of micronuclei (MN). We assessed the genotoxicity PTSO in rats following oral administration (doses: 5.5, 17.4, and 55mg/kg). The comet assay in liver and stomach (OECD 489) and the MN assay in bone marrow (OECD 474) were carried out. After necropsy, histopathological examinations of the liver and the stomach were performed. The results revealed no in vivo genotoxicity and the histopathological analysis showed only slight modifications, such as increased glycogen storage in the liver and a degenerative process in stomach, with vacuolization of cell membranes, only at the highest dose. Therefore, the present work confirms that this compound is not genotoxic and could be considered as a natural alternative to synthetic preservatives used in the food packaging industry.

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

    PubMed

    Mouchet, F; Gauthier, L; Mailhes, C; Ferrier, V; Devaux, A

    2005-02-01

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

  15. 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.

  16. DNA alkylation lesions and their repair in human cells: modification of the comet assay with 3-methyladenine DNA glycosylase (AlkD).

    PubMed

    Hašplová, Katarína; Hudecová, Alexandra; Magdolénová, Zuzana; Bjøras, Magnar; Gálová, Eliška; Miadoková, Eva; Dušinská, Mária

    2012-01-05

    3-methyladenine DNA glycosylase (AlkD) belongs to a new family of DNA glycosylases; it initiates repair of cytotoxic and promutagenic alkylated bases (its main substrates being 3-methyladenine and 7-methylguanine). The modification of the comet assay (single cell gel electrophoresis) using AlkD enzyme thus allows assessment of specific DNA alkylation lesions. The resulting baseless sugars are alkali-labile, and under the conditions of the alkaline comet assay they appear as DNA strand breaks. The alkylating agent methyl methanesulfonate (MMS) was used to induce alkylation lesions and to optimize conditions for the modified comet assay method with AlkD on human lymphoblastoid (TK6) cells. We also studied cellular and in vitro DNA repair of alkylated bases in DNA in TK6 cells after treatment with MMS. Results from cellular repair indicate that 50% of DNA alkylation is repaired in the first 60 min. The in vitro repair assay shows that while AlkD recognises most alkylation lesions after 60 min, a cell extract from TK6 cells recognises most of the MMS-induced DNA adducts already in the first 15 min of incubation, with maximum detection of lesions after 60 min' incubation. Additionally, we tested the in vitro repair capacity of human lymphocyte extracts from 5 individuals and found them to be able to incise DNA alkylations in the same range as AlkD. The modification of the comet assay with AlkD can be useful for in vitro and in vivo genotoxicity studies to detect alkylation damage and repair and also for human biomonitoring and molecular epidemiology studies.

  17. Reduction and analysis of photometric data on Comet Halley

    NASA Technical Reports Server (NTRS)

    Belton, Michael J. S.; Fink, U.; Wehinger, P.; Spinrad, H.; Meech, K.

    1988-01-01

    The discovery that periodic variations in the brightness of Comet Halley were characterized by two unrelated frequencies implies that the nucleus is in a complex state of rotation. It either nutates as a result of the random addition of small torque perturbations accumulated over many perihelion passages, or the jet activity torques are so strong that it precesses wildly at each perihelion passage. To diagnose the state of nuclear rotation, researchers began a program to acquire photometric time series of the comet as it recedes from the sun. The intention is to observe the decay of the comet's atmosphere and then, when it is unemcumbered by the light of the coma, follow the light variation of the nucleus itself. The latter will be compared with preperihelion time series and the orientation of the nucleus at the time of Vega and Giotto flybys and an accurate rotational ephemeris constructed. Halley was observed on 38 nights during 1987 and approximately 21 nights in 1988. The comet moved from 5 AU to 8.5 AU during this time. The brightness of the coma was found to rapidly decrease in 1988 as the coma and cometary activity collapses. The magnitude in April 1988 was 19 mag (visual) and it is predicted that the nucleus itself will be the major contributor to the brightness in the 1988 and 1989 season.

  18. In vivo gamma-rays induced initial DNA damage and the effect of famotidine in mouse leukocytes as assayed by the alkaline comet assay.

    PubMed

    Mozdarani, Hossein; Nasirian, Borzo; Haeri, S Abolghasem

    2007-03-01

    Ionizing radiation induces a variety of lesions in DNA, each of which can be used as a bio-indicator for biological dosimetry or the study of the radioprotective effects of substances. To assess gamma ray-induced DNA damage in vivo in mouse leukocytes at various doses and the effect of famotidine, blood was collected from Balb/c male mice after irradiation with 4 Gy gamma-rays at different time intervals post-irradiation. To assess the response, mice were irradiated with doses of gamma-rays at 1 to 4 Grays. Famotidine was injected intra-peritoneally (i.p) at a dose of 5 mg/kg at various time intervals before irradiation. Four slides were prepared from each sample and alkaline comet assay was performed using standard protocols. Results obtained show that radiation significantly increases DNA damage in leukocytes in a dose dependent manner (p < 0.01) when using appropriate sampling time after irradiation, because increasing sampling time after irradiation resulted in a time dependent disappearance of DNA damage. Treatment with only 5 mg/kg famotidine before 4 Gy irradiation led to almost 50% reduction in DNA damage when compared with those animals which received radiation alone. The radioprotective capability of famotidine might be attributed to radical scavenging properties and an anti-oxidation mechanism.

  19. Chlorination-induced genotoxicity in the mussel Perna viridis: assessment by single cell gel electrophoresis (comet) assay.

    PubMed

    Chavan, Pooja; Kumar, Rajesh; Kirubagaran, Ramalingam; Venugopalan, Vayalam P

    2016-08-01

    Mussels are important fouling organisms in the cooling water systems of coastal power plants. Continuous low-dose chlorination (CLDC) is being practiced as an effective method to control mussel biofouling in power plant cooling water systems. CLDC effectively controls mussel fouling by discouraging larval settlement rather than by killing the larvae or adults. Mussels are an integral part of the natural benthic community in the receiving water body where the coolant water is discharged. Hence, from a toxicological point of view, they can serve as both target and non-target organisms. Previous researchers have indicated that chlorine residual, rather than elevated temperature, can be the major stress factor in the effluents released from coastal power plants. However, very little data are available on the sub-lethal effects of low level chlorination on representative benthic fauna. In this study, we used native and transplanted mussels (Perna viridis) to study lethal and sub-lethal effects of chlorination in the cooling water circuit of an operating power plant. Experiments involving comet assay suggested that CLDC can cause DNA damage in treated mussels. However, activation of DNA repair appeared to get initiated after the accrued damage reached a threshold. The results indicate that, at chlorine residual levels observed at the discharge point, exposure to chlorinated effluents is unlikely to cause significant genetic damage to mussels in the recipient water body. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Genotoxicity of marine sediments in the fish hepatoma cell line PLHC-1 as assessed by the Comet assay.

    PubMed

    Šrut, Maja; Traven, Luka; Štambuk, Anamaria; Kralj, Sonja; Žaja, Roko; Mićović, Vladimir; Klobučar, Göran I V

    2011-02-01

    The main goal of this study was to test the usefulness of the Comet assay in the PLHC-1 hepatoma fish cell line as a tool for detecting the presence of genotoxic compounds in contaminated marine sediments. The system has been tested using both model chemicals (benzo[a]pyrene (B[a]P) and ethyl methanesulfonate (EMS)) and extracts of sediment samples obtained with solvent dichloromethane/methanol. For all of the analysed sediment extracts as well as for the model chemicals a concentration dependent genotoxic effect was observed. The sediment with the highest observed genotoxic potential was additionally extracted using various solvents in order to test which class of compounds, according to their polarity, is most responsible for the observed genotoxic effect. Non-polar solvents (cyclohexane and dichloromethane) yielded stronger genotoxic effect but the highest level of DNA damage was determined after exposure to sediment extract obtained with the solvent mixture dichloromethane/methanol which extracts a wide range of contaminants. Our results indicate that the PLHC-1 cell line is a suitable in vitro model in sediment genotoxicity assessment and encourage the use of fish cell lines as versatile tools in ecogenotoxicology.

  1. Use of the comet assay to measure DNA damage in cells exposed to photosensitizers and gamma radiation

    NASA Astrophysics Data System (ADS)

    Pouget, J.-P.; Ravanat, J.-L.; Douki, T.; Richard, M.-J.; Cadet, J.

    1999-01-01

    We used the comet assay associated with DNA-glycosylases to estimate DNA damage in cells exposed to gamma irradiation or photosensitized either with methylene blue or orange acridine. A calibration performed using irradiation allowed the measurement of the steady-state level and the yield of 8-oxodGuo as well as strand breaks and alkali-labile sites. Nous avons utilisé la méthode des comètes associée à des ADN-glycosylases, pour estimer les dommages de l'ADN dans des cellules après l'exposition à un rayonnement gamma ou après photosensibilisation par le bleu de méthylène ou l'acridine orange. Une calibration de la méthode des comètes a permis de mesurer le niveau basal et les taux de formation de 8-oxodGuo ainsi que le nombre de cassures de brins et de sites alcali labiles.

  2. DNA damage in barn swallows (Hirundo rustica) from the Chernobyl region detected by use of the comet assay.

    PubMed

    Bonisoli-Alquati, Andrea; Voris, Andrew; Mousseau, Timothy A; Møller, Anders Pape; Saino, Nicola; Wyatt, Michael D

    2010-04-01

    We investigated levels of DNA damage in blood cells of barn swallows (Hirundo rustica) inhabiting the Chernobyl region to evaluate whether chronic exposure to low-level radioactive contamination continues to induce genetic damage in free-living populations of animals. Blood samples were obtained from barn swallows collected at sites with different background levels of radiation, including a relatively uncontaminated area. The extent of DNA damage was evaluated using the alkaline (pH=12.1) comet assay, a robust and sensitive electrophoresis-based technique widely employed in research ranging from biomonitoring to clinical studies. We found that levels of DNA damage, as indexed by the extent of DNA migration, were increased in barn swallows living in areas surrounding Chernobyl when compared to swallows sampled at low-level sites. The results we obtained are consistent with previous findings on this same species, which showed that swallows breeding in areas heavily contaminated with radionuclides have increased mutation rates, higher oxidative stress and incidence of morphological aberrations and tumors. Overall, these results indicate that chronic exposure to radioactive contaminants, even 20years after the accident at the Chernobyl nuclear power plant, continues to induce DNA damage in cells of free-living animals.

  3. Assessing the genotoxicities of sparteine and compounds isolated from Lupinus mexicanus and L. montanus seeds by using comet assay.

    PubMed

    Silva, M R; Alvarez, C M; García, P M; Ruiz, M A

    2014-12-12

    The genus Lupinus is widely distributed. Its seeds are used for animal and human food, and Lupinus possesses pharmacological potential because of its high content of quinolizidine alkaloids and flavonoids; however, there is little available information about its genotoxicity. We used the comet assay and staminal nuclei of Tradescantia (clone 4430) to evaluate the in vitro genotoxicity of 4 concentrations (0.01, 0.1, 0.5, and 1.0 mM) of alkaloid extracts of Lupinus mexicanus and Lupinus montanus, flavonoids of L. mexicanus, and commercial sparteine; nitrosodiethylamine was used as a positive control and untreated nuclei were used as a negative control. All concentrations of L. mexicanus and L. montanus showed significant genotoxic activity (P ≤ 0.05). A similar behavior was observed for flavonoid extracts of L. montanus except the 1.0 mM concentration. Sparteine showed genotoxic activity only at 0.5 mM. The order of genotoxicity of the compounds studied was as follows: L. mexicanus > L. montanus > flavonoids of L. montanus > sparteine. There is evident genotoxic activity in the compounds that were studied, particularly at lower concentrations (0.01 and 0.1 mM). Given the limited information about the genotoxicity of the compounds of L. mexicanus and L. montanus, further studies are necessary.

  4. Comet assay measures of DNA damage as biomarkers of irinotecan response in colorectal cancer in vitro and in vivo.

    PubMed

    Wood, Joanna P; Smith, Andrew J O; Bowman, Karen J; Thomas, Anne L; Jones, George D D

    2015-09-01

    The use of irinotecan to treat metastatic colorectal cancer (CRC) is limited by unpredictable response and variable toxicity; however, no reliable clinical biomarkers are available. Here, we report a study to ascertain whether irinotecan-induced DNA damage measures are suitable/superior biomarkers of irinotecan effect. CRC-cell lines (HCT-116 and HT-29) were treated in vitro with irinotecan and peripheral blood lymphocytes (PBL) were isolated from patients before and after receiving irinotecan-based chemotherapy. Levels of in vitro-, in vivo-, and ex vivo-induced DNA damage were measured using the Comet assay; correlations between damage levels with in vitro cell survival and follow-up clinical data were investigated. Irinotecan-induced DNA damage was detectable in both CRC cell-lines in vitro, with higher levels of immediate and residual damage noted for the more sensitive HT-29 cells. DNA damage was not detected in vivo, but was measurable in PBLs upon mitogenic stimulation prior to ex vivo SN-38 treatment. Results showed that, following corrections for experimental error, those patients whose PBLs demonstrated higher levels of DNA damage following 10 h of SN-38 exposure ex vivo had significantly longer times to progression than those with lower damage levels (median 291 vs. 173 days, P = 0.014). To conclude, higher levels of irinotecan-induced initial and residual damage correlated with greater cell kill in vitro and a better clinical response. Consequently, DNA damage measures may represent superior biomarkers of irinotecan effect compared to the more often-studied genetic assays for differential drug metabolism. © 2015 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

  5. Use of the Comet-FISH Assay to Compare DNA Damage and Repair in p53 and hTERT Genes following Ionizing Radiation

    PubMed Central

    McKenna, Declan J.; Doherty, Bernadette A.; Downes, C. Stephen; McKeown, Stephanie R.; McKelvey-Martin, Valerie J.

    2012-01-01

    The alkaline single cell gel electrophoresis (comet) assay can be combined with fluorescent in situ hybridisation (FISH) methodology in order to investigate the localisation of specific gene domains within an individual cell. The number and position of the fluorescent signal(s) provides information about the relative damage and subsequent repair that is occurring in the targeted gene domain(s). In this study, we have optimised the comet-FISH assay to detect and compare DNA damage and repair in the p53 and hTERT gene regions of bladder cancer cell-lines RT4 and RT112, normal fibroblasts and Cockayne Syndrome (CS) fibroblasts following γ-radiation. Cells were exposed to 5Gy γ-radiation and repair followed for up to 60 minutes. At each repair time-point, the number and location of p53 and hTERT hybridisation spots was recorded in addition to standard comet measurements. In bladder cancer cell-lines and normal fibroblasts, the p53 gene region was found to be rapidly repaired relative to the hTERT gene region and the overall genome, a phenomenon that appeared to be independent of hTERT transcriptional activity. However, in the CS fibroblasts, which are defective in transcription coupled repair (TCR), this rapid repair of the p53 gene region was not observed when compared to both the hTERT gene region and the overall genome, proving the assay can detect variations in DNA repair in the same gene. In conclusion, we propose that the comet-FISH assay is a sensitive and rapid method for detecting differences in DNA damage and repair between different gene regions in individual cells in response to radiation. We suggest this increases its potential for measuring radiosensitivity in cells and may therefore have value in a clinical setting. PMID:23145163

  6. 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.

  7. Genotoxicity of the herbicide formulation Roundup (glyphosate) in broad-snouted caiman (Caiman latirostris) evidenced by the Comet assay and the Micronucleus test.

    PubMed

    Poletta, G L; Larriera, A; Kleinsorge, E; Mudry, M D

    2009-01-31

    The genotoxicity of pesticides is an issue of worldwide concern. The present study was undertaken to evaluate the genotoxic potential of a widely used herbicide formulation, Roundup (glyphosate), in erythrocytes of broad-snouted caiman (Caiman latirostris) after in ovo exposure. Caiman embryos were exposed at early embryonic stage to different sub-lethal concentrations of Roundup (50, 100, 200, 300, 400, 500, 750, 1000, 1250 and 1750microg/egg). At time of hatching, blood samples were obtained from each animal and two short-term tests, the Comet assay and the Micronucleus (MN) test, were performed on erythrocytes to assess DNA damage. A significant increase in DNA damage was observed at a concentration of 500microg/egg or higher, compared to untreated control animals (p<0.05). Results from both the Comet assay and the MN test revealed a concentration-dependent effect. This study demonstrated adverse effects of Roundup on DNA of C. latirostris and confirmed that the Comet assay and the MN test applied on caiman erythrocytes are useful tools in determining potential genotoxicity of pesticides. The identification of sentinel species as well as sensitive biomarkers among the natural biota is imperative to thoroughly evaluate genetic damage, which has significant consequences for short- and long-term survival of the natural species.

  8. The comets of 1999

    NASA Astrophysics Data System (ADS)

    Shanklin, J.

    2009-12-01

    This report is the tenth in the annual series which gives for each comet: the discovery details, orbital data and general information, magnitude parameters and BAA Comet Section observations. Further details of the analysis techniques used in this report are given in an earlier paper. Ephemerides for the comets predicted to return during the year can be found in the BAA or ICQ Handbooks.

  9. The comets of 2000

    NASA Astrophysics Data System (ADS)

    Shanklin, J. D.

    2010-08-01

    This report is the eleventh in the annual series which gives for each comet: the discovery details, orbital data and general information, magnitude parameters and BAA Comet Section observations. Further details of the analysis techniques used in this report are given in an earlier paper. Ephemerides for the comets predicted to return during the year can be found in the BAA or ICQ Handbooks.

  10. Mechanical and SEM analysis of artificial comet nucleus samples

    NASA Technical Reports Server (NTRS)

    Thiel, K.; Kochan, H.; Roessler, K.; Gruen, E.; Schwehm, G.; Hellmann, H.; Hsiung, P.; Koelzer, G.

    1989-01-01

    Since 1987 experiments dealing with comet nucleus phenomena have been carried out in the DFVLR space simulation chambers. The main objective of these experiments is a better understanding of thermal behavior, surface phenomena and especially the gas dust interaction. As a function of different sample compositions and exposure to solar irradiation (xenon-bulbs) crusts of different hardness and thickness were measured. The measuring device consists of a motor driven pressure foot (5 mm diameter), which is pressed into the sample. The applied compressive force is electronically monitored. The microstructure of the crust and dust residuals is investigated by scanning electron microscopy (SEM) techniques. Stress-depth profiles of an unirradiated and an irradiated model comet are given.

  11. 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.

  12. Integration of Pig-a, micronucleus, chromosome aberration and comet assay endpoints in a 28-day rodent toxicity study with urethane

    PubMed Central

    Stankowski, Leon F.; Aardema, Marilyn J.; Lawlor, Timothy E.; Pant, Kamala; Roy, Shambhu; Xu, Yong; Elbekai, Reem

    2015-01-01

    As part of the international Pig-a validation trials, we examined the induction of Pig-a mutant reticulocytes and red blood cells (RETCD59− and RBCCD59−, respectively) in peripheral blood of male Sprague Dawley® rats treated with urethane (25, 100 and 250mg/kg/day) or saline by oral gavage for 29 days. Additional endpoints integrated into this study were: micronucleated reticulocytes (MN-RET) in peripheral blood; chromosome aberrations (CAb) and DNA damage (%tail intensity via the comet assay) in peripheral blood lymphocytes (PBL); micronucleated polychromatic erythrocytes (MN-PCE) in bone marrow; and DNA damage (comet) in various organs at termination (the 29th dose was added for the comet endpoint at sacrifice). Ethyl methanesulfonate (EMS; 200mg/kg/day on Days 3, 4, 13, 14, 15, 27, 28 and 29) was evaluated as the concurrent positive control (PC). All animals survived to termination and none exhibited overt toxicity, but there were significant differences in body weight and body weight gain in the 250-mg/kg/day urethane group, as compared with the saline control animals. Statistically significant, dose-dependent increases were observed for urethane for: RETCD59− and RBCCD59− (on Days 15 and 29); MN-RET (on Days 4, 15 and 29); and MN-PCE (on Day 29). The comet assay yielded positive results in PBL (Day 15) and liver (Day 29), but negative results for PBL (Days 4 and 29) and brain, kidney and lung (Day 29). No significant increases in PBL CAb were observed at any sample time. Except for PBL CAb (likely due to excessive cytotoxicity), EMS-induced significant increases in all endpoints/tissues. These results compare favorably with earlier in vivo observations and demonstrate the utility and sensitivity of the Pig-a in vivo gene mutation assay, and its ability to be easily integrated, along with other standard genotoxicity endpoints, into 28-day rodent toxicity studies. PMID:25934985

  13. Protective effect of dry olive leaf extract in adrenaline induced DNA damage evaluated using in vitro comet assay with human peripheral leukocytes.

    PubMed

    Cabarkapa, Andrea; Zivković, Lada; Zukovec, Dijana; Djelić, Ninoslav; Bajić, Vladan; Dekanski, Dragana; Spremo-Potparević, Biljana

    2014-04-01

    Excessive release of stress hormone adrenaline is accompanied by generation of reactive oxygen species which may cause disruption of DNA integrity leading to cancer and age-related disorders. Phenolic-rich plant product dry olive leaf extract (DOLE) is known to modulate effects of various oxidants in human cells. The aim was to evaluate the effect of commercial DOLE against adrenaline induced DNA damage in human leukocytes by using comet assay. Peripheral blood leukocytes from 6 healthy subjects were treated in vitro with three final concentrations of DOLE (0.125, 0.5, and 1mg/mL) for 30 min at 37°C under two different protocols, pretreatment and post-treatment. Protective effect of DOLE was assessed from its ability to attenuate formation of DNA lesions induced by adrenaline. Compared to cells exposed only to adrenaline, DOLE displayed significant reduction (P<0.001) of DNA damage at all three concentrations and under both experimental protocols. Pearson correlation analysis revealed a significant positive association between DOLE concentration and leukocytes DNA damage (P<0.05). Antigenotoxic effect of the extract was more pronounced at smaller concentrations. Post-treatment with 0.125 mg/mL DOLE was the most effective against adrenaline genotoxicity. Results indicate genoprotective and antioxidant properties in dry olive leaf extract, strongly supporting further explorations of its underlying mechanisms of action.

  14. Detection of DNA strand breaks by comet assay in sputum leucocytes of bitumen-exposed workers: a pilot study.

    PubMed

    Marczynski, B; Raulf-Heimsoth, M; Pesch, B; Kendzia, B; Käfferlein, H U; Vosshans, B; Borowitzki, G; Lee, E-H; Bramer, R; Brüning, T

    2010-09-01

    DNA strand breaks were determined in leucocytes of induced sputum (IS) and compared with DNA strand breaks in blood lymphocytes from 42 bitumen-exposed workers pre and post shift. Comet assay results were expressed in arbitrary units based on visual scoring (sputum leucocytes) and Olive tail moment (OTM, blood lymphocytes). DNA damage in IS leucocytes was overall high but did not change during shift. Level of DNA strand breaks in IS samples correlated with total cell count and neutrophil content (Spearman rank correlation coefficient r(s) = 0.47, p = 0.001, r(s)= 0.48, p = 0.001, respectively) and with IL-8 concentration before and after shift (r(s) = 0.31, P = 0.048, and r(s) = 0.43, P = 0.005). DNA damage in IS was not associated with DNA strand breaks in blood lymphocytes (r(s) = -0.04, p = 0.802 before shift, r(s) = 0.27, p = 0.088 after shift). A higher level of DNA strand breaks was measured in blood lymphocytes before shift (median OTM 1.7 before and 1.3 after shift, p = 0.023). A strong correlation was found between the number of neutrophils and IL-8 concentration in IS before and after shift (r(s) = 0.77 and r(s)= 0.75, p < 0.001). This study showed an association between genotoxic and inflammatory effects in the lower airways and compared simultaneously DNA strand breaks in IS and blood of bitumen-exposed workers.

  15. Development of a miniature scanning electron microscope for in-flight analysis of comet dust

    NASA Technical Reports Server (NTRS)

    Conley, J. M.; Bradley, J. G.; Giffin, C. E.; Albee, A. L.; Tomassian, A. D.

    1983-01-01

    A description is presented of an instrument which was developed with the original goal of being flown on the International Comet Mission, scheduled for a 1985 launch. The Scanning Electron Microscope and Particle Analyzer (SEMPA) electron miniprobe is a miniaturized electrostatically focused electron microscope and energy dispersive X-ray analyzer for in-flight analysis of comet dust particles. It was designed to be flown on board a comet rendezvous spacecraft. Other potential applications are related to asteroid rendezvous and planetary lander missions. According to the development objectives, SEMPA miniprobe is to have the capability for imaging and elemental analysis of particles in the size range of 0.25 microns and larger.

  16. Meteoroid streams and comet disintegration

    NASA Astrophysics Data System (ADS)

    Guliyev, A.

    2016-01-01

    The results of the statistical analysis of the dynamic parameters of 114 comets that have undergone nuclear splitting are presented in the article. The list of the objects contains: comets that have split in the period of the observation; data of twin-comets; lost comets with designation D; comets with large-scale structure in the coma. We will describe these comets as "splitted". Some aspects of the following hypothesis are studied: disintegration of comet nuclei happens as the result of their collision with meteoroid streams. For the verification of this hypothesis, the position of splitted comet orbits relatively to 125 meteor streams from Kronk's list is analyzed. It was found that the total number of comet orbit nodes located close to the meteor stream planes (for the distances up to 0.1 AU) is N = 1041. It is shown that if these comets are replaced by randomly selected different comets, N will be reduced by a factor of approximately three.

  17. 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.

  18. Shortening of alkaline DNA unwinding time does not interfere with detecting DNA damage to mouse and human spermatozoa in the comet assay.

    PubMed

    Kusakabe, Hirokazu; Tateno, Hiroyuki

    2011-01-01

    The comet assay was performed on mouse and human spermatozoa to examine the effect of alkaline DNA unwinding time. The spermatozoa were treated in vitro with the DNA-damaging agents, methyl methanesulfonate (MMS) or hydrogen peroxide (H₂O₂), and then embedded in agarose gel on glass slides. The slides were immersed in alkaline solution (> pH 13) for 1, 5, 10 and 20 min, and then subjected to the electrophoresis under neutral conditions. In mouse spermatozoa, comet tails seen in solvent controls became brighter and longer as the alkaline DNA unwinding time increased. However, in the MMS-treated mouse spermatozoa, a smaller difference in the damage from that in the solvent control was seen with time within a dose. DNA damage induced by H₂O₂ could also be detected accurately after alkali treatment for 1-20 min. In human spermatozoa, DNA damage induced by MMS and H₂O₂ could be detected in a dose-dependent manner after alkali treatment for 1 min. The ability of the comet assay to detect DNA damage was not adversely affected by the short period (1 min) of the alkaline DNA unwinding time.

  19. 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

  20. Identification of irradiated wheat by germination test, DNA comet assay and electron spin resonance

    NASA Astrophysics Data System (ADS)

    Barros, Adilson C.; Freund, Maria Teresa L.; Villavicencio, Ana Lúcia C. H.; Delincée, Henry; Arthur, Valter

    2002-03-01

    In several countries, there has been an increase in the use of radiation for food processing thus improving the quality and sanitary conditions, inhibiting pathogenic microorganisms, delaying the natural aging process and so extending product lifetime. The need to develop analytical methods to detect these irradiated products is also increasing. The goal of this research was to identify wheat irradiated using different radiation doses. Seeds were irradiated with a gamma 60Co source (Gammacell 220 GC) in the Centro de Energia Nuclear na Agricultura and the Instituto de Pesquisas Energéticas e Nucleares. Dose rate used were 1.6 and 5.8kGy/h. Applied doses were 0.0, 0.10, 0.25, 0.50, 0.75, 1.0, and 2.0kGy. After irradiation, seeds were analysed over a 6 month period. Three different detection methods were employed to determine how irradiation had modified the samples. Screening methods consisted of a germination test measuring the inhibition of shooting and rooting and analysis of DNA fragmentation. The method of electron spin resonance spectroscopy allowed a better dosimetric evaluation. These techniques make the identification of irradiated wheat with different doses possible.

  1. The interactive astronomical data analysis facility - image enhancement techniques to Comet Halley

    NASA Technical Reports Server (NTRS)

    Kinglesmith, D. A., III

    1981-01-01

    PDP 11/40 computer is at the heart of a general purpose interactive data analysis facility designed to permit easy access to data in both visual imagery and graphic representations. The major components consist of: the 11/40 CPU and 256 K bytes of 16-bit memory; two TU10 tape drives; 20 million bytes of disk storage; three user terminals; and the COMTAL image processing display system. The application of image enhancement techniques to two sequences of photographs of Comet Halley taken in Egypt in 1910 provides evidence for eruptions from the comet's nucleus.

  2. Navigation accuracy analysis for the Halley flyby phase of a dual comet mission using ion drive

    NASA Technical Reports Server (NTRS)

    Wood, L. J.; Hast, S. L.

    1980-01-01

    A dual comet (Halley Flyby/Tempel 2 Rendezvous) mission, making use of the solar electric propulsion system, is under consideration for a 1985 launch. This paper presents navigation accuracy analysis results for the Halley flyby phase of this mission. Orbit determination and guidance accuracies are presented for the baseline navigation strategy, along with the results of a number of sensitivity studies involving parameters such as data frequencies, data accuracies, ion drive thrust vector errors, comet ephemeris uncertainties, time lags associated with data processing and command sequence generation, probe release time, and navigation coast arc duration.

  3. The interactive astronomical data analysis facility - image enhancement techniques to Comet Halley

    NASA Astrophysics Data System (ADS)

    Klinglesmith, D. A.

    1981-10-01

    PDP 11/40 computer is at the heart of a general purpose interactive data analysis facility designed to permit easy access to data in both visual imagery and graphic representations. The major components consist of: the 11/40 CPU and 256 K bytes of 16-bit memory; two TU10 tape drives; 20 million bytes of disk storage; three user terminals; and the COMTAL image processing display system. The application of image enhancement techniques to two sequences of photographs of Comet Halley taken in Egypt in 1910 provides evidence for eruptions from the comet's nucleus.

  4. Assessment by Ames test and comet assay of toxicity potential of polymer used to develop field-capable rapid-detection device to analyze environmental samples

    NASA Astrophysics Data System (ADS)

    Hebert, Amanda; Bishop, Michelle; Bhattacharyya, Dhiman; Gleason, Karen; Torosian, Stephen

    2015-08-01

    There is need for devices that decrease detection time of food-borne pathogens from days to real-time. In this study, a rapid-detection device is being developed and assessed for potential cytotoxicity. The device is comprised of melt-spun polypropylene coupons coated via oxidative chemical vapor deposition (oCVD) with 3,4-Ethylenedioxythiophene (EDOT), for conductivity and 3-Thiopheneethanol (3TE), allowing antibody attachment. The Ames test and comet assay have been used in this study to examine the toxicity potentials of EDOT, 3TE, and polymerized EDOT-co-3TE. For this study, Salmonella typhimurium strain TA1535 was used to assess the mutagenic potential of EDOT, 3TE and the copolymer. The average mutagenic potential of EDOT, 3TE and copolymer was calculated to be 0.86, 0.56, and 0.92, respectively. For mutagenic potential, on a scale from 0 to 1, close to 1 indicates low potential for toxicity, whereas a value of 0 indicates a high potential for toxicity. The comet assay is a single-cell gel electrophoresis technique that is widely used for this purpose. This assay measures toxicity based on the area or intensity of the comet-like shape that DNA fragments produce when DNA damage has occurred. Three cell lines were assessed; FRhK-4, BHK-21, and Vero cells. After averaging the results of all three strains, the tail intensity of the copolymer was 8.8 % and tail moment was 3.0, and is most similar to the untreated control, with average tail intensity of 5.7 % and tail moment of 1.7. The assays conducted in this study provide evidence that the copolymer is non-toxic to humans.

  5. Comet deflection by directed energy: a finite element analysis

    NASA Astrophysics Data System (ADS)

    Madajian, Jonathan; Griswold, Janelle; Gandra, Anush; Hughes, Gary B.; Zhang, Qicheng; Rupert, Nic; Lubin, Philip

    2016-09-01

    Comets and Asteroids are viable threats to our planet; if these space rocks are smaller than 25 meters, they burn up in the atmosphere, but if they are wider than 25 meters they can cause damage to the impact area. Anything more than one to two kilometers can have worldwide effects, furthermore a mile-wide asteroid travelling at 30,000 miles per hour has the energy equal to a megaton bomb and is very likely to wipe out most of the life on Earth. Residents near Chelyabinsk, Russia experienced the detrimental effects of a collision with a Near-Earth Asteroid (NEA) on 15 February 2013 as a 20 m object penetrated the atmosphere above that city. The effective yield from this object was approximately 1/2 Megaton TNT equivalent (Mt), or that of a large strategic warhead. The 1908 Tunguska event, also over Russia, is estimated to have had a yield of approximately 15 Mt and had the potential to kill millions of people had it come down over a large city1. In the face of such danger a planetary defense system is necessary and this paper proposes a design for such a system. DE-STAR (Directed Energy System for Targeting of Asteroids and exploRation) is a phased array laser system that can be used to oblate, deflect and de-spin asteroids and comets.

  6. Comets. [IUE

    NASA Technical Reports Server (NTRS)

    Ahearn, Michael F.

    1988-01-01

    The IUE was used to study comets including the first dynamically new comet to approach closer than 3 AU. Differences between old and new comets are studied. Results relevant to the nature of cometary nuclei are discussed. Identification of species in the spectra; relative abundances; variability of comets; and comet mass are considered.

  7. Trajectory analysis and performance for SEP Comet Encke missions

    NASA Technical Reports Server (NTRS)

    Sauer, C. G., Jr.

    1973-01-01

    A summary of the performance of Solar Electric Propulsion spacecraft for Comet Encke missions for the 1980, 1984 and 1987 mission opportunities is presented together with a description of the spacecraft trajectory for each opportunity. Included is data for rendezvous trajectories for all three opportunities and data for a slow flyby mission during the 1980 opportunity. A range of propulsion system input powers of 10 to 20 kW are considered together with a constant spacecraft power requirement of 400 watts. The performance presented in this paper is indicative of that using 30 cm Mercury electron bombardment thrusters that are currently being developed. Performance is given in terms of final spacecraft mass and is thus independent of any particular spacecraft design concept.

  8. Assessment of DNA damage of Lewis lung carcinoma cells irradiated by carbon ions and X-rays using alkaline comet assay

    NASA Astrophysics Data System (ADS)

    Li, Ping; Zhou, Li-Bin; Jin, Xiao-Dong; He, Jing; Dai, Zhong-Ying; Zhou, Guang-Ming; Gao, Qing-Xiang; Li, Sha; Li, Qiang

    2008-01-01

    DNA damage and cell reproductive death determined by alkaline comet and clonogenic survival assays were examined in Lewis lung carcinoma cells after exposure to 89.63 MeV/u carbon ion and 6 MV X-ray irradiations, respectively. Based on the survival data, Lewis lung carcinoma cells were verified to be more radiosensitive to the carbon ion beam than to the X-ray irradiation. The relative biological effectiveness (RBE) value, which was up to 1.77 at 10% survival level, showed that the DNA damage induced by the high-LET carbon ion beam was more remarkable than that induced by the low-LET X-ray irradiation. The dose response curves of “Tail DNA (%)” (TD) and “Olive tail moment” (OTM) for the carbon ion irradiation showed saturation beyond about 8 Gy. This behavior was not found in the X-ray curves. Additionally, the carbon ion beam produced a lower survival fraction at 2 Gy (SF2) value and a higher initial Olive tail moment 2 Gy (OTM2) than those for the X-ray irradiation. These results suggest that carbon ion beams having high-LET values produced more severe cell reproductive death and DNA damage in Lewis lung carcinoma cells in comparison with X-rays and comet assay might be an effective predictive test even combining with clonogenic assay to assess cellular radiosensitivity.

  9. Protective effects of aqueous and ethanolic extracts of Portulaca oleracea L. aerial parts on H2O2-induced DNA damage in lymphocytes by comet assay.

    PubMed

    Behravan, Javad; Mosafa, Fatemeh; Soudmand, Negar; Taghiabadi, Elahe; Razavi, Bibi Marjan; Karimi, Gholamreza

    2011-09-01

    The comet assay is a standard method for measuring DNA damage. In this study, the protective effects of ethanolic and aqueous extracts of Portulaca oleracea L. (P. oleracea) on human lymphocyte DNA lesions were evaluated with the comet assay. Lymphocytes were isolated from blood samples taken from healthy volunteers. Human lymphocytes were incubated in H(2)O(2) (50,100, and 200 μM), aqueous extract (0.05, 0.1, 0.5, 1, and 2.5mg/ml), and ethanolic extracts (0.05, 0.1, 0.5, 1, and 2.5mg/ml) of P. oleraceae aerial parts alone with a combination of H(2)O(2) (100 μM) with either 1 or 2.5mg/ml of both extracts at 4°C for 30 minutes. The extent of DNA migration was measured using the alkaline single cell gel electrophoresis approach assay, and DNA damage was expressed as percentage tail DNA. We found that the aqueous extract of P. oleracea significantly inhibited DNA damage, while there was no effect of the ethanolic extract. These data suggest that the aqueous extract of P. oleracea can prevent oxidative DNA damage to human lymphocytes, which is likely due to antioxidant constituents in the extract. Copyright © 2011. Published by Elsevier B.V.

  10. Comet assay study of DNA damage and repair of tumour cells following boron neutron capture irradiation with fast d(14) + Be neutrons.

    PubMed

    Pöller, F; Bauch, T; Sauerwein, W; Böcker, W; Wittig, A; Streffer, C

    1996-11-01

    We compared the amount of radiation-induced DNA damage and the extent of DNA repair in human melanoma cells (MeWo) using the 'comet assay' after neutron, boron neutron capture and X-irradiation. Using a colony-forming assay it was shown earlier that lethal effects in tumour cells treated with fast neutrons may be increased by the neutron capture reaction 10B(n, alpha)7Li. The effectiveness of boron neutron capture in killing tumour cells depends on the number of 10B atoms delivered to the tumour, the subcellular distribution of 10B and the thermal neutron fluence at the side of the tumour. Using the 'comet assay' the DNA damage of fast neutrons (mean energy 5.8 MeV) was shown to be significantly greater than for the same absorbed dose of X-rays. The presence of 600 ppm 10B (boric acid H5 10BO3) in the cell medium during irradiation with d(14) + Be neutrons in a phantom enhances the DNA damage by 20% compared with neutron irradiation alone. After DNA damage induction by neutrons and neutron capture of boron, the DNA repair capacity of the MeWo cells is significantly reduced in comparison with X-irradiation resulting in proportionally more residual DNA damage after 180 min of repair time.

  11. An investigation of the potential effect of sperm nuclear vacuoles in human spermatozoa on DNA fragmentation using a neutral and alkaline Comet assay.

    PubMed

    Pastuszek, E; Kiewisz, J; Skowronska, P; Liss, J; Lukaszuk, M; Bruszczynska, A; Jakiel, G; Lukaszuk, K

    2017-03-01

    Presence of vacuoles and degree of sperm DNA damage are considered to be the basic factors used for the assessment of sperm fertilization capacity. We aimed to investigate the link between these two parameters. According to our knowledge, this is the first study where the Comet assay was used to assess the degree of DNA fragmentation of sperm categorized by Motile Sperm Organelle Morphology Examination (MSOME) Grades. Semen samples from 10 patients were assessed. Spermatozoa were graded into four MSOME groups according to the Vanderzwalmen's criteria. A total of 3930 motile spermatozoa were selected one-by-one using an inverted microscope and transferred onto two different slides. The degree of DNA fragmentation was analyzed by alkaline and neutral Comet assay. Results of the neutral Comet assay showed that Grade I spermatozoa (absence of vacuoles) presented significantly lower dsDNA fragmentation level (mean: 3.13 ± 1.17%) than Grade II (maximum of two small vacuoles; mean: 10.34 ± 2.65%), Grade III (more than two small vacuoles or at least one large vacuole; mean: 23.88 ± 8.37%), and Grade IV (large vacuoles associated with abnormal head shapes or other abnormalities; mean: 36.94 ± 7.78%; p < 0.05). Results of the alkaline Comet assay showed that Grade I spermatozoa had significantly lower DNA (ssDNA + dsDNA) fragmentation level (mean: 8.33 ± 3.62%) than Grade III (mean: 25.64 ± 9.15%) and Grade IV (mean: 40.10 ± 9.10%, p < 0.05), but not significantly lower than Grade II (mean: 12.73 ± 5.06%; p > 0.05). Probably, the vacuoles may be responsible for double strand DNA breaks rather than single strand DNA breaks (only 2.39% spermatozoa in MSOME Grade II, 1.76% in III, and 3.16% in IV has single strand breaks). The results demonstrate that lower MSOME grading correlates with lower sperm DNA fragmentation. Therefore, the observation of sperm nuclear vacuoles using real-time optical microscopy without precise DNA fragmentation

  12. 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.

  13. Characterization of the Fine Component of Comet Wild 2: Analysis of 11 Stardust Craters from Foil C2010W

    NASA Astrophysics Data System (ADS)

    Haas, B. A.; Croat, T. K.; Floss, C.

    2016-08-01

    NASA's Stardust mission returned cometary material from comet Wild 2 in Al foil collectors. We report on SEM-EDX and Auger elemental analysis as well as FIB-TEM analysis performed on 11 craters from foil C2010W.

  14. Comet Odyssey: Comet Surface Sample Return

    NASA Astrophysics Data System (ADS)

    Weissman, Paul R.; Bradley, J.; Smythe, W. D.; Brophy, J. R.; Lisano, M. E.; Syvertson, M. L.; Cangahuala, L. A.; Liu, J.; Carlisle, G. L.

    2010-10-01

    Comet Odyssey is a proposed New Frontiers mission that would return the first samples from the surface of a cometary nucleus. Stardust demonstrated the tremendous power of analysis of returned samples in terrestrial laboratories versus what can be accomplished in situ with robotic missions. But Stardust collected only 1 milligram of coma dust, and the 6.1 km/s flyby speed heated samples up to 2000 K. Comet Odyssey would collect two independent 800 cc samples directly from the surface in a far more benign manner, preserving the primitive composition. Given a minimum surface density of 0.2 g/cm3, this would return two 160 g surface samples to Earth. Comet Odyssey employs solar-electric propulsion to rendezvous with the target comet. After 180 days of reconnaissance and site selection, the spacecraft performs a "touch-and-go” maneuver with surface contact lasting 3 seconds. A brush-wheel sampler on a remote arm collects up to 800 cc of sample. A duplicate second arm and sampler collects the second sample. The samples are placed in a return capsule and maintained at colder than -70 C during the return flight and at colder than -30 C during re-entry and for up to six hours after landing. The entire capsule is then refrigerated and transported to the Astromaterials Curatorial Facility at NASA/JSC for initial inspection and sample analysis by the Comet Odyssey team. Comet Odyssey's planned target was comet 9P/Tempel 1, with launch in December 2017 and comet arrival in June 2022. After a stay of 300 days at the comet, the spacecraft departs and arrives at Earth in May 2027. Comet Odyssey is a forerunner to a flagship Cryogenic Comet Sample Return mission that would return samples from deep below the nucleus surface, including volatile ices. This work was supported by internal funds from the Jet Propulsion Laboratory.

  15. Study of DNA damage via the comet assay and base excision repair activities in rat brain neurons and astrocytes during aging.

    PubMed

    Swain, Umakanta; Subba Rao, Kalluri

    2011-08-01

    Earlier we have used biochemical approach to assess the number of single (SSBs) and double (DSBs) strand breaks in brain cellular DNA. However, a quick method to obtain a reliable measure of DNA damage in cells was in need for population studies. Therefore, single cell gel electrophoresis technique (popularly known as "comet" assay) has been standardized using the Trevigen protocol. DNA damage was assessed in isolated neurons and astrocytes from the cortex of young (7 days), adult (6 months) and old (2 years). Marked increase is seen in DNA damage in terms SSBs and DSBs in both types of cells by 6 months of age, which increased further by 2 years of age. The number of 8-oxoguanine DNA glycosylase (OGG1) and uracil DNA glycosylase (UDG) sensitive sites also increased in DNA with age with the simultaneous decrease in OGG1, UDG and AP endonuclease (APE1) activities. Thus the comet assay adapted to our lab conditions has proven to be useful for a quick assessment of DNA damage in a large number of samples that constitute our future studies.

  16. Oxidative damage, bleomycin, and gamma radiation induce different types of DNA strand breaks in normal lymphocytes and thymocytes. A comet assay study.

    PubMed

    Benítez-Bribiesca, L; Sánchez-Suárez, P

    1999-01-01

    Most anticancer treatments such as chemo- and radiotherapy induce DNA damage and apoptosis in normal cells. The aim of this study was to assess the induction of single and double DNA strand breaks (ssb and dsb, respectively) and apoptosis in normal human lymphocytes and rat thymocytes subjected to the action of H2O2, bleomycin and ionizing radiation. Normal human peripheral thymocytes and young rat thymocytes were subjected to the following treatments: a) H2O2; b) bleomycin, and c) gamma-radiation, all with various doses. DNA strand breaks were studied with the alkaline and neutral comet assay for detection of ssb and dsb. Apoptosis was quantified morphologically and with DNA agarose gel electrophoresis. After H2O2 treatment, a dose-dependent increase of ssb was observed. Bleomycin treatment produced a moderate increase of ssb at lower concentrations and a striking increase of dsb at higher concentrations that coincided with the presence of apoptosis and DNA ladders. Gamma radiation initially induced the formation of ssb, and after three hours an increase of dsb in a dose-dependent manner. Apoptosis and DNA laddering appeared only 3 hours post-irradiation. The biomonitoring of DNA damage inflicted by antineoplastic agents can be easily performed with the comet assay and could be useful to monitor and modulate chemo- and radiotherapeutic regimes in cancer patients.

  17. 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.

  18. Evolution of DNA strand-breaks in cultured spermatocytes: the Comet Assay reveals differences in normal and gamma-irradiated germ cells.

    PubMed

    Perrin, J; Lussato, D; De Méo, M; Durand, P; Grillo, J-M; Guichaoua, M-R; Botta, A; Bergé-Lefranc, J-L

    2007-02-01

    In reproductive toxicity assessment, in vitro systems can be used to determine mechanisms of action of toxicants. However, they generally investigate the immediate effects of toxicants, on isolated germ cells or spermatozoa. We report here the usefulness of in vitro cultures of rat spermatocytes and Sertoli cells, in conjunction with the Comet Assay to analyze the evolution of DNA strand-breaks and thus to determine DNA damage in germ cells. We compared cultures of normal and gamma-irradiated germ cells. In non-irradiated spermatocytes, the Comet Assay revealed the presence of DNA strand-breaks, which numbers decreased with the duration of the culture, suggesting the involvement of DNA repair mechanisms related to the meiotic recombination. In irradiated cells, the evolution of DNA strand-breaks was strongly modified. Thus our model is able to detect genotoxic lesions and/or DNA repair impairment in cultured spermatocytes. We propose this model as an in vitro tool for the study of genotoxic injuries on spermatocytes.

  19. Determination of genotoxic effects of copper sulphate and cobalt chloride in Allium cepa root cells by chromosome aberration and comet assays.

    PubMed

    Yildiz, Mustafa; Ciğerci, Ibrahim Hakki; Konuk, Muhsin; Fidan, A Fatih; Terzi, Hakan

    2009-05-01

    We used the anaphase-telophase chromosome aberration and comet (Single Cell Gel Electrophoresis, SCGE) assays to evaluate the genotoxic effects of copper sulphate (CS) and cobalt chloride (CC) chemicals prepared in two concentrations (EC(50), 2xEC(50)), using methyl methanesulfonate (MMS) as a positive control and untreated cells as a negative control. In Allium root growth inhibition test, EC(50) values for CS and CC are 1.5 and 5.5 ppm, respectively. Mitotic index (MI) decreased in all concentrations tested of CS and CC compared to the control at each exposure time. The bridge, stickiness, vagrant chromosomes, fragments, c-anaphase and multipolarity chromosome aberrations were observed in anaphase-telophase cells. The total chromosome aberrations were more frequent with an increasing in the exposure time and the concentrations of both chemicals. The genotoxicity of CS and CC in Allium cepa root cells was analyzed using a mild alkaline comet assay at pH 12.3, which allows the detection of single strand breaks. In all the concentrations, CS and CC induced a significant increase (P<0.05) in DNA damage. No significant difference was found between positive control (300+/-5.81) and 3 ppm CS (280+/-4.61). The methods used are applicable for biological monitoring of environmental pollutants.

  20. Cordyceps sinensis: Genotoxic Potential in Human Peripheral Blood Cells and Antigenotoxic Properties Against Hydrogen Peroxide by Comet Assay.

    PubMed

    Vasiljevic, Jovana D; Zivkovic, Lada P; Cabarkapa, Andrea M; Bajic, Vladan P; Djelic, Ninoslav J; Spremo-Potparevic, Biljana M

    2016-06-01

    Context • Cordyceps sinensis (C sinensis) is a well-known, traditional, Chinese medicinal mushroom, valued for its beneficial properties for human health. C sinensis has been reported to have immunomodulatory, anticancer, antiaging, antioxidant and anti-inflammatory activity. Despite potential medicinal benefits, no previously published reports are available about the genotoxicity or antigenotoxicity of C sinensis, as detected by comet assay. Objective • The objective of the study was to evaluate both the genotoxic and antigenotoxic potential of an extract of C sinensis (CS extract) in human peripheral blood cells. Design • The research team designed a pilot study. Setting •The study was conducted at the Center for Biological Research, University of Belgrade, in Belgrade, Serbia. Participants • Participants were 6 healthy individuals (2 males and 4 females), between the ages of 20 and 45 y, recruited on a voluntary basis, who provided heparinized, peripheral blood samples. Intervention • Four concentrations of the CS extract-125 μg/mL, 250 μg/mL, 500 μg/mL, and 1000 μg/mL-were used in the treatment of tested blood cells from the blood samples. Three independent procedures were performed: (1) a genotoxicity assessment, (2) an antigenotoxicity assessment for pretreatment of human cells with the CS extract prior to their exposure to hydrogen peroxide (H2O2) (ie, an evaluation of the benefits of the CS extract as a preventive agent); and (3) posttreatment of human cells with the CS extract after their exposure to H2O2 (ie, an evaluation of the benefits of the CS extract as an interventional agent). Outcome Measures • Cells were graded by eye inspection into 5 classes, depending on the extent of DNA damage, representing: (1) class A-undamaged cells with no tail (<5% damaged DNA); (2) class B-low-level damage (5%-20%); (3) class C-medium-level damage (20%-40%); (4) class D-high-level damage (40%-95%), and (5) class E-total destruction (>95%).Results

  1. Expression of Inflammatory and Cell Death Program Genes and Comet DNA Damage Assay Induced by Escherichia coli in Layer Hens

    PubMed Central

    Mehaisen, Gamal M. K.; Eshak, Mariam G.; El Sabry, M. I.; Abass, Ahmed O.

    2016-01-01

    Modern methods of industrial poultry and egg production systems involve stressful practices that stimulate Escherichia coli (E. coli) activity causing endotoxic shock. This investigation was conducted to evaluate the expression of pro-inflammatory cytokines and cell death program genes and DNA damage induced by E. coli in the brain and liver tissues of laying hens. A total of two hundred and ten H&N brown layer hens with 20 week age, were used in this research. First, preliminary experiments were designed (60 hens in total) to establish the optimal exposure dose of E. coli and to determine the nearest time of notable response to be used in the remainder studies of this research. At 35-wk of age, 150 hens were randomly assigned into 2 groups with 3 replicates of 25 birds each; the first group was injected in the brachial wing vein with 107 E. coli colony/hen, while the second group was injected with saline and served as a control. The body temperature and plasma corticosterone concentration were measured 3 hr after injection. Specimens of liver and brain were obtained from each group and the gene expression of p38 mitogen-activated protein kinase, interlukin-1β (IL-1β), tumor necrosis factor alpha (TNF-α), Bax, and caspase-3 genes were measured by quantitative real-time PCR. DNA damage in the brain and liver tissues were also measured by comet assay. Hens treated with E. coli showed significant (P<0.05) increase of body temperature and plasma corticosterone (42.6°C and 14.5 ng/ml, respectively) compared to the control group (41.1°C and 5.5 ng/ml, respectively). Additional remarkable over-inflammation gene expression of p38, IL-1β and TNF-α.genes were also detected in the brain (2.2-fold, 2.0-fold and 3.3-fold, respectively) and the liver (2.1-fold, 1.9-fold and 3.0-fold, respectively) tissues of the infected chickens. It is also important to note that hens injected with E. coli showed an increase in DNA damage in the brain and liver cells (P<0.05). These

  2. Nicotine derived genotoxic effects in human primary parotid gland cells as assessed in vitro by comet assay, cytokinesis-block micronucleus test and chromosome aberrations test.

    PubMed

    Ginzkey, Christian; Steussloff, Gudrun; Koehler, Christian; Burghartz, Marc; Scherzed, Agmal; Hackenberg, Stephan; Hagen, Rudolf; Kleinsasser, Norbert H

    2014-08-01

    Genotoxic effects of nicotine were described in different human cells including salivary gland cells. Based on the high nicotine concentration in saliva of smokers or patients using therapeutic nicotine patches, the current study was performed to evaluate the genotoxic potential of nicotine in human salivary gland cells. Therefore, primary salivary gland cells from 10 patients undergoing parotid gland surgery were exposed to nicotine concentrations between 1 μM and 1000 μM for 1 h in the absence of exogenous metabolic activation. The acinar phenotype was proven by immunofluorescent staining of alpha-amylase. Genotoxic effects were evaluated using the Comet assay, the micronucleus test and the chromosome aberration test. Cytotoxicity and apoptosis were determined by trypan blue exclusion test and Caspase-3 assay. Nicotine was able to induce genotoxic effects in all three assays. The chromosome aberration test was the most sensitive and increases in numerical and structural (chromatid-type and chromosome-type) aberrations were seen at ≥1 μM, whereas increases in micronuclei frequency were detected at 10 μM and DNA damage as measured in the Comet assay was noted at >100 μM. No cytotoxic damage or influence of apoptosis could be demonstrated. Nicotine as a possible risk factor for tumor initiation in salivary glands is still discussed controversially. Our results demonstrated the potential of nicotine to induce genotoxic effects in salivary gland cells. These results were observed at saliva nicotine levels similar to those found after oral or transdermal exposure to nicotine and suggest the necessity of careful monitoring of the use of nicotine in humans.

  3. Relative sensitivity of fish and mammalian cells to the antibiotic, trimethoprim: cytotoxic and genotoxic responses as determined by neutral red retention, Comet and micronucleus assays.

    PubMed

    Papis, Elena; Davies, Simon J; Jha, Awadhesh N

    2011-01-01

    Relative cytotoxicity and genotoxicity of a widely used antibiotic, trimethoprim (TRIMP) was evaluated under in vitro conditions using rainbow trout gonad-2 (RTG-2) and Chinese hamster ovary-K1 (CHO-K1) cells. Whilst cytotoxicity was determined using neutral red retention (NRR) assay, the genotoxicity was determined using single cell gel electrophoresis or the Comet assay and cytokinesis-block micronucleus (CBMN) assay. For NRR assay, concentration-dependent cytotoxic effect was observed for both the cell lines (estimated EC(50) values: 671.82 ± 21.78 and 611.6 ± 20.4 μg ml(-1) for RTG-2 and CHO-K1 cells, respectively). There was no statistically significant difference between the two cell lines for this assay. For the Comet assay, standard 6 h exposure to TRIMP did not show any positive response for any of the cell types used. However, 48 h exposure to RTG-2 cells showed a concentration-dependent induction of DNA damage (r = 0.86). The highest concentration of TRIMP used (i.e. 100 μg ml(-1)) showed relatively higher DNA damage, compared to ethyl methane sulfonate (EMS; 1 μg ml(-1) or 8 mM), a reference genotoxic agent, used concurrently. In contrast, 24 h exposure time for CHO-K1 cells did not show any concentration-dependent increase for this assay. For MN assay, a significant correlation was found between the MN induction and TRIMP concentration for both the cell lines (RTG-2: r = 0.68; CHO-K1: r = 0.79), although only the highest concentration used showed a significant increase for binucleated (BN) cell with micronuclei (BNMN). The study suggests that whilst the cells of different origin could exhibit similar cytotoxicity, they could display differential genotoxic effects. Furthermore, genotoxic effects of TRIMP are primarily exposure period dependent phenomena and, in addition to inhibiting the action of dihydrofolate reductase, oxidative stress could also contribute for the observed toxic effects, fish cells in general being more sensitive for genotoxic

  4. 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

  5. Detailed analysis of Seth's circular niches on comet 67P Churyumov-Gerasimenko

    NASA Astrophysics Data System (ADS)

    Lucchetti, Alice; Pajola, Maurizio; Fornasier, Sonia; Mottola, Stefano; Penasa, Luca; Jorda, Laurent; Cremonese, Gabriele; Feller, Clement; Hasselmann, Pedro; Massironi, Matteo; Naletto, Giampiero; Deshapriya, Prasanna

    2017-04-01

    We performed a detailed geomorphological and spectrophotometric analysis of the circular niches located on Seth region on comet 67P Churyumov-Gerasimenko (67P): they are flat-floored and steep-walled circular depressions present on the Northern hemisphere of 67P (Thomas et al., 2015). The aim is to determine if these features are associated to landslides events or if they are correlated to comet's activity. We selected high resolution OSIRIS images acquired in August 2016 (scale of 31 cm/px) focusing on Seth's niches with unprecedented detail. First, we counted boulders in order to compare the boulders size-frequency distribution of niches with previous results (Pajola et al., 2015), finding that no changes occurred after comet perihelion passage. Consequently, we characterized the region by considering the erosion and insolation models covering the area (Keller et al., 2015), and using a high resolution DTM that allowed us to perform a more detailed geomorphological analysis regarding the height of niches' walls and the volume of the deposit material located at their feet. In addition, we performed a color analysis of the region identifying the presence of few bright patches, probably composed of ices. The next step will be the comparison of these results with pre-perihelion images to find if there are any compositional changes that occurred in that region. From this preliminary analysis we conclude that niches can be correlated to past landslide events that occurred on the comet. To strengthen our conclusion we will compare the fractures distribution on the niches' wall with those characterizing the boulders of the landslide deposit (with image of 6 - 10 cm/px in scale) in order to understand wether the fracture pattern is the same or fragmentation processes occurred after the landslide event.

  6. DNA staining with the fluorochromes EtBr, DAPI and YOYO-1 in the comet assay with tobacco plants after treatment with ethyl methanesulphonate, hyperthermia and DNase-I.

    PubMed

    Gichner, Tomás; Mukherjee, Anita; Velemínský, Jirí

    2006-06-16

    We applied the alkaline version of the single-cell gel electrophoresis (comet) assay to roots and leaves of tobacco (Nicotiana tabacum var. xanthi) seedlings or isolated leaf nuclei treated with: (1) the alkylating agent ethyl methanesulphonate, (2) necrotic heat treatments at 50 degrees C, and (3) DNase-I. All three treatments induced a dose-dependent increase in DNA migration, expressed as percentage of tail DNA. A comparison of the fluorochrome DNA dyes ethidium bromide, DAPI and YOYO-1 demonstrated that for the alkaline version of the comet assay in plants, the commonly used fluorescent dye ethidium bromide can be used with the same efficiency as DAPI or YOYO-1.

  7. Measurement Protocols for In Situ Analysis of Organic Compounds at Mars and Comets

    NASA Technical Reports Server (NTRS)

    Mahaffy, P. R.; Brinckerhoff, W. B.; Buch, A.; Cabane, M.; Coll, P.; Demick, J.; Glavin, D. P.; Navarro-Gonzalez, R.

    2005-01-01

    The determination of the abundance and chemical and isotopic composition of organic molecules in comets and those that might be found in protected environments at Mars is a first step toward understanding prebiotic chemistries on these solar system bodies. While future sample return missions from Mars and comets will enable detailed chemical and isotopic analysis with a wide range of analytical techniques, precursor insitu investigations can complement these missions and facilitate the identification of optimal sites for sample return. Robust automated experiments that make efficient use of limited spacecraft power, mass, and data volume resources are required for use by insitu missions. Within these constraints we continue to explore a range of instrument techniques and measurement protocols that can maximize the return from such insitu investigations.

  8. Measurement Protocols for In situ Analysis of Organic Compounds at Mars and Comets

    NASA Technical Reports Server (NTRS)

    Mahaffy, P. R.; Brinckerhuff, W. B.; Buch, A.; Cabane, M.; Coll, P.; Demick, J.; Glavin, D. P.; Navarro-Gonzalez, R.

    2005-01-01

    The determination of the abundance and chemical and isotopic composition of organic molecules in comets and those that might be found in protected environments at Mars is a first step toward understanding prebiotic chemistries on these solar system bodies. While future sample return missions from Mars and comets will enable detailed chemical and isotopic analysis with a wide range of analytical techniques, precursor insitu investigations can complement these missions and facilitate the identification of optimal sites for sample return. Robust automated experiments that make efficient use of limited spacecraft power, mass, and data volume resources are required for use by insitu missions. Within these constraints we continue to explore a range of instrument techniques and measurement protocols that can maximize the return from such insitu investigations.

  9. Comparative evaluation of genotoxicity of captan in amphibian larvae (Xenopus laevis and Pleurodeles waltl) using the comet assay and the micronucleus test.

    PubMed

    Mouchet, F; Gauthier, L; Mailhes, C; Ferrier, V; Devaux, A

    2006-06-01

    Captan (N-trichloromethylthio-4-cyclohexene-1,2-dicarboximide) is a fungicide used to inhibit the growth of many types of fungi on plants used as foodstuffs. The toxic and genotoxic potentials of captan were evaluated with the micronucleus test (MNT; AFNOR,2000) and the comet assay (CA) using amphibian larvae (Xenopus laevis and Pleurodeles waltl). Acute toxicity results showed that captan was toxic (1) to Xenopus larvae exposed to from 2 mg/L to 125 or 62.5 microg/L, depending on the nature of the water [reconstituted water containing mineral salts or mineral water (MW; Volvic, Danone, France)] and (2) to Pleurodeles exposed to from 2 mg/L to 125 microg/L in both types of water. The MNT results obtained in MW showed that captan (62.5 microg/L) was genotoxic to Xenopus but not genotoxic to Pleurodeles at all concentrations tested. CA established that the genotoxicity of captan to Xenopus and Pleurodeles larvae depended on the concentration, the exposure times, and the comet parameters (tail DNA, TEM, OTM, and TL). The CA and MNT results were compared for their ability to detect DNA damage at the concentrations of captan and the exposure times applied. CA showed captan to be genotoxic from the first day of exposure. In amphibians, CA appears to be a sensitive and suitable method for detecting genotoxicity such as that caused by captan. Copyright 2006 Wiley Periodicals, Inc.

  10. Evaluation of DNA Single and Double Strand Breaks in Women with Cervical Neoplasia Based on Alkaline and Neutral Comet Assay Techniques

    PubMed Central

    Cortés-Gutiérrez, Elva I.; Hernández-Garza, Fernando; García-Pérez, Jorge O.; Dávila-Rodríguez, Martha I.; Aguado-Barrera, Miguel E.; Cerda-Flores, Ricardo M.

    2012-01-01

    A hospital-based unmatched case-control study was performed in order to determine the relation of DNA single (ssb) and double (dsb) strand breaks in women with and without cervical neoplasia. Cervical epithelial cells of 30 women: 10 with low grade squamous intraepithelial lesions (LG-SIL), 10 with high-grade SIL (HG-SIL), and 10 without cervical lesions were evaluated using alkaline and neutral comet assays. A significant increase in global DNA damage (ssb + dsb) and dsb was observed in patients with HG-SIL (48.90 ± 12.87 and 23.50 ± 13.91), patients with LG-SIL (33.60 ± 14.96 and 11.20 ± 5.71), and controls (21.70 ± 11.87 and 5.30 ± 5.38; resp.). Pearson correlation coefficient reveled a strong relation between the levels ssb and dsb (r2 = 0.99, P = 0.03, and r2 = 0.94, P = 0.16, resp.) and progression of neoplasia. The increase of dsb damage in patients with HG-SIL was confirmed by DNA breakage detection-FISH (DBD-FISH) on neutral comets. Our results argue in favor of a real genomic instability in women with cervical neoplasia, which was strengthened by our finding of a higher proportion of DNA dsb. PMID:23093842

  11. Analysis of dust in the coma of comet 67P using VIRTIS-M observations

    NASA Astrophysics Data System (ADS)

    Rinaldi, G.; Tozzi, G. P.; Fink, U.; Doose, L.; Capaccioni, F.; Filacchione, G.; Bockelée-Morvan, D.; Leyrat, C.; Piccioni, G.; Blecka, M.; Ciarniello, M.; Irwin, P.; Combi, M.; Palomba, E.; Migliorini, A.; Capria, M. T.; Faggi, S.; Tosi, F.

    2015-10-01

    We present a preliminary overview of the analysis on the dust spectrophotometry in the inner coma of comet 67/P that was obtained during the escort phase (started on December 2014) with the imaging spectrometer VIRTIS-M onboard the Rosetta mission [1]. The morphology and behavior of the dust coma has been monitored by VIRTIS-M from the arrival at the comet (~August 2014) throughout the early escort phase. The data reveal intricate details and numerous radial jets coming from different regions on the surface. On March 15, 2015, VIRTIS-M performed a set of 22 coma observations, each about 23 minutes in duration and offset from the nucleus by about 1 km. The 22 observations lasted about 12 hours and thus covered a complete rotation of the comet. The maps of the dust distribution in the coma reveal three major structures: a roughly uniform background dusty coma, several enhanced radiance jet features and a region that shows a thermal radiation component between 3.5 and 5.0 μm. (Figure 1 and Figure 2) The jets features can be traced back to several region of the comet, neck,body and head. We shall analyse the three major structures to provide the basis to understand coma composition and properties and the relation between gas and dust. We will discuss the morphology of the background coma, the jet and the enhanced thermal radiation. We will also examine correlations between the water vapor column density and the coma/ jet /thermal radiation intensity. For the thermal radiation component there are several explanations, viz: stray instrumental scattered light or instrumental ghosts from heated part of the nucleus, or thermal rad iation emanating from the nucleus and scattered by the dust in closest proximity or a region of small particles in the coma heated by solar radiation.

  12. In vitro cytotoxic and non-genotoxic effects of gutta-percha solvents on mouse lymphoma cells by single cell gel (comet) assay.

    PubMed

    Ribeiro, Daniel Araki; Marques, Mariângela Esther Alencar; Salvador, Daisy Maria Fávero

    2006-01-01

    Chloroform and eucalyptol are widely used in clinical dentistry as gutta-percha solvents. However, these compounds may represent a hazard to human health, especially by causing injury to genetic apparatus and/or inducing cellular death. In this study, the genotoxic and cytotoxic potentials associated with exposure to chloroform and eucalyptol were assessed on mouse lymphoma cells in vitro by the single cell gel (comet) assay and trypan blue exclusion test, respectively. Both gutta-percha solvents proved to be cytotoxic at the same levels in concentrations of 2.5, 5 and 10 microL/mL (p<0.05). On the other hand, neither of the solvents induced DNA breakage. Taken together, these results suggest that although both tested compounds (chloroform and eucalyptol) are strong cytotoxicants, it seems that they are not likely to increase the level of DNA damage on mammalian cells.

  13. DNA damage in mononuclear leukocytes of farmers measured using the alkaline comet assay: discussion of critical parameters and evaluation of seasonal variations in relation to pesticide exposure.

    PubMed

    Lebailly, P; Vigreux, C; Lechevrel, C; Ledemeney, D; Godard, T; Sichel, F; LeTalaër, J Y; Henry-Amar, M; Gauduchon, P

    1998-10-01

    The alkaline comet assay was used to quantify, using visual and image analyses, the level of DNA damage in mononuclear leukocytes of farmers who were occupationally exposed to pesticides. Hematological parameters were also measured on the same samples. Enrollment of farmers was based on handling of heavily used pesticides at particular periods during one spraying season. Forty-one blood samples from 29 different farmers were collected at the beginning of the season (n = 11) and at the intermediate (n = 14) and final (n = 16) periods of intense spraying activity. The mean numbers of lymphocytes and eosinophils were nonsignificantly higher in groups 3, 1, and 4 than they were in group 2. No individual characteristics significantly influenced the mean number of lymphocytes or eosinophils, and no correlation was observed between pesticide exposure-related parameters and hematological parameters. The level of DNA damage was significantly (P < 0.01) higher in groups 3, 1, and 4 than it was in group 2. In addition, DNA damage quantification was not significantly different among investigators or among slides. Prescription medicine, alcohol consumption, and age had no statistically significant effect on DNA damage level. Conversely, smoking (smokers versus non- and ex-smokers) significantly influenced DNA damage level (P < 0.0001). A significant (P < 0.05) negative correlation was detected between the number of days without pesticide spraying and DNA damage level, particularly among non- and ex-smokers. DNA damage detected by the alkaline comet assay seems to reflect ongoing exposure to genotoxic agents but not an accumulation of damage.

  14. Genotoxicity evaluation of pesticide formulations containing alachlor and atrazine in multiple mouse tissues (blood, kidney, liver, bone marrow, spleen) by comet assay.

    PubMed

    Zeljezic, D; Garaj-Vrhovac, V

    2004-01-01

    Every year, in the European countries more than 2 million tons of pesticides are released into the environment. More than 60% of those substances appear to be herbicides. Due to extensive production and application of this chemical their putative detrimental effect on life should be known and minimized. In this study we applied the comet assay on blood and 4 mouse organs (kidney, liver, bone marrow, and spleen) to evaluate possible genome damage caused by two pesticide formulations (Bravo and Gesaprim) containing alachlor and atrazine as active ingredients. Five male CBA mice were assigned to each of 4 treatment groups and control group. Bravo and Gesaprim were injected intraperitoneally once. Two different doses of Bravo were used: 0.031 ml/kg and 0.021 microl/kg, so that doses of alachlor mice received within the pesticide formulation given were 15 mg/kg and 0.01 mg/kg. Also Gesaprim was given at two different doses: 1.08 ml/kg and 0.07 microl/kg so that the doses of atrazine contained within the pesticide formulation given were 540 mg/kg and 3.5 x 10(-2) mg/kg. Mice were sacrificed 24 hours after treatment. Alkaline comet assay on the blood samples, kidney, liver, bone marrow and spleen was performed. Statistically significant (p<0.01) increase of tail length for all 5 tissues examined in mice treated with both Bravo and Gesaprim compared to the control was found. For both pesticides DNA of kidney and liver showed largest increase in migration. Also, distribution of tail length values for Bravo and Gesaprim for all mouse tissues examined showed a shift to the right when compared to the controls.

  15. Allelopathic effects of volatile organic compounds from Eucalyptus grandis rhizosphere soil on Eisenia fetida assessed using avoidance bioassays, enzyme activity, and comet assays.

    PubMed

    Zhiqun, Tang; Jian, Zhang; Junli, Yu; Chunzi, Wang; Danju, Zhang

    2017-04-01

    Allelopathy has been identified as an underlying mechanism of detrimental environmental impacts within commercial plantations. Eucalyptus spp. are known to generate huge amounts of volatile organic compounds (VOCs) that can function as phytotoxins and thus inhibit other plants. In the present study, biochemical markers, including activities of acetylcholinesterase (AChE) and oxidative stress enzymes, such as superoxide dismutase (SOD) and glutathione S-transferase (GST), were assayed to assess changes in Eisenia fetida at the physiological level induced by different doses of VOCs as part of an acute toxicity test over 7 and 14-day exposures. In addition, the toxicities of VOCs were investigated using a soil avoidance test and comet assay. The results revealed that E. fetida exhibited significant avoidance behavior towards the highest concentrations of undecane, decane, 2,4-dimethyl heptane, and 2,2,4,6,6-pentametyl heptane. The tail DNA percentages were significantly increased for all experimental treatments relative to control. However, under the treatments of VOCs, Olive tail moment content and comet tail length also display an obvious increase compared to control, except for that of octane, undecane and decane treatments. As VOC concentrations and durations increased in the soil, activities of AChE, SOD, and GST were either stimulated or inhibited. Among the VOCs, decane, 2,4-dimethyl heptane, 2,2,4,6,6-pentamethyl heptane, and 2,4-di tert buyl phenol exerted stronger effects on enzymatic activities. In summary, VOCs in rhizosphere soils of E. grandis might exert a toxic impact on E. fetida, among which 2,4-dimethyl heptane, 2,2,4,6,6-pentamethyl heptane, and 2,4-di tert buyl phenol have the strongest effects. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. 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...

  17. 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...

  18. Analysis of Gold Ores by Fire Assay

    ERIC Educational Resources Information Center

    Blyth, Kristy M.; Phillips, David N.; van Bronswijk, Wilhelm

    2004-01-01

    Students of an Applied Chemistry degree course carried out a fire-assay exercise. The analysis showed that the technique was a worthwhile quantitative analytical technique and covered interesting theory including acid-base and redox chemistry and other concepts such as inquarting and cupelling.

  19. Analysis of Gold Ores by Fire Assay

    ERIC Educational Resources Information Center

    Blyth, Kristy M.; Phillips, David N.; van Bronswijk, Wilhelm

    2004-01-01

    Students of an Applied Chemistry degree course carried out a fire-assay exercise. The analysis showed that the technique was a worthwhile quantitative analytical technique and covered interesting theory including acid-base and redox chemistry and other concepts such as inquarting and cupelling.

  20. Atlas of Great Comets

    NASA Astrophysics Data System (ADS)

    Stoyan, Ronald; Dunlop, Storm

    2015-01-01

    Foreword; Using this book; Part I. Introduction: Cometary beliefs and fears; Comets in art; Comets in literature and poetry; Comets in science; Cometary science today; Great comets in antiquity; Great comets of the Middle Ages; Part II. The 30 Greatest Comets of Modern Times: The Great Comet of 1471; Comet Halley 1531; The Great Comet of 1556; The Great Comet of 1577; Comet Halley, 1607; The Great Comet of 1618; The Great Comet of 1664; Comet Kirch, 1680; Comet Halley, 1682; The Great Comet of 1744; Comet Halley, 1759; Comet Messier, 1769; Comet Flaugergues, 1811; Comet Halley, 1835; The Great March Comet of 1843; Comet Donati, 1858; Comet Tebbutt, 1861; The Great September Comet of 1882; The Great January Comet of 1910; Comet Halley, 1910; Comet Arend-Roland, 1956; Comet Ikeya-Seki, 1965; Comet Bennett, 1970; Comet Kohoutek, 1973-4; Comet West, 1976; Comet Halley, 1986; Comet Shoemaker-Levy 9, 1994; Comet Hyakutake, 1996; Comet Hale-Bopp, 1997; Comet McNaught, 2007; Part III. Appendices; Table of comet data; Glossary; References; Photo credits; Index.

  1. Giotto data analysis: Electron plasma and heavy ion composition measurements at Comet Halley

    NASA Technical Reports Server (NTRS)

    Lin, Robert P.

    1992-01-01

    This investigation involved the analysis of electron plasma and heavy ion composition measurements made by the COPERNIC (COmplete Positive ion, Electron, and Ram Negative Ion measurements near Comet Halley) plasma experiment during the close fly-by of Halley by the European Space Agency's Giotto spacecraft. The experiment provided measurements of the full 3-dimensional distribution of 10 eV-30 keV electrons, and mass analysis of cold cometary ions from 10-210 amu. The analysis of the COPERNIC data has yielded some remarkable results, including: The discovery of negatively charged ions in the inner coma; the discovery of far heavier (mass is greater than 50 amu) ions than predicted, dominated by complex molecular ions made up of C, H, O, and N; the discovery of an adiabatic heating effect on electrons from the compression of the solar wind plasma; the identification of several organic and sulfur bearing ions; and the discovery of a new 'mystery region' where electrons are accelerated to high energies. These discoveries were in addition to the detailed analysis of 'expected' features at Comet Halley. Although this grant has expired, analysis continues on the data at a low (unfunded) level, and it is expected that more significant results will be obtained. A bibliography of the papers resulting from this research is attached, and a copy of each paper is included.

  2. Cosima - Cometary Dust Analysis Next to Comet 67P/Churyumov-Gerasimenko

    NASA Astrophysics Data System (ADS)

    Hilchenbach, M.; Kissel, J.; Briois, C.; Henkel, H.; Langevin, Y.; Schulz, R.; Silen, J. V.; Altwegg, K.; Colangeli, L.; Cottin, H.; Engrand, C.; Glasmachers, A.; Grün, E.; Haerendel, G.; Höfner, H.; Hornung, K.; Jessberger, E.; Koch, A.; Lehto, H.; Lehto, K.; Raulin, F.; Le Roy, L.; Rynö, J.; Steiger, W.; Stephan, T.; Thirkell, L.; Thomas, R.; Torkar, K.; Varmuza, K.; Wanczek, K. P.

    2014-12-01

    After a long journey through the inner solar system, ESA's corner stone mission ROSETTA has arrived at comet 67P/Churyumov-Gerasimenko. COSIMA or the COmetary Secondary Ion Mass Analyzer onboard ROSETTA is a secondary ion mass spectrometer focussing on in-situ measurements of the composition of cometary grains collected next to the nucleus and inner coma. High resolution mass spectra will contain complex mixtures of mineral and organic elements and molecules as well as molecular fragments representing the elements and molecules on the surface of the cometary grains. We will report on first results of the in-situ analysis of cometary grains as captured, imaged and analysed by COSIMA .

  3. Great Comets

    NASA Astrophysics Data System (ADS)

    Burnham, Robert

    2000-05-01

    Spectacular and mysterious objects that come and go in the night sky, comets have dwelt in our popular culture for untold ages. As remnants from the formation of the Solar system, they are objects of key scientific research and space missions. As one of nature's most potent and dramatic dangers, they pose a threat to our safety--and yet they were the origin of our oceans and perhaps even life itself. This beautifully illustrated book tells the story of the biggest and most awe-inspiring of all comets: those that have earned the title "Great." Robert Burnham focuses on the Great comets Hyakutake in 1996 and Hale-Bopp in 1997, which gripped attention worldwide because, for many, they were the first comets ever seen. He places these two recent comets in the context of their predecessors from past ages, among them the famous Comet Halley. Great Comets explains the exciting new discoveries that have come from these magnificent objects and profiles the spaceprobes to comets due for launch in the next few years. The book even takes a peek behind Hollywood's science-fiction fantasies to assess the real risks humanity faces from potential impacts of both comets and asteroids. For everyone interested in astronomy, this exciting book reveals the secrets of the Great Comets and provides essential tools for keeping up to date with comet discoveries in the future. Robert Burnham has been an amateur astronomer since the mid-1950s. He has been a senior editor of Astronomy magazine (1986-88) and is the author of many books and CD-ROMS, including Comet Hale-Bopp: Find and Enjoy the Great Comet and Comet Explorer.

  4. Genotoxicity study with special reference to DNA damage by comet assay in fission yeast, Schizosaccharomyces pombe exposed to drinking water.

    PubMed

    Banerjee, Pamela; Talapatra, Soumendra N; Mandal, Nivedita; Sundaram, Geetanjali; Mukhopadhyay, Aniruddha; Chattopadhyay, Dhrubajyoti; Banerjee, Sudip K

    2008-01-01

    The objective of this study was to investigate genotoxicity, especially DNA damage, in drinking water samples collected from tap by using fission yeast Schizosaccharomyces pombe as a model organism. Generally raw water potabolization is done by treatment with polymeric coagulant, alum, chlorine, etc. In the comet test, highly significant (P<0.001) effects of DNA damage were detected in treated water (tap water) when compared to negative control (raw water) as well as laboratory control (distilled water) samples for both 1 h and 2 h exposure. In the water treatment plant, raw water treatment is done by the process of prechlorination, alum and polymeric coagulant (CatflocT) dosing, postchlorination, filtration and final discharge for consumption. In conclusion it can be stated from the results that chlorinated disinfectant, alum and polymeric coagulant (CatflocT) mixture used in drinking water has a potent cumulative genotoxic effect in the eukaryotic cells and may pose potential genotoxic risk for human health following long-term consumption.

  5. Radiation-induced DNA damage in canine hemopoietic cells and stromal cells as measured by the comet assay

    SciTech Connect

    Kreja, L.; Selig, C.; Plappert, U.; Nothdurft, W.

    1996-12-31

    Stromal cell progenitors (fibroblastoid colony-forming unit; CFU-Fs) are representative of the progenitor cell population of the hemopoietic microenvironment in bone marrow (BM). Previous studies of the radiation dose-effect relationships for colony formation have shown that canine CFU-Fs are relatively radioresistant as characterized by a D{sub 0} value of about 2.4 Gy. In contrast, hemopoietic progenitors are particularly radiosensitive (D{sub 0} values = 0.12-0.60 Gy). In the present study, the alkaline single-cell gel electrophoresis technique for the in situ quantitation of DNA strand breaks and alkalilabile site was employed. Canine buffy coat cells from BM aspirates and cells harvested from CFU-F colonies or from mixed populations of adherent BM stromal cell (SC) layers were exposed to increasing doses of X-rays, embedded in agarose gel on slides, lysed with detergents, and placed in an electric field. DNA migrating from single cells in the gel was made visible as {open_quotes}comets{close_quotes} by ethidium bromide staining. Immediate DNA damage was much less in cultured stromal cells than in hemopoietic cells in BM aspirates. These results suggest that the observed differences in clonogenic survival could be partly due to differences in the type of the initial DNA damage between stromal cells and hemopoietic cells. 37 refs., 2 figs., 1 tab.

  6. Entry Dispersion Analysis for the Stardust Comet Sample Return Capsule

    NASA Technical Reports Server (NTRS)

    Desai, Prasun N.; Mitcheltree, Robert A.; Cheatwood, F. McNeil

    1997-01-01

    Stardust will be the first mission to return samples from beyond the Earth-Moon system. The sample return capsule, which is passively controlled during the fastest Earth entry ever, will land by parachute in Utah. The present study analyzes the entry, descent, and landing of the returning sample capsule. The effects of two aerodynamic instabilities are revealed (one in the high altitude free molecular regime and the other in the transonic/subsonic flow regime). These instabilities could lead to unacceptably large excursions in the angle-of-attack near peak heating and main parachute deployment, respectively. To reduce the excursions resulting from the high altitude instability, the entry spin rate of the capsule is increased. To stabilize the excursions from the transonic/subsonic instability, a drogue chute with deployment triggered by an accelerometer and timer is added prior to main parachute deployment. A Monte Carlo dispersion analysis of the modified entry (from which the impact of off-nominal conditions during the entry is ascertained) shows that the capsule attitude excursions near peak heating and drogue chute deployment are within Stardust program limits. Additionally, the size of the resulting 3-sigma landing ellipse is 83.5 km in downrange by 29.2 km in crossrange, which is within the Utah Test and Training Range boundaries.

  7. Automated Protein Assay Using Flow Injection Analysis

    NASA Astrophysics Data System (ADS)

    Wolfe, Carrie A. C.; Oates, Matthew R.; Hage, David S.

    1998-08-01

    The technique of flow injection analysis (FIA) is a common instrumental method used in detecting a variety of chemical and biological agents. This paper describes an undergraduate laboratory that uses FIA to perform a bicinchoninic acid (BCA) colorimetric assay for quantitating protein samples. The method requires less than 2 min per sample injection and gives a response over a broad range of protein concentrations. This method can be used in instrumental analysis labs to illustrate the principles and use of FIA, or as a means for introducing students to common methods employed in the analysis of biological agents.

  8. The nucleus of Comet 67P/Churyumov-Gerasimenko. A new shape model and thermophysical analysis

    NASA Astrophysics Data System (ADS)

    Lowry, S.; Duddy, S. R.; Rozitis, B.; Green, S. F.; Fitzsimmons, A.; Snodgrass, C.; Hsieh, H. H.; Hainaut, O.

    2012-12-01

    Context. Comet 67P/Churyumov-Gerasimenko is the target of the European Space Agency Rosetta spacecraft rendez-vous mission. Detailed physical characteristation of the comet before arrival is important for mission planning as well as providing a test bed for ground-based observing and data-analysis methods. Aims: To conduct a long-term observational programme to characterize the physical properties of the nucleus of the comet, via ground-based optical photometry, and to combine our new data with all available nucleus data from the literature. Methods: We applied aperture photometry techniques on our imaging data and combined the extracted rotational lightcurves with data from the literature. Optical lightcurve inversion techniques were applied to constrain the spin state of the nucleus and its broad shape. We performed a detailed surface thermal analysis with the shape model and optical photometry by incorporating both into the new Advanced Thermophysical Model (ATPM), along with all available Spitzer 8-24 μm thermal-IR flux measurements from the literature. Results: A convex triangular-facet shape model was determined with axial ratios b/a = 1.239 and c/a = 0.819. These values can vary by as much as 7% in each axis and still result in a statistically significant fit to the observational data. Our best spin state solution has Psid = 12.76137 ± 0.00006 h, and a rotational pole orientated at Ecliptic coordinates λ = 78°(±10°), β = + 58°(±10°). The nucleus phase darkening behaviour was measured and best characterized using the IAU HG system. Best fit parameters are: G = 0.11 ± 0.12 and HR(1,1,0) = 15.31 ± 0.07. Our shape model combined with the ATPM can satisfactorily reconcile all optical and thermal-IR data, with the fit to the Spitzer 24 μm data taken in February 2004 being exceptionally good. We derive a range of mutually-consistent physical parameters for each thermal-IR data set, including effective radius, geometric albedo, surface thermal inertia

  9. Advanced analysis techniques for uranium assay

    SciTech Connect

    Geist, W. H.; Ensslin, Norbert; Carrillo, L. A.; Beard, C. A.

    2001-01-01

    Uranium has a negligible passive neutron emission rate making its assay practicable only with an active interrogation method. The active interrogation uses external neutron sources to induce fission events in the uranium in order to determine the mass. This technique requires careful calibration with standards that are representative of the items to be assayed. The samples to be measured are not always well represented by the available standards which often leads to large biases. A technique of active multiplicity counting is being developed to reduce some of these assay difficulties. Active multiplicity counting uses the measured doubles and triples count rates to determine the neutron multiplication (f4) and the product of the source-sample coupling ( C ) and the 235U mass (m). Since the 35U mass always appears in the multiplicity equations as the product of Cm, the coupling needs to be determined before the mass can be known. A relationship has been developed that relates the coupling to the neutron multiplication. The relationship is based on both an analytical derivation and also on empirical observations. To determine a scaling constant present in this relationship, known standards must be used. Evaluation of experimental data revealed an improvement over the traditional calibration curve analysis method of fitting the doubles count rate to the 235Um ass. Active multiplicity assay appears to relax the requirement that the calibration standards and unknown items have the same chemical form and geometry.

  10. 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.

  11. Multivariate statistical analysis of OSIRIS/Rosetta spectrophotometric data of comet 67P/Churyumov-Gerasimenko

    NASA Astrophysics Data System (ADS)

    Perna, D.; Fulchignoni, M.; Barucci, M. A.; Fornasier, S.; Feller, C.; Deshapriya, J. D. P.; Hasselmann, P. H.; Sierks, H.; Barbieri, C.; Lamy, P. L.; Rodrigo, R.; Koschny, D.; Rickman, H.; A'Hearn, M.; Bertaux, J.-L.; Bertini, I.; Cremonese, G.; Da Deppo, V.; Davidsson, B.; Debei, S.; Deller, J.; De Cecco, M.; El-Maarry, M. R.; Fulle, M.; Groussin, O.; Gutierrez, P. J.; Güttler, C.; Hofmann, M.; Hviid, S. F.; Ip, W.-H.; Jorda, L.; Keller, H. U.; Knollenberg, J.; Kramm, R.; Kührt, E.; Küppers, M.; Lara, L. M.; Lazzarin, M.; Lopez Moreno, J. J.; Marzari, F.; Naletto, G.; Oklay, N.; Thomas, N.; Tubiana, C.; Vincent, J.-B.

    2017-04-01

    Context. The ESA Rosetta mission explored comet 67P/Churyumov-Gerasimenko in 2014-2016, following its target before and after the perihelion passage on 13 August 2015. The NAC camera of the OSIRIS imaging system allowed to map the nucleus surface acquiring images with different filters in the visible wavelength range. Aims: Here we study the spectrophotometric behaviour of the nucleus by a multivariate statistical analysis, aiming to distinguish homogeneous groups and to constrain the bulk composition. Methods: We applied the G-mode clustering algorithm to 16 OSIRIS data cubes acquired on 5-6 August 2014 (mostly covering the northern hemisphere) and 2 May 2015 (mostly covering the southern hemisphere), selected to have complete coverage of the comet's surface with similar observing conditions. Results: We found four similar homogeneous groups for each of the analysed cubes. The first group corresponds to the average spectrophotometric behaviour of the nucleus. The second (spectrally redder) and the third (spectrally bluer) groups are found in regions that were already found to deviate from the average terrain of the comet by previous studies. A fourth group (characterised by enhancements of the flux at 700-750 nm and 989 nm, possibly due to H2O+ and/or NH2 emissions) seems connected with the cometary activity rather than with the bulk composition. Conclusions: While our aim in this work was to study the spectrophotometric behaviour of the nucleus of 67P/Churyumov-Gerasimenko as a whole, we found that a follow-up application of the G-mode to smaller regions of the surface could be useful in particular to identify and study the temporal evolution of ice patches, as well as to constrain the composition and physical processes behind the emission of dust jets.

  12. Stable isotope dilution assays in mycotoxin analysis.

    PubMed

    Rychlik, Michael; Asam, Stefan

    2008-01-01

    The principle and applications of stable isotope dilution assays (SIDAs) in mycotoxin analysis are critically reviewed. The general section includes historical aspects of SIDAs, the prerequisites and limitations of the use of stable isotopically labelled internal standards, and possible calibration procedures. In the application section actual SIDAs for the analysis of trichothecenes, zearalenone, fumonisins, patulin, and ochratoxin A are presented. The syntheses and availability of labelled mycotoxins for use as internal standards is reviewed and specific advances in food analysis and toxicology are demonstrated. The review indicates that LC-MS applications, in particular, require the use of stable isotopically labelled standards to compensate for losses during clean-up and for discrimination due to ion suppression. As the commercial availability of these compounds continues to increase, SIDAs can be expected to find expanding use in mycotoxin analysis.

  13. A comparative analysis of water ice on the surface of comets Tempel 1 and 67P/Churyumov-Gerasimenko

    NASA Astrophysics Data System (ADS)

    Raponi, A.; Capaccioni, F.; De Sanctis, M. C.; Ciarniello, M.; Filacchione, G.; Tosi, F.; Palomba, E.; Capria, M. T.; Piccioni, G.; Cerroni, P.; Longobardo, A.; Migliorini, A.; Rosetta VIRTIS Team

    In this work we compare data of two spectrometers onboard space missions directed to comets: HRI (High Resolution Imager) on board Deep Impact \\citep{AHearn2005} which overflown Tempel 1 on 2005 July 4th, and VIRTIS (Visible InfraRd and Thermal Imaging Spectrometer) onboard Rosetta which nowadays is orbiting around Comet Churyumov-Gerasimenko \\citep{Coradini2007}. This work is focused on the detection of water ice on the surface, which seems to be present on both comets in two distinct modalities: - small grain size (1-2 mu m), as derived in the material ejected from the surface of Tempel 1 after the impact \\citep{Sunshine2007}, and on the surface of 67P/C-G as result of vapour recondensation \\citep{DeSanctis2015}. - large grain size (>30 mu m), in minor amounts, detected as exposed ice on both comets \\citep{Sunshine2006, Raponi2013, Filacchione2015}. These two modalities are related to different spectral features. To retrieve the physical properties of the surface we apply the Hapke scattering model to the measured spectra. The data are corrected for artifacts and thermal emission before comparing them with the model. Moreover the estimated signal to noise ratio is taken into account by a least square optimization algorithm in the fitting procedure. This comparative analysis could reveal common processes for comets, which have implication on their formation and evolution. Authors acknowledge the funding from Italian, French and German Space Agencies.

  14. Organics in comet 67P - a first comparative analysis of mass spectra from ROSINA-DFMS, COSAC and Ptolemy

    NASA Astrophysics Data System (ADS)

    Altwegg, Kathrin; Balsiger, H.; Berthelier, J. J.; Bieler, A.; Calmonte, U.; Fuselier, S. A.; Goesmann, F.; Gasc, S.; Gombosi, T. I.; Le Roy, L.; de Keyser, J.; Morse, A.; Rubin, M.; Schuhmann, M.; Taylor, M. G. G. T.; Tzou, C.-Y.; Wright, I.

    2017-07-01

    The ESA Rosetta spacecraft followed comet 67P at a close distance for more than 2 yr. In addition, it deployed the lander Philae on to the surface of the comet. The (surface) composition of the comet is of great interest to understand the origin and evolution of comets. By combining measurements made on the comet itself and in the coma, we probe the nature of this surface material and compare it to remote sensing observations. We compare data from the double focusing mass spectrometer (DFMS) of the ROSINA experiment on ESA's Rosetta mission and previously published data from the two mass spectrometers COSAC (COmetary Sampling And Composition) and Ptolemy on the lander. The mass spectra of all three instruments show very similar patterns of mainly CHO-bearing molecules that sublimate at temperatures of 275 K. The DFMS data also show a great variety of CH-, CHN-, CHS-, CHO2- and CHNO-bearing saturated and unsaturated species. Methyl isocyanate, propanal and glycol aldehyde suggested by the earlier analysis of the measured COSAC spectrum could not be confirmed. The presence of polyoxymethylene in the Ptolemy spectrum was found to be unlikely. However, the signature of the aromatic compound toluene was identified in DFMS and Ptolemy data. Comparison with remote sensing instruments confirms the complex nature of the organics on the surface of 67P, which is much more diverse than anticipated.

  15. Trajectory analysis for the nucleus and dust of comet C/2013 A1 (Siding Spring)

    SciTech Connect

    Farnocchia, Davide; Chesley, Steven R.; Chodas, Paul W.; Tricarico, Pasquale; Kelley, Michael S. P.; Farnham, Tony L.

    2014-08-01

    Comet C/2013 A1 (Siding Spring) will experience a high velocity encounter with Mars on 2014 October 19 at a distance of 135,000 km ± 5000 km from the planet center. We present a comprehensive analysis of the trajectory of both the comet nucleus and the dust tail. The nucleus of C/2013 A1 cannot impact on Mars even in the case of unexpectedly large nongravitational perturbations. Furthermore, we compute the required ejection velocities for the dust grains of the tail to reach Mars as a function of particle radius and density and heliocentric distance of the ejection. A comparison between our results and the most current modeling of the ejection velocities suggests that impacts are possible only for millimeter to centimeter size particles released more than 13 AU from the Sun. However, this level of cometary activity that far from the Sun is considered extremely unlikely. The arrival time of these particles spans a 20-minute time interval centered at 2014 October 19 at 20:09 TDB, i.e., around the time that Mars crosses the orbital plane of C/2013 A1. Ejection velocities larger than currently estimated by a factor >2 would allow impacts for smaller particles ejected as close as 3 AU from the Sun. These particles would reach Mars from 19:13 TDB to 20:40 TDB.

  16. Evaluation of the mutagenicity and genotoxic potential of carvacrol and thymol using the Ames Salmonella test and alkaline, Endo III- and FPG-modified comet assays with the human cell line Caco-2.

    PubMed

    LLana-Ruiz-Cabello, Maria; Maisanaba, Sara; Puerto, Maria; Prieto, Ana I; Pichardo, Silvia; Jos, Ángeles; Cameán, Ana M

    2014-10-01

    Currently, direct antimicrobial and antioxidant additives derived from essential oils are used in food packaging and are perceived by consumers as low-health-risk compounds. In this study, we investigated the potential mutagenicity and genotoxicity of carvacrol and thymol, major compounds in several essential oils, using the Ames Salmonella test and the alkaline, Endo III- and formamidopyrimidine glycosylase (FPG)-modified comet assays, respectively. Thymol did not show any mutagenic activity at any concentration assayed (0-250 μM), whereas carvacrol exhibited mutagenic potential, displaying greater activity in presence of the metabolic fraction (29-460 μM). The genotoxic effects were evaluated in the human colon carcinoma cell line Caco-2, and the standard comet assay revealed that neither carvacrol (0-460 μM) nor thymol (0-250 μM) had any affects at 24 and 48 h. The FPG-modified comet assay showed that the highest concentration of carvacrol (460 μM) caused DNA damage, indicating damage to the purine bases. These results should be used to identify the appropriate concentrations of carvacrol and thymol as additives in food packaging. Moreover, further studies are necessary to explore the safety and/or the toxicity mechanisms of these compounds. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Genotoxicity of the herbicide imazethapyr in mammalian cells by oxidative DNA damage evaluation using the Endo III and FPG alkaline comet assays.

    PubMed

    Soloneski, Sonia; Ruiz de Arcaute, Celeste; Nikoloff, Noelia; Larramendy, Marcelo L

    2017-03-07

    We evaluated the role of oxidative stress in the genotoxic damage induced by imazethapyr (IMZT) and its formulation Pivot® in mammalian CHO-K1 cell line. Using the alkaline comet assay, we observed that a concentration of 0.1 μg/mL of IMZT or Pivot® was able to induce DNA damage by increasing the frequency of damaged nucleoids. To test whether the DNA lesions were caused by oxidative stress, the DNA repair enzymes endonuclease III (Endo III) and formamidopyrimidine-DNA glycosylase (Fpg), which convert base damage to strand breaks, were used. Our results demonstrate that after treatment of CHO-K1 cells with the pure active ingredient as well as the commercial formulation Pivot®, an increase in DNA strand breaks was observed after incubation of both Endo III and Fpg enzymes, indicating that both compounds induce DNA damage involving both pyrimidine and purine-based oxidations, at least in CHO-K1 cells. Our findings confirm the genotoxic potential of IMZT and suggest that this herbicide formulation must be employed with great caution, especially not only for exposed occupational workers but also for other living species.

  18. Use of DNA strand damage (Comet assay) and embryo hatching effects to assess contaminant exposure in blue crab (Callinectes sapidus) embryos

    SciTech Connect

    Lee, R.F.; Steinert, S.A.; Nakayama, K.; Oshima, Y.

    1999-07-01

    After fertilization, blue crab eggs are embedded in a sponge which is attached to the female abdomen during embryo development. Embryos after 9 stages in the egg sac hatch into a swimming zoea stage (stage 10). The authors have developed a bioassay where embryo development is monitored in culture plates with and without toxicants in the water. Toxicant effects are based on determining the percentage of embryos which hatch to zoea. Hatching EC{sub 50} (toxicant concentration at which 50% of the embryos fail to hatch) for a number of pesticides, organometallics and metals were determined. The test takes from 2 to 6 days depending on the embryo stage selected for the study. In addition to embryo development effects the prevalence of DNA single-strand breaks in individual embryo cells were determined using the single cell gel electrophoresis method (Comet assay). A good correlation between DNA strand breakage and embryo defects was found after exposure to genotoxic contaminants. Thus, the bioassay linking DNA damage to embryo hatching effects is rapid, sensitive and mechanistically relevant.

  19. Assessment of the genotoxic potential along the Danube River by application of the comet assay on haemocytes of freshwater mussels: The Joint Danube Survey 3.

    PubMed

    Kolarević, Stoimir; Kračun-Kolarević, Margareta; Kostić, Jovana; Slobodnik, Jaroslav; Liška, Igor; Gačić, Zoran; Paunović, Momir; Knežević-Vukčević, Jelena; Vuković-Gačić, Branka

    2016-01-01

    In this study we assessed the level of genotoxic pollution along the Danube River by measuring the level of DNA damage in the haemocytes of freshwater mussels of Unio sp. (Unio pictorum/Unio tumidus) and Sinanodonta woodiana. The comet assay was used for the assessment of DNA damage. The research was performed on 34 out of 68 sites analysed within the Joint Danube Survey 3 - the world's biggest river research expedition of its kind in 2013. During research, 2285 river kilometres were covered with an average distance of 68 km between the sites. The complex data set on concentrations of various substances present in water, suspended particulate matter and sediment on investigated sites gave the opportunity to identify the groups of xenobiotics which mostly affect the studied biomarker - DNA damage. The highest levels of DNA damage were recorded in the section VI (Panonnian Plain), which is under the impact of untreated wastewater discharges. Both positive and negative influences of the large tributaries on the level of genotoxicity in the Danube River were evident. Significant correlation in response was detected between the studied species of freshwater mussels. The level of DNA damage in mussels correlated with concentrations of compounds from the group of hazardous priority substances (polycyclic aromatic hydrocarbons), persistent organic pollutants (dioxins) and emerging pollutants (Oxazepam, Chloridazon-desphenyl).

  20. The effect of insecticides chlorpyrifos, α-cypermethrin and imidacloprid on primary DNA damage, TP 53 and c-Myc structural integrity by comet-FISH assay.

    PubMed

    Zeljezic, Davor; Vinkovic, Benjamin; Kasuba, Vilena; Kopjar, Nevenka; Milic, Mirta; Mladinic, Marin

    2017-09-01

    In parallel with the continuous use of conventional insecticides, introduction of more environmentally friendly substances continues to grow in modern agriculture. In the present study, we evaluated chlorpyrifos, and imidacloprid and α-cypermethrin as two representatives of green insecticides for their genotoxic activity. We conducted a 14-day treatment in extended human lymphocytes cultures using real life exposure relevant concentrations. An alkaline comet assay was used to detect primary DNA damage. Simultaneously, the effect on the specific action towards the TP 53 and c-Myc genes in terms of fragmentation and copy number were determined. Both genes are responsible for cell cycle regulation; thus playing an active role in carcinogenesis. Contrary to what was expected, imidacloprid showed the highest genotoxicity potential, irrespective of the fact that none of the insecticides induced a significant level of primary DNA damage at all tested concentrations. Similar, no significant effect towards the TP 53 and c-Myc gene was recorded. The present study indicates that low level use of chlorpyrifos as a conventional insecticide and imidacloprid and α-cypermethrin as green insecticides does not pose a risk to DNA in general, nor to the TP 53 and c-Myc gene structural integrity. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Iron oxide nanoparticles show no toxicity in the comet assay in lymphocytes: A promising vehicle as a nitric oxide releasing nanocarrier in biomedical applications

    NASA Astrophysics Data System (ADS)

    de Lima, R.; Oliveira, J. L.; Murakami, P. S. K.; Molina, M. A. M.; Itri, R.; Haddad, P.; Seabra, A. B.

    2013-04-01

    This work reports the synthesis and toxicological evaluation of surface modified magnetic iron oxide nanoparticles as vehicles to carry and deliver nitric oxide (NO). The surface of the magnetic nanoparticles (MNPs) was coated with two thiol-containing hydrophilic ligands: mercaptosuccinic acid (MSA) or dimercaptosuccinic acid (DMSA), leading to thiolated MNPs. Free thiols groups on the surface of MSA- or DMSA-MNPs were nitrosated leading to NO-releasing MNPs. The genotoxicity of thiolated-coated MNPs was evaluated towards human lymphocyte cells by the comet assay. No genotoxicity was observed due to exposure of human lymphocytes to MSA- or DMSA-MNPs, indicating that these nanovectors can be used as inert vehicles in drug delivery, in biomedical applications. On the other hand, NO-releasing MPNs showed genotoxicity and apoptotic activities towards human lymphocyte cell cultures. These results indicate that NO-releasing MNPs may result in important biomedical applications, such as the treatment of tumors, in which MNPs can be guided to the target site through the application of an external magnetic field, and release NO directly to the desired site of action.

  2. Validation of Comet assay in Oregon-R and Wild type strains of Drosophila melanogaster exposed to a natural radioactive environment in Brazilian semiarid region.

    PubMed

    Verçosa, Cícero Jorge; Moraes Filho, Aroldo Vieira de; Castro, Ícaro Fillipe de Araújo; Santos, Robson Gomes Dos; Cunha, Kenya Silva; Silva, Daniela de Melo E; Garcia, Ana Cristina Lauer; Navoni, Julio Alejandro; Amaral, Viviane Souza do; Rohde, Claudia

    2017-07-01

    Natural radiation of geological origin is a common phenomenon in Brazil, a country where radioactive agents such as uranium may be often found. As an unstable atom, uranium undergoes radioactive decay with the generation of a series of decay by-products, including radon, which may be highly genotoxic and trigger several pathological processes, among which cancer. Because it is a gas, radon may move freely between cracks and gaps in the ground, seeping upwards into the buildings and in the environment. In this study, two Drosophila melanogaster Meigen (Diptera, Drosophilidae) strains called Oregon-R and Wild (collected in a non-radioactive environment) were exposed to atmospheric radiation in the Lajes Pintadas city, in the semiarid zone of northeastern Brazil. After six days of environmental exposure, the organisms presented genetic damage significantly higher than that of the negative control group. The genotoxic effects observed reinforce the findings of other studies carried out in the same region, which warn about the environmental risks related to natural radioactivity occurrence. The results also validate the use of the Comet assay in hemocytes of D. melanogaster as a sensitive test to detect genotoxicity caused by natural radiation, and the use of a recently collected D. melanogaster strain in the environmental of radon. Copyright © 2017. Published by Elsevier Inc.

  3. Physical Exercise and Redox Balance in Type 2 Diabetics: Effects of Moderate Training on Biomarkers of Oxidative Stress and DNA Damage Evaluated through Comet Assay

    PubMed Central

    Pittaluga, Monica; Sgadari, Antonio; Dimauro, Ivan; Tavazzi, Barbara; Parisi, Paolo; Caporossi, Daniela

    2015-01-01

    Objective. Hyperglycemia leads to increased production of reactive oxygen species (ROS) in type 2 diabetes, which reduces cellular antioxidant defenses and induces DNA lesions. The aim of this study was to investigate the effects on redox homeostasis and DNA oxidative damage of exercise training in patients with type 2 diabetes compared with nondiabetic individuals. Methods and Results. 12 sedentary type 2 diabetic males (62.1 ± 4.3 yrs) and 12 sedentary healthy males (61.7 ± 3.9 yrs) were exposed to 4-month moderate training, 3 times per week, to evaluate the effect on plasma biomarkers of oxidative stress malondialdehyde and antioxidant status (GSSG, GSH/GSSG, and ascorbic acid) as well as basal and H2O2-induced DNA damage trough alkaline comet assay in peripheral blood lymphocytes. After training, glutathione and ascorbic acid levels increased in both groups, but only in diabetics the malondialdehyde as well as the DNA damage decreased. Conclusion. Our study demonstrates for the first time that moderate exercise training is not only effective in improving the redox homeostasis, through an increase of the endogenous antioxidant defences in healthy as well as in diabetic patients, but also, specifically in diabetic patients, effective in lowering the susceptibility to oxidative DNA damage and the lipid peroxidation levels. PMID:25789083

  4. Physical exercise and redox balance in type 2 diabetics: effects of moderate training on biomarkers of oxidative stress and DNA damage evaluated through comet assay.

    PubMed

    Pittaluga, Monica; Sgadari, Antonio; Dimauro, Ivan; Tavazzi, Barbara; Parisi, Paolo; Caporossi, Daniela

    2015-01-01

    Hyperglycemia leads to increased production of reactive oxygen species (ROS) in type 2 diabetes, which reduces cellular antioxidant defenses and induces DNA lesions. The aim of this study was to investigate the effects on redox homeostasis and DNA oxidative damage of exercise training in patients with type 2 diabetes compared with nondiabetic individuals. 12 sedentary type 2 diabetic males (62.1 ± 4.3 yrs) and 12 sedentary healthy males (61.7 ± 3.9 yrs) were exposed to 4-month moderate training, 3 times per week, to evaluate the effect on plasma biomarkers of oxidative stress malondialdehyde and antioxidant status (GSSG, GSH/GSSG, and ascorbic acid) as well as basal and H2O2-induced DNA damage trough alkaline comet assay in peripheral blood lymphocytes. After training, glutathione and ascorbic acid levels increased in both groups, but only in diabetics the malondialdehyde as well as the DNA damage decreased. Our study demonstrates for the first time that moderate exercise training is not only effective in improving the redox homeostasis, through an increase of the endogenous antioxidant defences in healthy as well as in diabetic patients, but also, specifically in diabetic patients, effective in lowering the susceptibility to oxidative DNA damage and the lipid peroxidation levels.

  5. Comparison of post-thaw DNA integrity of boar spermatozoa assessed with the neutral comet assay and Sperm-Sus Halomax test kit.

    PubMed

    Fraser, L; Parda, A; Filipowicz, K; Strzeżek, J

    2010-10-01

    In this study, we tested the hypothesis whether the neutral Comet assay (NCA) and the Sperm-Sus-Halomax (SSH) test kit could provide similar measurements of post-thaw DNA fragmentation of boar spermatozoa. Whole ejaculates or sperm-rich fractions of boar semen were frozen in an extender containing lactose, lipoprotein fractions isolated from ostrich egg yolk (LPFo), glycerol (lactose-LPFo-G) or in a standard boar semen extender (K3), without the addition of cryoprotective substances. In all boars, both the NCA and SSH test showed similar levels of post-thaw sperm DNA fragmentation in samples of the same ejaculates, regardless of the ejaculate collection procedure and extender. Yet, the levels of post-thaw sperm DNA damage, detected by the NCA and SSH test, were more accentuated in spermatozoa frozen in the absence of cryoprotective substances. Both the NCA and SSH detected variations among individual boars in terms of post-thaw sperm DNA fragmentation. Agreement between the measurements of the NCA and SSH was confirmed by scatter plots of differences, suggesting that the DNA integrity tests could detect the same sperm populations, which were susceptible to cryo-induced DNA damage. The findings of this study indicate that the NCA and the SSH test are effective in detecting similar levels of sperm DNA fragmentation and reinforce their importance in the assessment of frozen-thawed boar semen quality.

  6. Radiation-induced DNA double-strand breaks produced in histone-depleted tumor cell nuclei measured using the neutral comet assay

    SciTech Connect

    Olive, P.L.; Banath, J.P.

    1995-05-01

    Removal of histones and other nuclear proteins greatly enhances the sensitivity of mammalian cells to DNA damage by ionizing radiation. We examined the possibility that the ease of dissociation of histones, or the association of other nuclear proteins with DNA, may differ between radioresistant and sensitive human tumor cells. Cells embedded in agarose were exposed to increasing salt concentrations prior to irradiation and examination using a microscopic gel electrophoresis method, the neutral comet assay. Induction of double-strand breaks increased by a factor of about 20 when cells of four human tumor cell line HT144 melanoma, HT29 adenocarcinoma, DU145 prostate carcinoma and U87 glioma, were exposed to 2 M NaCl; however, no correlation with radiosensitivity was apparent. While a significant number of histone and non-histone proteins are present after extraction with 1.2 M NaCL, these proteins apparently have only a minor influence on radiosensitivity. However, if they are allowed to remain with DNA during electrophoresis, about 15 times more strand breaks are required to produce a similar amount of DNA migration in both DU145 and HT144 cells. These results suggest that the association between proteins and DNA within the nucleus, as probed by extraction with sodium chloride, does not help to explain differences in intrinsic radiosensitivity among cells of these diverse tumor cell lines. 33 refs., 11 figs.

  7. 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.

  8. Comet composition and Lab

    NASA Astrophysics Data System (ADS)

    Bockelée-Morvan, Dominique; Biver, Nicolas

    2016-10-01

    Comet composition and properties provide information on chemical and physical processes that occurred in the early Solar system, 4.6 Gyr ago. The study of comets and of star-forming regions both help for a better understanding of the formation of planetary systems. A review of our present knowledge of cometary composition is presented. We also discuss laboratory studies that would be helpful for data analysis.

  9. Halley's Comet.

    ERIC Educational Resources Information Center

    Carey, Tom

    1985-01-01

    Provides tips for viewing Comet Halley in the Northeast including best viewing dates from November 1985-January 1986. Discusses going south to view the comet in March-April 1986 and gives specific information about accommodations for the Halley Rally in Everglades National Park, southernmost site in the contiguous 48 states. (JHZ)

  10. Halley's Comet.

    ERIC Educational Resources Information Center

    Carey, Tom

    1985-01-01

    Provides tips for viewing Comet Halley in the Northeast including best viewing dates from November 1985-January 1986. Discusses going south to view the comet in March-April 1986 and gives specific information about accommodations for the Halley Rally in Everglades National Park, southernmost site in the contiguous 48 states. (JHZ)

  11. A new analysis of Spitzer observations of Comet 29P/Schwassmann-Wachmann 1

    NASA Astrophysics Data System (ADS)

    Schambeau, Charles A.; Fernández, Yanga R.; Lisse, Carey M.; Samarasinha, Nalin; Woodney, Laura M.

    2015-11-01

    We present a new analysis of Spitzer observations of Comet 29P/Schwassmann-Wachmann 1 taken on UT 2003 November 21, 23, and 24, similar to a previous investigation of the observations (Stansberry et al., 2004), but using the most recent Spitzer data pipeline products and intensive image processing techniques. Analysis of images from the IRAC 5.8 and 8.0 μ m bands and the MIPS 24.0 and 70.0 μ m bands resulted in photometry measurements of the nucleus after a suite of coma modeling and removal processes were implemented. SW1 was not identified in the 5.8 μ m image from the previous work so its incorporation into this analysis is entirely new. Using the Near Earth Asteroid Thermal Model (Harris, 1998) resulted in a nucleus radius measurement of R =30.2-2.9+3.7 km and an infrared beaming parameter value of η =0.99-0.19+0.26 . We also measured an infrared geometric albedo, p5.8 = 0.5 ± 0.5 . Extrapolating a 0.04 V-band albedo and using a normalized reflectivity gradient S‧ = 14.94 ± 1.09 [% (1000 Å)-1] (Duffard, R., et al. [2014]. Astron. Astrophys. 564, A92) we recover an infrared albedo of p5.8 = 0.31 in the near infrared consistent with the value recovered from thermal modeling. The dust composition extracted from IRS spectra are very comet-like, containing mainly amorphous ferromagnesian silicates (but with a minority of crystalline silicates as well), water ice, and metal sulfides.

  12. The World of Comets

    NASA Astrophysics Data System (ADS)

    Guillemin, Amédée; Glaisher, James

    2010-10-01

    1. Beliefs and superstitions relative to comets; 2. Cometary astronomy up to the time of Newton; 3. The motions and orbits of comets; 4. Periodical comets; 5. Periodical comets; 6. The world of comets and cometary systems; 7. Physical and chemical constitution of comets; 8. Physical transformations of comets; 9. Mass and density of comets; 10. The light of comets; 11. Theory of cometary phenomena; 12. Comets and shooting stars; 13. Comets and the earth; 14. Physical influences of comets; 15. Some questions about comets; Tables.

  13. 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

    -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

  14. Comet fluorescence in situ hybridization analysis for oxidative stress-induced DNA damage in colon cancer relevant genes.

    PubMed

    Glei, Michael; Schaeferhenrich, Anja; Claussen, Uwe; Kuechler, Alma; Liehr, Thomas; Weise, Anja; Marian, Brigitte; Sendt, Wolfgang; Pool-Zobel, Beatrice L

    2007-04-01

    Our objective was to study whether products of oxidative stress, such as hydrogen peroxide (H(2)O(2)), trans-2-hexenal, and 4-hydroxy-2-nonenal (HNE), cause DNA damage in genes, relevant for human colon cancer. For this, total DNA damage was measured in primary human colon cells and colon adenoma cells (LT97) using the single-cell gel electrophoresis assay, known as "Comet Assay." APC, KRAS, and TP53 were marked in the comet images using fluorescence in situ hybridization (Comet FISH). The migration of APC, KRAS, or TP53 signals into the comet tails was quantified and compared to total DNA damage. All three substances were clearly genotoxic for APC, KRAS, and TP53 genes and total DNA in both types of cells. In primary colon cells, TP53 gene was more sensitive toward H(2)O(2), trans-2-hexenal, and HNE than total DNA was. In LT97 cells, the TP53 gene was more sensitive only toward trans-2-hexenal and HNE. APC and KRAS genes were more susceptible than total DNA to both lipid peroxidation products but only in primary colon cells. This suggests genotoxic effects of lipid peroxidation products in APC, KRAS, and TP53 genes. In LT97 cells, TP53 was more susceptible than APC and KRAS toward HNE. Based on the reported gatekeeper properties of TP53, which in colon adenoma is frequently altered to yield carcinoma, this implies that HNE is likely to contribute to cancer progression. This new experimental approach facilitates studies on effects of nutrition-related carcinogens in relevant target genes.

  15. Low-thrust mission risk analysis, with application to a 1980 rendezvous with the comet Encke

    NASA Technical Reports Server (NTRS)

    Yen, C. L.; Smith, D. B.

    1973-01-01

    A computerized failure process simulation procedure is used to evaluate the risk in a solar electric space mission. The procedure uses currently available thrust-subsystem reliability data and performs approximate simulations of the thrust sybsystem burn operation, the system failure processes, and the retargeting operations. The method is applied to assess the risks in carrying out a 1980 rendezvous mission to the comet Encke. Analysis of the results and evaluation of the effects of various risk factors on the mission show that system component failure rates are the limiting factors in attaining a high mission relability. It is also shown that a well-designed trajectory and system operation mode can be used effectively to partially compensate for unreliable thruster performance.

  16. COSIMA - Cometary Dust Analysis in the inner coma of Comet 67P/Churyumov-Gerasimenko

    NASA Astrophysics Data System (ADS)

    Hilchenbach, Martin; Kissel, Jochen; Briois, Christelle; von Hoerner, Hanna; Langevin , Yves; Schulz, Rita; Silen, Johan; Altwegg, Kathrin; Colangeli, Luigi; Cottin, Herve; Engrand, Cecile; Glasmachers, Albrecht; Gruen, Eberhard; Haerendel, Gerhard; Henkel, Hartmut; Höfner, Herwig; Hornung, Klaus; Jessberger, Elmar; Koch, Andreas; Letho, Harry; Letho, Kirsi; Raulin, Francois; Le Roy, Lena; Rynö, Jouni; Steiger , Wolfgang; Stephan , Thomas; Laurent, Thirkell; Thomas, Roger.; Torkar, Klaus; Varmuza, Kurt; Wanczek, Klaus Peter

    2014-11-01

    After a long journey through the inner solar system, ESA’s corner stone mission ROSETTA has arrived at comet 67P/Churyumov-Gerasimenko. COSIMA or the COmetary Secondary Ion Mass Analyzer onboard ROSETTA is a secondary ion mass spectrometer focussing on in-situ measurements of the composition of cometary grains collected near the nucleus and inner coma. High resolution mass spectra will contain ions of complex mixtures of mineral compounds and organic molecules as well as molecular fragments representing the elements and molecules on the surface of the cometary grains. We will report on our envisaged in-situ analysis goals of cometary grains as captured, imaged and analysed by COSIMA.

  17. Photometrical analysis of the neck-line structure of Comet Halley

    SciTech Connect

    Cremonese, G.; Fulle, M.; Osservatorio Astronomico, Trieste )

    1989-08-01

    The Fulle and Sedmak (1988) quantitative analysis of Neck-Line Structures (NLSs) is presently applied to the dust streamer and the real antitail noted in images obtained for Comet Halley during April-June, 1986. Results for the dust size distribution at very large sizes, the time-dependent dust velocity ejection velocity from the inner coma, and the anisotropy of dust are related to the January 10-18, 1986 interval. The power index of the size distribution fitting the NLS isophotes is -4.0 for 0.2-2.0 mm, and -0.4 for 2-10 mm; the strong excess of large dust suggested by McDonnell, et al. (1987) to account for the Giotto data is therefore confirmed. 18 refs.

  18. A Comet's Missing Light

    NASA Astrophysics Data System (ADS)

    Kohler, Susanna

    2016-05-01

    On 28 November 2013, comet C/2012 S1 better known as comet ISON should have passed within two solar radii of the Suns surface as it reached perihelion in its orbit. But instead of shining in extreme ultraviolet (EUV) wavelengths as it grazed the solar surface, the comet was never detected by EUV instruments. What happened to comet ISON?Missing EmissionWhen a sungrazing comet passes through the solar corona, it leaves behind a trail of molecules evaporated from its surface. Some of these molecules emit EUV light, which can be detected by instruments on telescopes like the space-based Solar Dynamics Observatory (SDO).Comet ISON, a comet that arrived from deep space and was predicted to graze the Suns corona in November 2013, was expected to cause EUV emission during its close passage. But analysis of the data from multiple telescopes that tracked ISON in EUV including SDO reveals no sign of it at perihelion.In a recent study, Paul Bryans and DeanPesnell, scientists from NCARs High Altitude Observatory and NASA Goddard Space Flight Center, try to determine why ISON didnt display this expected emission.Comparing ISON and LovejoyIn December 2011, another comet dipped into the Suns corona: comet Lovejoy. This image, showingthe orbit Lovejoy took around the Sun, is a composite of SDO images of the pre- and post-perihelion phases of the orbit. Click for a closer look! The dashed part of the curve represents where Lovejoy passed out of view behind the Sun. [Bryans Pesnell 2016]This is not the first time weve watched a sungrazing comet with EUV-detecting telescopes: Comet Lovejoy passed similarly close to the Sun in December 2011. But when Lovejoy grazed the solar corona, it emitted brightly in EUV. So why didnt ISON? Bryans and Pesnell argue that there are two possibilities:the coronal conditions experienced by the two comets were not similar, orthe two comets themselves were not similar.To establish which factor is the most relevant, the authors first demonstrate that both

  19. Low-frequency electromagnetic plasma waves at comet P/Grigg-Skjellerup: Analysis and interpretation

    NASA Technical Reports Server (NTRS)

    Neubauer, Fritz M.; Glassmeier, Karl-Heinz; Coates, A. J.; Johnstone, A. D.

    1993-01-01

    The propagation and polarization characteristic of low-frequency electromagnetic wave fields near comet P/Grigg-Skjellerup (P/GS) are analyzed using magnetic field and plasma observations obtained by the Giotto magnetometer experiment and the Johnstone plasma analyzer during the encounter at the comet on July 10, 1992. The results have been physically interpreted.

  20. Comet Odyssey: Comet Nucleus Orbiter

    NASA Astrophysics Data System (ADS)

    Weissman, P. R.; Smythe, W. D.; Spitz, S. J.; Bernard, D. E.; Bailey, R. W.

    2004-11-01

    Comet Odyssey is a comet nucleus orbiter mission, proposed to NASA's Discovery program in 2004. The goal of the mission is to completely characterize a cometary nucleus, both physically and compositionally, as can only be done during an extended rendezvous and not with a fast flyby. Comet Odyssey will launch in October 2009 on a Delta II 7925 and use a solar-electric powered spacecraft to effect a rendezvous with periodic comet 46P/Wirtanen in October 2013. Arrival is 96 days after perihelion at a heliocentric distance of 1.61 AU. Comet Odyssey's science payload includes narrow- and wide-angle CCD cameras, an infrared thermal imager, a gas chromatograph/mass spectrometer, an XRD/XRF dust compositional analyzer, and a dust counter and accumulation sensors. The Comet Odyssey spacecraft implementation uses a high heritage approach of flight proven and redundant hardware. The 3-engine ion propulsion subsystem is derived from that on Dawn but includes the capability for multi-engine thrusting. Comet Odyssey will approach the Wirtanen nucleus and make repeated slow flybys through the active cometary coma for a period of three months. It will then be placed in a ˜100-km radius orbit around the nucleus, with a plan to eventually orbit at 40-km altitude or less. From that altitude the narrow-angle camera will map the entire nucleus surface at 1 meter/pixel and the thermal imager will map at 19 meter/pixel. The orbital portion of the nominal mission will last 4.5 months, following the comet outward from the Sun to 3.3 AU as the comet evolves from an active to a quiescent state. En route to P/Wirtanen, the