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...

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 (H 2 O 2 ) 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 H 2 O 2 , 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.V. All rights reserved.

The comet moment as a measure of DNA damage in the comet assay.

PubMed

Kent, C R; Eady, J J; Ross, G M; Steel, G G

1995-06-01

The development of rapid assays of radiation-induced DNA damage requires the definition of reliable parameters for the evaluation of dose-response relationships to compare with cellular endpoints. We have used the single-cell gel electrophoresis (SCGE) or 'comet' assay to measure DNA damage in individual cells after irradiation. Both the alkaline and neutral protocols were used. In both cases, DNA was stained with ethidium bromide and viewed using a fluorescence microscope at 516-560 nm. Images of comets were stored as 512 x 512 pixel images using OPTIMAS, an image analysis software package. Using this software we tested various parameters for measuring DNA damage. We have developed a method of analysis that rigorously conforms to the mathematical definition of the moment of inertia of a plane figure. This parameter does not require the identification of separate head and tail regions, but rather calculates a moment of the whole comet image. We have termed this parameter 'comet moment'. This method is simple to calculate and can be performed using most image analysis software packages that support macro facilities. In experiments on CHO-K1 cells, tail length was found to increase linearly with dose, but plateaued at higher doses. Comet moment also increased linearly with dose, but over a larger dose range than tail length and had no tendency to plateau.

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

PubMed

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

2014-01-01

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

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.

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

PubMed

McNamee, J P; Bellier, P V

2015-07-01

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

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

PubMed

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

2014-05-01

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

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

PubMed

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

2015-07-01

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

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

PubMed

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

2016-08-01

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

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

PubMed Central

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

2014-01-01

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

A quantitative comet infection assay for influenza virus

PubMed Central

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

2011-01-01

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

The effect of flash-freezing temperature on stallion sperm DNA structure.

PubMed

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

2017-06-01

The effect of flash-freezing storage temperature on stallion sperm DNA has not been evaluated. Commonly, sperm are flash-frozen at various temperatures to preserve sperm DNA prior to analysis. It is unclear whether the temperature at which sperm are frozen and stored may affect the results of DNA assays. In this study, the neutral comet assay was used to evaluate the effect of flash-freezing storage temperature (freezer [-60 °C], dry ice [-78.5 °C], liquid nitrogen [-196 °C]) compared to fresh sperm DNA structure. In addition, intra- and inter-assay and intra- and inter-stallion variabilities were determined. All comet tail measures were higher following any flash-freezing method, as compared to fresh sperm DNA (P < 0.05), with no difference among flash-frozen treatments (P > 0.05). For most comet variables, intra- and inter-assay variabilities were <10%. Intra- and inter-stallion variabilities revealed that comet head length (HL) and width (CW) were less variable as compared to comet tail values, i.e., % comet tail DNA (T-DNA), tail length (TL), tail moment (OTM), and tail migration (TM). Certain comet tail values in fresh (% T-DNA, and OTM) and flash-frozen sperm (OTM, % T-DNA, TL, and TM) were correlated to the Sperm Chromatin Structure Assay (SCSA) variable, COMP-α t . The comet tail measures were negatively correlated to % morphologically normal sperm (P < 0.05) and positively correlated to % abnormal heads and premature germ cells (P < 0.05). Variables COMP-α t and % total sperm motility were not correlated to any morphologic sperm feature in this group of stallions (P > 0.05). While significant differences in the structure of the sperm DNA were identified in the flash-frozen as compared to the fresh sperm DNA with the neutral comet assay, it cannot be assumed that these changes are fertility limiting. Copyright © 2017. Published by Elsevier Inc.

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

PubMed

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

2018-03-01

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

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

PubMed

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

2017-01-01

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

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

PubMed

Gajski, Goran; Garaj-Vrhovac, Vera

2008-01-01

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

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

PubMed

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

2008-08-15

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

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

PubMed

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

2017-05-01

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

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

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

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

2008-08-15

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

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

PubMed

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

2016-01-01

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

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

2018-01-01

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.

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

PubMed Central

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

2014-01-01

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

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

PubMed

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

2014-01-01

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

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

PubMed

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

2015-03-01

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

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

PubMed

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

2014-07-01

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

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

PubMed

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

2014-12-01

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

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

PubMed

Bajpayee, Mahima; Kumar, Ashutosh; Dhawan, Alok

2013-01-01

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

Recommendations for safety testing with the in vivo comet assay.

PubMed

Vasquez, Marie Z

2012-08-30

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

The comet assay: ready for 30 more years.

PubMed

Møller, Peter

2018-02-24

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

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

PubMed

Frötschl, Roland

2015-01-01

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

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

PubMed

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

2014-11-01

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

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

PubMed

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

2014-03-01

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

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

PubMed Central

Karbaschi, Mahsa; Cooke, Marcus S.

2014-01-01

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

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

PubMed

Karbaschi, Mahsa; Cooke, Marcus S

2014-11-26

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

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

DNA comet Giemsa staining for conventional bright-field microscopy.

PubMed

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

2014-04-10

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

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

PubMed

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

2017-03-01

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

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.

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

PubMed Central

2014-01-01

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

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

PubMed

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

2015-07-01

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

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.

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

PubMed

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

2016-07-01

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

Alkaline Comet Assay for Assessing DNA Damage in Individual Cells.

PubMed

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

2015-08-06

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

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. © 2012 Elsevier B.V. All rights reserved.

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

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

PubMed Central

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

2016-01-01

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

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

PubMed

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

2016-05-04

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

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.

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

PubMed

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

2018-01-01

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

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

PubMed

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

2017-08-01

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

Epithelial cells as alternative human biomatrices for comet assay.

PubMed

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

2014-01-01

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

Epithelial cells as alternative human biomatrices for comet assay

PubMed Central

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

2014-01-01

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

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

PubMed Central

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

2014-01-01

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

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

PubMed

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

2014-09-01

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

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

PubMed

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

2016-01-01

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

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.

Assessment of occupational genotoxic risk among Brazilian hairdressers.

PubMed

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

2008-10-01

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

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

PubMed

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

2001-01-01

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

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

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

PubMed

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

2014-11-23

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

Comet assay: an essential tool in toxicological research.

PubMed

Glei, M; Schneider, T; Schlörmann, W

2016-10-01

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

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

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

PubMed

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

2015-12-01

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

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

PubMed Central

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

2013-01-01

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

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

PubMed

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

2018-03-01

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

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

PubMed

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

2015-07-01

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

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

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

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.

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

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

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

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

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

PubMed

Augustyniak, Maria; Gladysz, Marcin; Dziewięcka, Marta

2016-01-01

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

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

PubMed

Speit, Günter; Schütz, Petra; Bausinger, Julia

2016-06-01

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

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

Evaluating In Vitro DNA Damage Using Comet Assay.

PubMed

Lu, Yanxin; Liu, Yang; Yang, Chunzhang

2017-10-11

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

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

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

PubMed

Martins, Marta; Costa, Pedro M

2015-01-01

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

Worldwide interest in the comet assay: a bibliometric study.

PubMed

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

2015-01-01

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

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

PubMed

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

2017-01-01

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

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

PubMed Central

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

2017-01-01

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

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).

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

PubMed

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

2017-03-01

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

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. Copyright © 2015 Elsevier Inc. All rights reserved.

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

PubMed

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

2013-05-01

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

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

PubMed

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

2015-05-01

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

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

PubMed

Bausinger, Julia; Speit, Günter

2014-11-01

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

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

PubMed

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

2013-01-20

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

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

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

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

1995-12-01

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

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

PubMed

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

2016-09-01

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

Anti-proliferative effect of biogenic gold nanoparticles against breast cancer cell lines (MDA-MB-231 & MCF-7)

NASA Astrophysics Data System (ADS)

K. S., Uma Suganya; Govindaraju, K.; Ganesh Kumar, V.; Prabhu, D.; Arulvasu, C.; Stalin Dhas, T.; Karthick, V.; Changmai, Niranjan

2016-05-01

Breast cancer is a major complication in women and numerous approaches are being developed to overcome this problem. In conventional treatments such as chemotherapy and radiotherapy the post side effects cause an unsuitable effect in treatment of cancer. Hence, it is essential to develop a novel strategy for the treatment of this disease. In the present investigation, a possible route for green synthesis of gold nanoparticles (AuNPs) using leaf extract of Mimosa pudica and its anticancer efficacy in the treatment of breast cancer cell lines is studied. The synthesized nanoparticles were found to be effective in killing cancer cells (MDA-MB-231 & MCF-7) which were studied using various anticancer assays (MTT assay, cell morphology determination, cell cycle analysis, comet assay, Annexin V-FITC/PI staining and DAPI staining). Cell morphological analysis showed the changes occurred in cancer cells during the treatment with AuNPs. Cell cycle analysis revealed apoptosis in G0/G1 to S phase. Similarly in Comet assay, there was an increase in tail length in treated cells in comparison with the control. Annexin V-FITC/PI staining assay showed prompt fluorescence in treated cells indicating the translocation of phosphatidylserine from the inner membrane. PI and DAPI staining showed the DNA damage in treated cells.

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

PubMed

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

2014-10-01

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

Genotoxicity of Water Contaminants from the Basin of Lake Sevan, Armenia Evaluated by the Comet Assay in Gibel Carp (Carassius auratus gibelio) and Tradescantia Bioassays.

PubMed

Simonyan, Anna; Gabrielyan, Barduch; Minasyan, Seyran; Hovhannisyan, Galina; Aroutiounian, Rouben

2016-03-01

Combination of bioassays and chemical analysis was applied to determine the genotoxic/mutagenic contamination in four different sites of the basin of Lake Sevan in Armenia. Water genotoxicity was evaluated using the single cell gel electrophoresis technique (comet assay) in erythrocytes of gibel carp (Carassius auratus gibelio), Tradescantia micronucleus (Trad-MCN) and Tradescantia stamen hair mutation (Trad-SHM) assays. Significant inter-site differences in the levels of water genotoxicity according to fish and Trad-MCN bioassays have been revealed. Two groups of locations with lower (south-southwest of the village Shorzha and Peninsula of Lake Sevan) and higher (estuaries of Gavaraget and Dzknaget rivers) levels of water genotoxicity were distinguished. Correlation analysis support the hypothesis that the observed genetic alterations in fish and plant may be a manifestation of the effects of water contamination by nitrate ions, Si, Al, Fe, Mn and Cu. Increase of DNA damage in fish also correlated with content of total phosphorus.

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

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

PubMed

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

2013-03-01

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

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

PubMed

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

2015-07-01

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

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

PubMed

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

2015-10-01

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

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

PubMed

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

2017-01-01

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

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

PubMed Central

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

2011-01-01

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

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

PubMed

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

2011-10-01

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

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

PubMed

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

2015-07-01

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

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

PubMed

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

2014-01-01

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

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

PubMed

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

2013-06-01

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

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

PubMed

Collins, Andrew R

2014-02-01

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

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

PubMed

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

2014-01-01

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

Comet Assay in Cancer Chemoprevention.

PubMed

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

2016-01-01

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

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.

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

PubMed

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

2014-03-01

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

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

PubMed

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

2017-01-01

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

[Essential oil from Artemisia lavandulaefolia induces apoptosis and necrosis of HeLa cells].

PubMed

Zhang, Lu-min; Lv, Xue-wei; Shao, Lin-xiang; Ma, Yan-fang; Cheng, Wen-zhao; Gao, Hai-tao

2013-12-01

To investigate the effects of Artemisia lavandulaefolia essential oil on apoptosis and necrosis of HeLa cells. Cell viability was assayed using MTT method. The morphological and structure alterations in HeLa cells were observed by microscopy. Furthermore, cell apoptosis was measured by DNA Ladder and flow cytometry. DNA damage was measured by comet assay, and the protein expression was examined by Western blot analysis. MTT assay displayed essential oil from Artemisia lavandulaefolia could inhibit the proliferation of HeLa cells in a dose-dependent manner. After treated with essential oil of Artemisia lavadulaefolia for 24 h, HeLa cells in 100 and 200 microg/mL experiment groups exhibited the typical morphology changes of undergoing apoptosis, such as cell shrinkage and nucleus chromatin condensed. However, the cells in the 400 microg/mL group showed the necrotic morphology changes including cytomembrane rupture and cytoplasm spillover. In addition, DNA Ladder could be demonstrated by DNA electrophoresis in each experiment group. Apoptosis peak was also evident in flow cytometry in each experiment group. After treating the HeLa cells with essential oil of Artemisia lavadulaefolia for 6 h, comet tail was detected by comet assay. Moreover, western blotting analysis showed that caspase-3 was activated and the cleavage of PARP was inactivated. Essential oil from Artemisia lavadulaefolia can inhibit the proliferation of HeLa cells in vitro. Low concentration of essential oil from Artemisia lavadulaefolia can induce apoptosis, whereas high concentration of the compounds result in necrosis of HeLa cells. And,the mechanism may be related to the caspase-3-mediated-PARP apoptotic signal pathway.

Measuring Sperm DNA Fragmentation and Clinical Outcomes of Medically Assisted Reproduction: A Systematic Review and Meta-Analysis.

PubMed

Cissen, Maartje; Wely, Madelon van; Scholten, Irma; Mansell, Steven; Bruin, Jan Peter de; Mol, Ben Willem; Braat, Didi; Repping, Sjoerd; Hamer, Geert

2016-01-01

Sperm DNA fragmentation has been associated with reduced fertilization rates, embryo quality, pregnancy rates and increased miscarriage rates. Various methods exist to test sperm DNA fragmentation such as the sperm chromatin structure assay (SCSA), the sperm chromatin dispersion (SCD) test, the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labelling (TUNEL) assay and the single cell gel electrophoresis (Comet) assay. We performed a systematic review and meta-analysis to assess the value of measuring sperm DNA fragmentation in predicting chance of ongoing pregnancy with IVF or ICSI. Out of 658 unique studies, 30 had extractable data and were thus included in the meta-analysis. Overall, the sperm DNA fragmentation tests had a reasonable to good sensitivity. A wide variety of other factors may also affect the IVF/ICSI outcome, reflected by limited to very low specificity. The constructed hierarchical summary receiver operating characteristic (HSROC) curve indicated a fair discriminatory capacity of the TUNEL assay (area under the curve (AUC) of 0.71; 95% CI 0.66 to 0.74) and Comet assay (AUC of 0.73; 95% CI 0.19 to 0.97). The SCSA and the SCD test had poor predictive capacity. Importantly, for the TUNEL assay, SCD test and Comet assay, meta-regression showed no differences in predictive value between IVF and ICSI. For the SCSA meta-regression indicated the predictive values for IVF and ICSI were different. The present review suggests that current sperm DNA fragmentation tests have limited capacity to predict the chance of pregnancy in the context of MAR. Furthermore, sperm DNA fragmentation tests have little or no difference in predictive value between IVF and ICSI. At this moment, there is insufficient evidence to recommend the routine use of sperm DNA fragmentation tests in couples undergoing MAR both for the prediction of pregnancy and for the choice of treatment. Given the significant limitations of the evidence and the methodological weakness and design of the included studies, we do urge for further research on the predictive value of sperm DNA fragmentation for the chance of pregnancy after MAR, also in comparison with other predictors of pregnancy after MAR.

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, ...

Irradiation influence on the detection of genetic-modified soybeans

NASA Astrophysics Data System (ADS)

Villavicencio, A. L. C. H.; Araújo, M. M.; Baldasso, J. G.; Aquino, S.; Konietzny, U.; Greiner, R.

2004-09-01

Three soybean varieties were analyzed to evaluate the irradiation influence on the detection of genetic modification. Samples were treated in a 60Co facility at dose levels of 0, 500, 800, and 1000Gy. The seeds were at first analyzed by Comet Assay as a rapid screening irradiation detection method. Secondly, germination test was performed to detect the viability of irradiated soybeans. Finally, because of its high sensitivity, its specificity and rapidity the polimerase chain reaction was the method applied for genetic modified organism detection. The analysis of DNA by the single technique of microgel electrophoresis of single cells (DNA Comet Assay) showed that DNA damage increased with increasing radiation doses. No negative influence of irradiation on the genetic modification detection was found.

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

PubMed

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

2010-05-01

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

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

PubMed

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

2011-12-01

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

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

PubMed

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

2004-02-01

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

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

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

PubMed

Singh, Narendra P

2016-01-01

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

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

PubMed

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

2017-05-01

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

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

PubMed Central

Vandghanooni, Somayeh; Eskandani, Morteza

2011-01-01

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

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

PubMed

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

2007-12-01

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

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

PubMed

Jost, Petr; Svobodova, Hana; Stetina, Rudolf

2015-07-25

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

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. © The Author 2016. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.

Occupational risk assessment of paint industry workers

PubMed Central

de Oliveira, Hugo M.; Dagostim, Gracilene P.; da Silva, Arielle Mota; Tavares, Priscila; da Rosa, Luiz A. Z. C.; de Andrade, Vanessa M.

2011-01-01

Background: Thousands of chemical compounds are used in paint products, like pigments, extenders, binders, additives, and solvents (toluene, xylene, ketones, alcohols, esters, and glycol ethers). Paint manufacture workers are potentially exposed to the chemicals present in paint products although the patterns and levels of exposure to individual agents may differ from those of painters. The aim of the present study was to evaluate genome damage induced in peripheral blood lymphocytes and oral mucosa cells of paint industry workers. Materials and Methods: Genotoxicity was evaluated using the alkaline Comet assay in blood lymphocytes and oral mucosa cells, and the Micronucleus test in oral mucosa cells. For the micronucleus test in exfoliated buccal cells, no significant difference was detected between the control and paint industry workers. Results: The Comet assay in epithelia buccal cells showed that the damage index (DI) and damage frequency (DF) observed in the exposed group were significantly higher relative to the control group (P≤0.05). In the same way, the Comet assay data in peripheral blood leukocytes showed that both analysis parameters (DI and DF) were significantly greater than that for the control group (P≤0.05). Conclusions: Chronic occupational exposure to paints may lead to a slightly increased risk of genetic damage among paint industry workers. PMID:22223950

Evaluation of cytogenetic and DNA damage in human lymphocytes treated with adrenaline in vitro.

PubMed

Djelić, Ninoslav; Radaković, Milena; Spremo-Potparević, Biljana; Zivković, Lada; Bajić, Vladan; Stevanović, Jevrosima; Stanimirović, Zoran

2015-02-01

Catechol groups can be involved in redox cycling accompanied by generation of reactive oxygen species (ROS) which may lead to oxidative damage of cellular macromolecules including DNA. The objective of this investigation was to evaluate possible genotoxic effects of a natural catecholamine adrenaline in cultured human lymphocytes using cytogenetic (sister chromatid exchange and micronuclei) and the single cell gel electrophoresis (Comet) assay. In cytogenetic tests, six experimental concentrations of adrenaline were used in a range from 0.01-500 μM. There were no indications of genotoxic effects of adrenaline in sister chromatid exchange and micronucleus tests. However, at four highest concentrations of adrenaline (5 μM, 50 μM, 150 μM and 300 μM) we observed a decreased mitotic index and cell-cycle delay. In addition, in the Comet assay we used adrenaline in a range from 0.0005-500 μM, at two treatment times: 15 min or 60 min. In contrast to cytogenetic analysis, there was a dose-dependent increase of DNA damage detected in the Comet assay. These effects were significantly reduced by concomitant treatment with quercetin or catalase. Therefore, the obtained results indicate that adrenaline may exhibit genotoxic effects in cultured human lymphocytes, most likely due to production of reactive oxygen species. Copyright © 2014 Elsevier Ltd. All rights reserved.

Chemical composition and genotoxicity assessment of sanitary landfill leachate from Rovinj, Croatia.

PubMed

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

2012-04-01

Chemical analysis and an in vitro approach were performed to assess elemental composition and genotoxic effects of the samples of landfill leachate taken from Lokva Vidotto sanitary landfill the official landfill for Rovinj town, Croatia. Two samples of landfill leachate were collected and analyzed in order to evaluate macro, micro and trace elements by atomic absorption spectroscopy, energy dispersive X-ray spectrometry and colorimetry. Genotoxicity of sanitary landfill leachate was evaluated in human lymphocytes by the use of the micronucleus test and comet assay. Samples were characterized with relatively low concentrations of heavy metals while organic component level exceeded upper permissible limit up to 39 times. Observed genotoxic effects should be connected with high concentrations of ammonia nitrogen, which exceeded permissible limit up to 180 times. Leachate samples of both sanitary landfills increased the frequency of micronuclei, nucleoplasmic bridges and nuclear buds. Increase of DNA damage in human lymphocytes was also detected by virtue of measuring comet assay parameters. All parameters showed statistically significant difference compared to negative control. Increased micronucleus and comet assay parameters indicate that both samples of sanitary landfill leachate are genotoxic and could pose environmental and human health risk if discharged to an aquatic environment. Copyright © 2011 Elsevier Inc. All rights reserved.

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

EPA Science Inventory

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

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

The Use of Bacterial Repair Endonucleases in the Comet Assay.

PubMed

Collins, Andrew R

2017-01-01

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

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

PubMed

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

2016-02-01

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

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

PubMed

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

2013-07-01

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

Centered reduced moments and associate density functions applied to alkaline comet assay.

PubMed

Castaneda, Roman; Pelaez, Alejandro; Marquez, Maria-Elena; Abad, Pablo

2005-01-01

The single cell gel electrophoresis assay is a sensitive, rapid, and visual technique for deoxyribonucleic acid (DNA) strand-break detection in individual mammalian cells, whose application has significantly increased in the past few years. The cells are embedded in agarose on glass slides followed by lyses of the cell membrane. Thereafter, damaged DNA strands are electrophoresed away from the nucleus towards the anode giving the appearance of a comet tail. Nowadays, charge coupled device cameras are attached at optical microscopes for recording the images of the cells, and digital image processing is applied for obtaining quantitative descriptors. However, the conventional software is usually expensive, inflexible and, in many cases, can only provide low-order descriptors based in image segmentation, determination of centers of mass, and Euclidean distances. Associated density functions and centered reduced moments offer an effective and flexible alternative for quantitative analysis of the comet cells. We will show how the position of the center of mass, the lengths and orientation of the main semiaxes, and the eccentricity of such images can be accurately determined by this method.

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

USGS Publications Warehouse

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

2006-01-01

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

The comet assay in human biomonitoring.

PubMed

Anderson, Diana; Dhawan, Alok; Laubenthal, Julian

2013-01-01

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

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

PubMed

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

2015-06-01

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

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

PubMed

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

2016-05-01

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

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

PubMed

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

2015-07-01

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

A direct view by immunofluorescent comet assay (IFCA) of DNA damage induced by nicking and cutting enzymes, ionizing (137)Cs radiation, UV-A laser microbeam irradiation and the radiomimetic drug bleomycin.

PubMed

Grigaravicius, Paulius; Rapp, Alexander; Greulich, Karl Otto

2009-03-01

In DNA repair research, DNA damage is induced by different agents, depending on the technical facilities of the investigating researchers. A quantitative comparison of different investigations is therefore often difficult. By using a modified variant of the neutral comet assay, where the histone H1 is detected by immunofluorescence [immunofluorescent comet assay (IFCA)], we achieve previously unprecedented resolution in the detection of fragmented chromatin and show that trillions of ultraviolet A photons (of a few eV), billions of bleomycin (BLM) molecules and thousands of gamma quanta (of 662 keV) generate, in first order, similar damage in the chromatin of HeLa cells. A somewhat more detailed inspection shows that the damage caused by 20 Gy ionizing radiation and by a single laser pulse of 10 microJ are comparable, while the damage caused by 12 microg/ml BLM depends highly on the individual cell. Taken together, this work provides a detailed view of DNA fragmentation induced by different treatments and allows comparing them to some extent, especially with respect to the neutral comet assay.

Bovine Papillomavirus Clastogenic Effect Analyzed in Comet Assay

PubMed Central

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

2013-01-01

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

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

PubMed

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

2008-05-01

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

The use of comet assay to assess DNA integrity of boar spermatozoa following liquid preservation at 5 degrees C and 16 degrees C.

PubMed

Fraser, L; Strzezek, J

2004-01-01

The comet assay, under neutral conditions, allows the assessment of DNA integrity influenced by sperm ageing, which is manifested in DNA double-strand breaks. Here, we attempted to use a modified neutral comet assay test (single-cell gel electrophoresis), to our knowledge for the first time, to assess DNA integrity of boar spermatozoa during liquid storage for 96 h at 5 degrees C and 16 degrees C. In this comet assay protocol we used 2% beta-mercaptoethanol prior to the lysis procedure, to aid in removing nuclear proteins. Ejaculates from 3 boars (designated A, C and G) were diluted with a standard semen extender, Kortowo-3 (K-3), which was supplemented with lipoprotein fractions extracted from hen egg yolk (LPFh) or ostrich egg yolk (LPFo). Irrespective of the extender type, the percentage of comet-detected spermatozoa with damaged DNA increased gradually during prolonged storage at 5 degrees C and 16 degrees C. Spermatozoa stored in K-3 extender exhibited elevated levels of DNA damage at both storage temperatures. Significant differences in DNA damage among the boars were more pronounced during storage in LPF-based extenders at 5 degrees C: spermatozoa of boars A and G were less susceptible to DNA damage. The percent of tail DNA in comets was lower in LPF-based extenders, and there were individual variations among the boars. We observed that changes in DNA integrity were dependent on the extender type and storage temperature. A higher level of DNA instability was observed in K-3 extended semen compared with K-3/LPFh or K-3/LPFo extended semen during storage at 5 degrees C. No significant difference in the level of DNA damage between K-3/LPFh and K-3/LPFo was observed. It seems that a long-term storage can affect genomic integrity of boar spermatozoa. The modified neutral comet assay can be used to detect low levels of DNA damage in boar spermatozoa during liquid preservation. Therefore, screening for sperm DNA damage may be used as an additional test of sperm function that can have diagnostic value in practice.

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

PubMed

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

2011-05-18

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

Detection of ozone-induced DNA single strand breaks in murine bronchoalveolar lavage cells acutely exposed in vivo.

PubMed

Haney, J T; Connor, T H; Li, L

1999-04-01

Single-strand breaks (SSBs) in DNA have been used a biomarker of oxidative damage. The comet assay, also known as single-cell gel electrophoresis, was used to investigate the ability of ozone (O(3)) to induce DNA SSBs in murine bronchoalveolar lavage (BAL) cells. The comet assay is more sensitive than other techniques currently utilized for detecting SSBs and requires fewer cells. In the present study, 3 mice were exposed for 3 h to 0.25 ppm of O(3), and 3 to 0.5 ppm of O(3) for 3 h. Two air-exposed mice served as negative controls. All mice were euthanized 3 h after exposure, at which time BAL cells were recovered from the lungs and stained with ethidium bromide. BAL cells recovered from an air-exposed mouse were exposed to various concentrations of H(2)O(2) in vitro for 1 h at 4 degrees C. Excluding cells from the H(2)O(2) group (n = 25), 50 randomly selected BAL cells were graded by comet tail length into 1 of 4 categories: no damage (0 mm), low damage (1-10 mm), medium damage (11-30 mm), and high damage (31 + mm). The nonparametric Wilcoxon rank-sum test was used for statistical analysis, and p values lower than .05 were considered significant. The H(2)O(2) and the 0.25 and 0.5 ppm O3 groups showed statistically significant increases in DNA SSBs as compared to air-exposed controls. The results of this study indicate that (1) O(3) induces DNA strand breaks in murine BAL cells at 0.25 and 0.5 ppm, as evidenced by statistically significant increases in the length of comet tails for O(3)-exposed groups, and (2) the comet assay can be used to assess O(3)-induced SSBs for in vivo exposures. Therefore, it has the potential as a biomarker for in vivo oxidant exposures.

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

PubMed

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

2014-05-01

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

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

PubMed

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

2015-02-01

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

Genotoxicity assessment of nanomaterials: recommendations on best practices, assays and methods.

PubMed

Elespuru, Rosalie; Pfuhler, Stefan; Aardema, Marilyn; Chen, Tao; Doak, Shareen H; Doherty, Ann; Farabaugh, Christopher S; Kenny, Julia; Manjanatha, Mugimane; Mahadevan, Brinda; Moore, Martha M; Ouédraogo, Gladys; Stankowski, Leon F; Tanir, Jennifer Y

2018-04-26

Nanomaterials (NMs) present unique challenges in safety evaluation. An international working group, the Genetic Toxicology Technical Committee of the International Life Sciences Institute's Health and Environmental Sciences Institute, has addressed issues related to the genotoxicity assessment of NMs. A critical review of published data has been followed by recommendations on methods alterations and best practices for the standard genotoxicity assays: bacterial reverse mutation (Ames); in vitro mammalian assays for mutations, chromosomal aberrations, micronucleus induction, or DNA strand breaks (comet); and in vivo assays for genetic damage (micronucleus, comet and transgenic mutation assays). The analysis found a great diversity of tests and systems used for in vitro assays; many did not meet criteria for a valid test, and/or did not use validated cells and methods in the Organization for Economic Co-operation and Development Test Guidelines, and so these results could not be interpreted. In vivo assays were less common but better performed. It was not possible to develop conclusions on test system agreement, NM activity, or mechanism of action. However, the limited responses observed for most NMs were consistent with indirect genotoxic effects, rather than direct interaction of NMs with DNA. We propose a revised genotoxicity test battery for NMs that includes in vitro mammalian cell mutagenicity and clastogenicity assessments; in vivo assessments would be added only if warranted by information on specific organ exposure or sequestration of NMs. The bacterial assays are generally uninformative for NMs due to limited particle uptake and possible lack of mechanistic relevance, and are thus omitted in our recommended test battery for NM assessment. Recommendations include NM characterization in the test medium, verification of uptake into target cells, and limited assay-specific methods alterations to avoid interference with uptake or endpoint analysis. These recommendations are summarized in a Roadmap guideline for testing.

Evaluation of 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.

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.

Monitoring of DNA breakage in embryonic stages of the African catfish Clarias gariepinus (Burchell, 1822) after exposure to lead nitrate using alkaline comet assay.

PubMed

Osman, Alaa G M; Mekkawy, Imam A; Verreth, Johan; Wuertz, Sven; Kloas, Werner; Kirschbaum, Frank

2008-12-01

Increasing lead contamination in Egyptian ecosystems and high lead concentrations in food items have raised concern for human health and stimulated studies on monitoring ecotoxicological impact of lead-caused genotoxicity. In this work, the alkaline comet assay was modified for monitoring DNA strand breakage in sensitive early life stages of the African catfish Clarias gariepinus. Following exposure to 100, 300, and 500 microg/L lead nitrate, DNA strand breakage was quantified in embryos at 30, 48, 96, 144, and 168 h post-fertilization (PFS). For quantitative analysis, four commonly used parameters (tail % DNA, %TDNA; head % DNA, %HDNA; tail length, TL; tail moment, TM) were analyzed in 96 nuclei (in triplicates) at each sampling point. The parameter %TDNA revealed highest resolution and lowest variation. A strong correlation between lead concentration, time of exposure, and DNA strand breakage was observed. Here, genotoxicity detected by comet assay preceded the manifested malformations assessed with conventional histology. Qualitative evaluation was carried out using five categories are as follows: undamaged (%TDNA < or = 10%), low damaged (10% < %TDNA < or = 25%), median damaged (25 < %TDNA < or = 50%), highly damaged (50 < %TDNA < or = 75%), and extremely damaged (%TDNA > 75%) nuclei confirming a dose and time-dependent shift towards increased frequencies of highly and extremely damaged nuclei. A protective capacity provided by a hardened chorion is a an interesting finding in this study as DNA damage in the prehatching stages 30 h-PFS and 48 h-PFS was low in all treatments (qualitative and quantitative analyses). These results clearly show that the comet assay is a sensitive tool for the detection of genotoxicity in vulnerable early life stages of the African catfish and is a method more sensitive than histological parameters for monitoring genotoxic effects. 2008 Wiley Periodicals, Inc.

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

PubMed

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

2017-08-01

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

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

PubMed

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

2014-07-15

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

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

PubMed

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

2017-12-01

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

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

PubMed

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

2016-04-01

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

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

PubMed

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

2011-11-01

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

Comet assay with gill cells of Mytilus galloprovincialis end point tools for biomonitoring of water antibiotic contamination: Biological treatment is a reliable process for detoxification.

PubMed

Mustapha, Nadia; Zouiten, Amina; Dridi, Dorra; Tahrani, Leyla; Zouiten, Dorra; Mosrati, Ridha; Cherif, Ameur; Chekir-Ghedira, Leila; Mansour, Hedi Ben

2016-04-01

This article investigates the ability of Pseudomonas peli to treat industrial pharmaceuticals wastewater (PW). Liquid chromatography-mass spectrometry (MS)/MS analysis revealed the presence, in this PW, of a variety of antibiotics such as sulfathiazole, sulfamoxole, norfloxacine, cloxacilline, doxycycline, and cefquinome.P. peli was very effective to be grown in PW and inducts a remarkable increase in chemical oxygen demand and biochemical oxygen demand (140.31 and 148.51%, respectively). On the other hand, genotoxicity of the studied effluent, before and after 24 h of shaking incubation with P. peli, was evaluated in vivo in the Mediterranean wild mussels Mytilus galloprovincialis using comet assay for quantification of DNA fragmentation. Results show that PW exhibited a statistically significant (p< 0.001) genotoxic effect in a dose-dependent manner; indeed, the percentage of genotoxicity was 122.6 and 49.5% after exposure to 0.66 ml/kg body weight (b.w.); 0.33 ml/kg b.w. of PW, respectively. However, genotoxicity decreased strongly when tested with the PW obtained after incubation with P. peli We can conclude that using comet assay genotoxicity end points are useful tools to biomonitor the physicochemical and biological quality of water. Also, it could be concluded that P. peli can treat and detoxify the studied PW. © The Author(s) 2013.

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.

Development of a Test Method for the Evaluation of DNA Damage in Mouse Spermatogonial Stem Cells

PubMed Central

Jeon, Hye Lyun; Yi, Jung-Sun; Kim, Tae Sung; Oh, Youkyung; Lee, Hye Jeong; Lee, Minseong; Bang, Jin Seok; Ko, Kinarm; Ahn, Il Young; Ko, Kyungyuk; Kim, Joohwan; Park, Hye-Kyung; Lee, Jong Kwon; Sohn, Soo Jung

2017-01-01

Although alternative test methods based on the 3Rs (Replacement, Reduction, Refinement) are being developed to replace animal testing in reproductive and developmental toxicology, they are still in an early stage. Consequently, we aimed to develop alternative test methods in male animals using mouse spermatogonial stem cells (mSSCs). Here, we modified the OECD TG 489 and optimized the in vitro comet assay in our previous study. This study aimed to verify the validity of in vitro tests involving mSSCs by comparing their results with those of in vivo tests using C57BL/6 mice by gavage. We selected hydroxyurea (HU), which is known to chemically induce male reproductive toxicity. The 50% inhibitory concentration (IC50) value of HU was 0.9 mM, as determined by the MTT assay. In the in vitro comet assay, % tail DNA and Olive tail moment (OTM) after HU administration increased significantly, compared to the control. Annexin V, PI staining and TUNEL assays showed that HU caused apoptosis in mSSCs. In order to compare in vitro tests with in vivo tests, the same substances were administered to male C57BL/6 mice. Reproductive toxicity was observed at 25, 50, 100, and 200 mg/kg/day as measured by clinical measures of reduction in sperm motility and testicular weight. The comet assay, DCFH-DA assay, H&E staining, and TUNEL assay were also performed. The results of the test with C57BL/6 mice were similar to those with mSSCs for HU treatment. Finally, linear regression analysis showed a strong positive correlation between results of in vitro tests and those of in vivo. In conclusion, the present study is the first to demonstrate the effect of HU-induced DNA damage, ROS formation, and apoptosis in mSSCs. Further, the results of the current study suggest that mSSCs could be a useful model to predict male reproductive toxicity. PMID:28443181

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.

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

Drosophila comet assay: insights, uses, and future perspectives

PubMed Central

Gaivão, Isabel; Sierra, L. María

2014-01-01

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

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

PubMed

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

2011-09-18

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

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

PubMed

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

2015-07-01

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

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

PubMed Central

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

2015-01-01

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

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

PubMed

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

2015-07-01

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

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

NASA Astrophysics Data System (ADS)

Khan, Hasan M.; Delincée, Henry

1998-06-01

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

The comet assay for the evaluation of genotoxic potential of landfill leachate.

PubMed

Widziewicz, Kamila; Kalka, Joanna; Skonieczna, Magdalena; Madej, Paweł

2012-01-01

Genotoxic assessment of landfill leachate before and after biological treatment was conducted with two human cell lines (Me45 and NHDF) and Daphnia magna somatic cells. The alkali version of comet assay was used to examine genotoxicity of leachate by DNA strand breaks analysis and its repair dynamics. The leachate samples were collected from Zabrze landfill, situated in the Upper Silesian Industrial District, Poland. Statistically significant differences (Kruskal-Wallice ANOVA rank model) were observed between DNA strand breaks in cells incubated with leachate before and after treatment (P < 0.001). Nonparametric Friedman ANOVA confirmed time-reliable and concentration-reliable cells response to leachate concentration. Examinations of chemical properties showed a marked decrease in leachate parameters after treatment which correlate to reduced genotoxicity towards tested cells. Obtained results demonstrate that biological cotreatment of leachate together with municipal wastewater is an efficient method for its genotoxic potential reduction; however, treated leachate still possessed genotoxic character.

The Comet Assay for the Evaluation of Genotoxic Potential of Landfill Leachate

PubMed Central

Widziewicz, Kamila; Kalka, Joanna; Skonieczna, Magdalena; Madej, Paweł

2012-01-01

Genotoxic assessment of landfill leachate before and after biological treatment was conducted with two human cell lines (Me45 and NHDF) and Daphnia magna somatic cells. The alkali version of comet assay was used to examine genotoxicity of leachate by DNA strand breaks analysis and its repair dynamics. The leachate samples were collected from Zabrze landfill, situated in the Upper Silesian Industrial District, Poland. Statistically significant differences (Kruskal-Wallice ANOVA rank model) were observed between DNA strand breaks in cells incubated with leachate before and after treatment (P < 0.001). Nonparametric Friedman ANOVA confirmed time-reliable and concentration-reliable cells response to leachate concentration. Examinations of chemical properties showed a marked decrease in leachate parameters after treatment which correlate to reduced genotoxicity towards tested cells. Obtained results demonstrate that biological cotreatment of leachate together with municipal wastewater is an efficient method for its genotoxic potential reduction; however, treated leachate still possessed genotoxic character. PMID:22666120

DNA Damage among Wood Workers Assessed with the Comet Assay

PubMed Central

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

2016-01-01

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

Identification of gamma-irradiated papaya, melon and watermelon

NASA Astrophysics Data System (ADS)

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

2004-09-01

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

Explanation for excessive DNA single-strand breaks and endogenous repair foci in pluripotent mouse embryonic stem cells.

PubMed

Banáth, J P; Bañuelos, C A; Klokov, D; MacPhail, S M; Lansdorp, P M; Olive, P L

2009-05-01

Pluripotent mouse embryonic stem cells (mES cells) exhibit approximately 100 large gammaH2AX repair foci in the absence of measurable numbers of DNA double-strand breaks. Many of these cells also show excessive numbers of DNA single-strand breaks (>10,000 per cell) when analyzed using the alkaline comet assay. To understand the reasons for these unexpected observations, various methods for detecting DNA strand breaks were applied to wild-type mES cells and to mES cells lacking H2AX, ATM, or DNA-PKcs. H2AX phosphorylation and expression of other repair complexes were measured using flow and image analysis of antibody-stained cells. Results indicate that high numbers of endogenous gammaH2AX foci and single-strand breaks in pluripotent mES cells do not require ATM or DNA-PK kinase activity and appear to be associated with global chromatin decondensation rather than pre-existing DNA damage. This will limit applications of gammaH2AX foci analysis in mES cells to relatively high levels of initial or residual DNA damage. Excessive numbers of single-strand breaks in the alkaline comet assay can be explained by the vulnerability of replicating chromatin in mES cells to osmotic shock. This suggests that caution is needed in interpreting results with the alkaline comet assay when applied to certain cell types or after treatment with agents that make chromatin vulnerable to osmotic changes. Differentiation of mES cells caused a reduction in histone acetylation, gammaH2AX foci intensity, and DNA single-strand breakage, providing a link between chromatin structural organization, excessive gammaH2AX foci, and sensitivity of replicating mES cell chromatin to osmotic shock.

Detection of radiation treatment of beans using DNA comet assay

NASA Astrophysics Data System (ADS)

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

2002-03-01

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

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

EPA Science Inventory

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

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

Genotoxicity evaluation of HMG CoA reductase inhibitor rosuvastatin.

PubMed

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

2014-07-01

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

Antioxidants and the Comet assay.

PubMed

Cemeli, Eduardo; Baumgartner, Adolf; Anderson, Diana

2009-01-01

It is widely accepted that antioxidants, either endogenous or from the diet, play a key role in preserving health. They are able to quench radical species generated in situations of oxidative stress, either triggered by pathologies or xenobiotics, and they protect the integrity of DNA from genotoxicants. Nevertheless, there are still many compounds with unclear or unidentified prooxidant/antioxidant activities. This is of concern since there is an increase in the number of compounds synthesized or extracted from vegetables to which humans might be exposed. Despite the well-established protective effects of fruit and vegetables, the antioxidant(s) responsible have not all been clearly identified. There might also be alternative mechanisms contributing to the protective effects for which a comprehensive description is lacking. In the last two decades, the Comet assay has been extensively used for the investigation of the effects of antioxidants and many reports can be found in the literature. The Comet assay, a relatively fast, simple, and sensitive technique for the analysis of DNA damage in all cell types, has been applied for the screening of chemicals, biomonitoring and intervention studies. In the present review, several of the most well-known antioxidants are considered. These include: catalase, superoxide dismutase, glutathione peroxidase, selenium, iron chelators, melatonin, melanin, vitamins (A, B, C and E), carotenes, flavonoids, isoflavones, tea polyphenols, wine polyphenols and synthetic antioxidants. Investigations showing beneficial as well as non-beneficial properties of the antioxidants selected, either at the in vitro, ex vivo or in vivo level are discussed.

New nanostructural biomaterials based on active silicate systems and hydroxyapatite: characterization and genotoxicity in human peripheral blood lymphocytes.

PubMed

Opačić-Galić, V; Petrović, V; Zivković, S; Jokanović, V; Nikolić, B; Knežević-Vukčević, J; Mitić-Ćulafić, D

2013-06-01

To characterize and investigate the genotoxic effect of a new endodontic cement based on dicalcium- and tricalcium-silicate (CS) with hydroxyapatite (HA) on human lymphocytes. Hydrothermal treatment was applied for synthesis of CS and HA. The final mixture HA-CS, with potential to be used in endodontic practice, is composed of CS (34%) and HA (66%). Human lymphocytes were incubated with HA, HA-CS and CS for 1 h, at 37 °C and 5% CO2. Cell viability was determined using the trypan blue exclusion assay. To evaluate the level of DNA damage comet assay (single cell gel electrophoresis) was performed. For the statistical analysis anova and Duncan's Post Hoc Test were used. The SEM analysis indicated that CS consisted mostly of agglomerates of several micrometers in size, built up from smaller particles, with dimensions between 117 and 477 nm. This is promising because dimensions of agglomerates are not comparable with channels inside the cell membranes, whereas their nano-elements provide evident activity, important for faster setting of these mixtures compared to MTA. Values of DNA damage obtained in the comet assay indicated low genotoxic risk of the new endodontic materials. The significantly improved setting characteristics and low genotoxic risk of the new material support further research. © 2012 International Endodontic Journal.

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

PubMed Central

Nickson, Catherine M.; Parsons, Jason L.

2014-01-01

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

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

PubMed

Bausinger, Julia; Speit, Günter

2016-09-01

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

A Bayesian, generalized frailty model for comet assays.

PubMed

Ghebretinsae, Aklilu Habteab; Faes, Christel; Molenberghs, Geert; De Boeck, Marlies; Geys, Helena

2013-05-01

This paper proposes a flexible modeling approach for so-called comet assay data regularly encountered in preclinical research. While such data consist of non-Gaussian outcomes in a multilevel hierarchical structure, traditional analyses typically completely or partly ignore this hierarchical nature by summarizing measurements within a cluster. Non-Gaussian outcomes are often modeled using exponential family models. This is true not only for binary and count data, but also for, example, time-to-event outcomes. Two important reasons for extending this family are for (1) the possible occurrence of overdispersion, meaning that the variability in the data may not be adequately described by the models, which often exhibit a prescribed mean-variance link, and (2) the accommodation of a hierarchical structure in the data, owing to clustering in the data. The first issue is dealt with through so-called overdispersion models. Clustering is often accommodated through the inclusion of random subject-specific effects. Though not always, one conventionally assumes such random effects to be normally distributed. In the case of time-to-event data, one encounters, for example, the gamma frailty model (Duchateau and Janssen, 2007 ). While both of these issues may occur simultaneously, models combining both are uncommon. Molenberghs et al. ( 2010 ) proposed a broad class of generalized linear models accommodating overdispersion and clustering through two separate sets of random effects. Here, we use this method to model data from a comet assay with a three-level hierarchical structure. Although a conjugate gamma random effect is used for the overdispersion random effect, both gamma and normal random effects are considered for the hierarchical random effect. Apart from model formulation, we place emphasis on Bayesian estimation. Our proposed method has an upper hand over the traditional analysis in that it (1) uses the appropriate distribution stipulated in the literature; (2) deals with the complete hierarchical nature; and (3) uses all information instead of summary measures. The fit of the model to the comet assay is compared against the background of more conventional model fits. Results indicate the toxicity of 1,2-dimethylhydrazine dihydrochloride at different dose levels (low, medium, and high).

The genotoxic effect of oxcarbazepine on mice blood lymphocytes.

PubMed

Akbar, Huma; Khan, Ajmal; Mohammadzai, Imdadullah; Khisroon, Muhammad; Begum, Ilham

2018-04-01

This study was conducted to assess the amount of DNA damage caused by Oxcarbazepine (OXC) through single cell gel electrophoresis (SCGE) technique/comet assay. OXC derived from dibenzazepine series is an effective second generation antiepileptic drug (AED) for both children and adults. Side effects like genotoxic effects of AEDs are of prime importance resulting from toxic metabolites, free radicals and reactive oxygen species (ROS). Forty Eight adult male Bagg's albino mice (BALB/c) were randomly classified into eight groups, each comprising of six animals. Two of these groups were control and six were tested groups. Control groups were injected with 1% tween 80 while tested groups were injected with 10, 20, and 40 mg/kg-day OXC for seven days (acute therapy) and 28 days (subchronic therapy) in peritoneal cavity. Blood samples were collected by cardiac puncture and subjected to comet assay for the analysis of DNA damage. Per sample 100 cells were scored and classified according to comet tail length. The results showed that OXC in acute and long term therapies had significantly higher (p < 0.05) genotoxicity in treated groups as compared to control groups. Our study suggests that OXC may cause significant DNA damage in both acute as well as in subchronic therapies.

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

PubMed

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

2015-07-01

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

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

PubMed

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

2009-03-01

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

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

PubMed

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

2009-01-01

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

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

PubMed

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

2017-01-01

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

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

PubMed Central

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

2013-01-01

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

Genotoxicity assessment of amaranth and allura red using Saccharomyces cerevisiae.

PubMed

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

2013-01-01

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

Comparative analysis of three sperm DNA damage assays and sperm nuclear protein content in couples undergoing assisted reproduction treatment.

PubMed

Simon, L; Liu, L; Murphy, K; Ge, S; Hotaling, J; Aston, K I; Emery, B; Carrell, D T

2014-05-01

Is there an association between sperm DNA damage, measured by three different assays, sperm nuclear protein content and clinical outcomes in assisted reproduction treatment (ART)? Sperm DNA damage measured by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling (TUNEL) and the Comet assay were significantly associated with ART outcomes in our single institution study. Abnormal protamine expression is known to be associated with sperm DNA damage and male infertility. A number of studies have shown a significant relationship between sperm DNA damage and ART outcomes. To date, there are no large studies providing direct comparisons of DNA damage tests within the same study population. Thus, the prognostic value for each method remains unknown. Cross-sectional study of 238 men from infertile couples undergoing ART at the University Center for Reproductive Medicine, Utah, USA, between April 2011 and March 2013. Sperm from men undergoing ART were tested for DNA damage using the alkaline Comet assay, TUNEL and flow cytometric chromatin evaluation (FCCE) assays. Histone retention was analysed using the aniline blue staining method, whereas protamine content (proteins P1 and P2) and ratio were analysed using acid urea gel electrophoresis. The prognostic value of each sperm DNA test to predict clinical pregnancy was calculated. Histone retention was associated with sperm DNA damage (P < 0.001), reduced embryo quality (P = 0.005) and clinical pregnancies (P < 0.001). The mean percentage of sperm with DNA damage was significantly higher in sperm from non-pregnant couples compared with that from pregnant couples, as measured by TUNEL assay (15.04 ± 1.16% versus 8.79 ± 0.56%; P < 0.001) and alkaline Comet assay (72.79 ± 2.49% versus 55.86 ± 2.29%; P < 0.001). There was no association between clinical pregnancies and DNA fragmentation index measured by FCCE (12.97 ± 1.46 versus 14.93 ± 1.65; P = 0.379). Of the protamine parameters analysed, only the P1/P2 ratio was associated with sperm count (P = 0.013), men's age (P = 0.037), maturity (P = 0.049) and blastocyst quality (P = 0.012). Histone retention and sperm DNA damage measured by Comet and TUNEL assays were associated with fertilization rate (P < 0.05), embryo quality (P < 0.05) and implantation rate (P < 0.05). A potential drawback of this study is that it is cross-sectional. Generally in such studies there is more than one variable that could cause the effect. Analysing sperm is one part of the equation; there are also a number of female factors that have the potential to influence ART outcomes. Therefore, given the large and well-established role of female factors in infertility, normal sperm DNA integrity and protamination do not necessarily ensure clinical pregnancy in ART. Thus, female factors can reduce the prognostic value of sperm DNA tests. Further, our use of native semen instead of prepared sperm may have iatrogenically increased the DNA damage. Alteration in sperm nuclear protein affects sperm DNA integrity. Further, with the current dataset, TUNEL and Comet assays appeared more predictive of ART success than FCCE. No personal or direct financial support has been received for any of this work. The authors declare no competing interests. N/A.

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

PubMed

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

2017-02-01

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

Comet Assay on Daphnia magna in eco-genotoxicity testing.

PubMed

Pellegri, Valerio; Gorbi, Gessica; Buschini, Annamaria

2014-10-01

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

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

PubMed Central

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

2012-01-01

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

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

PubMed

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

2017-12-20

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

Evaluation of DNA damage in flight personnel by Comet assay.

PubMed

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

2002-04-26

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

Genotoxic Potential and Physicochemical Parameters of Sinos River, Southern Brazil

PubMed Central

Scalon, Madalena C. S.; Rechenmacher, Ciliana; Siebel, Anna Maria; Kayser, Michele L.; Rodrigues, Manoela T.; Maluf, Sharbel W.; Rodrigues, Marco Antonio S.

2013-01-01

The present study aimed to evaluate the physicochemical parameters and the genotoxic potential of water samples collected in the upper, middle, and lower courses of the Sinos River, southern Brazil. The comet assay was performed in the peripheral blood of fish Hyphessobrycon luetkenii exposed under laboratory conditions to water samples collected in summer and winter in three sampling sites of Sinos River. Water quality analysis demonstrated values above those described in Brazilian legislation in Parobé and Sapucaia do Sul sites, located in the middle and in the lower courses of the Sinos River, respectively. The Caraá site, located in the upper river reach, presented all the physicochemical parameters in accordance with the allowed limits in both sampling periods. Comet assay in fish revealed genotoxicity in water samples collected in the middle course site in summer and in the three sites in winter when compared to control group. Thus, the physicochemical parameters indicated that the water quality of the upper course complies with the limits set by the national guidelines, and the ecotoxicological assessment, however, indicated the presence of genotoxic agents. The present study highlights the importance of combining water physicochemical analysis and bioassays to river monitoring. PMID:24285934

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

PubMed

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

2018-06-09

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

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

PubMed

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

2009-06-01

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

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

DTIC Science & Technology

2007-10-01

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

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

PubMed

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

2016-12-01

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

Evaluation of Genotoxic Pressure along the Sava River

PubMed Central

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

2016-01-01

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

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

PubMed

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

2016-04-15

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

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

PubMed

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

2017-02-01

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

Genotoxicity of drinking water disinfection by-products (bromoform and chloroform) by using both Allium anaphase-telophase and comet tests.

PubMed

Khallef, Messaouda; Liman, Recep; Konuk, Muhsin; Ciğerci, İbrahim Hakkı; Benouareth, Djameleddine; Tabet, Mouna; Abda, Ahlem

2015-03-01

Genotoxic effects of bromoform and chloroform, disinfection by-products of the chlorination of drinking water, were examined by using mitotic index (MI), mitotic phase, chromosome aberrations (CAs) and comet assay on root meristematic cells of Allium cepa. Different concentrations of bromoform (25, 50, 75 and 100 μg/mL) and chloroform (25, 50, 100 and 200 μg/mL) were introduced to onion tuber roots. Distilled water was used as a negative control and methyl methansulfonate (MMS-10 μg/mL) as positive control. All obtained data were subjected to statistical analyses by using SPSS 15.0 for Windows software. For comparison purposes, Duncan multiple range tests by using one-way analysis of variance were employed and p < 0.05 was accepted as significant value. Exposure of both chemicals (except 25 μg/mL applications of bromoform) significantly decreased MI. Bromoform and chloroform (except 25 μg/mL applications) increased total CAs in Allium anaphase-telophase test. A significant increase in DNA damage was also observed at all concentrations of both bromoform and chloroform examined by comet assay. The damages were higher than that of positive control especially at 75-100 μg/mL for bromoform and 100-200 μg/mL for chloroform.

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

PubMed

Beevers, Carol; Henderson, Debbie; Lillford, Lucinda

2015-07-01

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

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

PubMed

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

2011-01-01

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

HPLC-DAD-ESI-MS/MS analysis of fruits from Firmiana simplex (L.) and evaluation of their antioxidant and antigenotoxic properties.

PubMed

Ghareeb, Mosad Ahmed; Mohamed, Tamer; Saad, Amal Mohamed; Refahy, Laila Abdel-Ghany; Sobeh, Mansour; Wink, Michael

2018-01-01

The secondary metabolites of the fruits of Firmiana simplex (L.) were analysed by LC-DAD-ESI-MS/MS; furthermore, we evaluated their antioxidant and antigenotoxic properties. The antioxidant activity was investigated using the 2,2'-diphenyl-1-picrylhydrazyl radical (DPPH), the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) and the ferric reducing antioxidant power (FRAP) assays. The antigenotoxic potential was determined via the comet assay. The ethyl acetate fraction (EtOAc) was analysed by LC-DAD-ESI-MS/MS: phenolic acids and flavonoids were the main polyphenols of the fruits. The EtOAc fraction yielded the highest content of polyphenols with 314.61 mg GAE/g extract, followed by 297.51, 153.75, 101.47, 97.19 for dichloromethane, butanol, methanol and water extracts, respectively. As expected, a strong correlation exists between the antioxidant activity of the investigated extracts and their total phenolic content. In the DPPH assay, the IC 50 value of the most active EtOAc fraction was 6.79 μg/ml, relative to 2.92 μg/ml of the standard ascorbic acid. ABTS and FRAP assays supported the results of DPPH assay. Moreover, using the comet assay, we could show that the phenol-rich EtOAc extract exhibits an antigenotoxic potential in human liver cancer cells (Hep-G2) treated with hydrogen peroxide (H 2 O 2 ) as a genotoxic agent. The fruits of Firmiana simplex may be a good natural source of antioxidant and antigenotoxic agents. © 2017 Royal Pharmaceutical Society.

Assessment of the in vitro cytotoxicity and in vivo anti-tumor activity of the alcoholic stem bark extract/fractions of Mimusops elengi Linn.

PubMed

Kumar, Harish; Savaliya, Mihir; Biswas, Subhankar; Nayak, Pawan G; Maliyakkal, Naseer; Manjunath Setty, M; Gourishetti, Karthik; Pai, K Sreedhara Ranganath

2016-08-01

Various parts of Mimusops elengi Linn. (Sapotaceae) have been used widely in traditional Indian medicine for the treatment of pain, inflammation and wounds. The study was conducted to explore the use of stem bark of M. elengi on pharmacological grounds and to evaluate the scientific basis of cytotoxic and anti-tumor activity. Extract/fractions were prepared and in vitro cytotoxicity was assessed using SRB assay. Most effective fractions were subjected to fluorescence microscopy based acridine orange/ethidium bromide (AO/EB) and Hoechst 33342 staining to determine apoptosis induction and DNA fragmentation assay. Comet and micronuclei assay were performed to assess genotoxicity. Cell cycle analysis was also performed. In vivo anti-tumor potential was evaluated by Ehrlich ascites carcinoma (EAC) model in mice. The alcoholic stem bark extract of M. elengi along with four fractions showed potential in vitro cytotoxicity in SRB assay. Of these, dichloromethane and ethyl acetate fractions were selected for further studies. The fractions revealed apoptosis inducing potential in AO/EB and Hoechst 33342 staining, which was further confirmed by DNA fragmentation assay. Genotoxic potential was revealed by comet and micronuclei assay. Fractions also exhibited specific cell cycle inhibition in G0/G1 phase. In EAC model, ethyl acetate fraction along with the standard (cisplatin) effectively reduced the increase in body weight compared to control and improved mean survival time. Both fractions were able to restore the altered hematological and biochemical parameters. Hence, M. elengi stem bark may be a possible therapeutic candidate having cytotoxic and anti-tumor potential.

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

PubMed

Laffon, B; Pásaro, E; Méndez, J

2001-04-05

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

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

PubMed

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

2015-09-01

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

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

PubMed

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

2002-10-01

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

Automated detection of irradiated food with the comet assay.

PubMed

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

2008-01-01

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

The 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.

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

PubMed

Patnaik, Rajashree; Padhy, Rabindra N

2018-05-11

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

Comet assay and micronucleus tests on Oreochromis niloticus (Perciforme: Cichlidae) exposed to raw sugarcane vinasse and to phisicochemical treated vinasse by pH adjustment with lime (CaO).

PubMed

Correia, Jorge E; Christofoletti, Cintya Ap; Ansoar-Rodríguez, Yadira; Guedes, Thays A; Fontanetti, Carmem S

2017-04-01

In Brazil vinasse, a main sugarcane distillery residue, stands out because every liter of alcohol generates 10-15 L of vinasse as waste. An alternative for the disposal of this waste is the fertirrigation of the sugarcane culture itself. However, the high amount released can saturate the soil and through leaching/percolation contaminate water resources. The aim of this study is verifying the toxic potential of vinasse in tilapias and effectiveness of the physicalchemical treatment of this waste with pH adjustment with lime (CaO). The comet assay and the micronucleus test were applied on animals exposed to dilutions of raw vinasse and vinasse adjusted to neutral pH. Bioassays with raw vinasse dilutions indicated a toxic and genotoxic potential; fish exposed to the highest concentration died less than 48 h after the exposure; the incidence of micronucleus was significantly higher when compared to negative control for all dilutions. For the comet assay, the scores of damage were statistically higher for all dilutions, with the exception of the 1% dillution. However, in the bioassay with the chemically treated vinasse (neutral pH), most fish in the 10% dilution survived and there was no significant difference when compared to the control. Damage scores in the comet assay were similar to the results of the untreated vinasse. The chemical treatment of vinasse with lime to neutralize the pH proved to be an effective alternative for the toxicity reduction of this residue, since it reduced the mortality of fish at higher concentrations and the incidence of damage to DNA. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

PubMed

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

2017-09-01

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

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

PubMed

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

2014-03-25

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

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.

Intermediate frequency magnetic field generated by a wireless power transmission device does not cause genotoxicity in vitro.

PubMed

Shi, Dejing; Zhu, Chunbo; Lu, Rengui; Mao, Shitong; Qi, Yanhua

2014-10-01

The aim of this study was to evaluate effects of intermediate frequency magnetic fields (IFMF) generated by a wireless power transmission (WPT) based on magnetic resonance from the perspective of cellular genotoxicity on cultured human lens epithelial cells (HLECs). We evaluated the effects of exposure to 90 kHz magnetic fields at 93.36 µT on cellular genotoxicity in vitro for 2 and 4 h. The magnetic flux density is approximately 3.5 times higher than the reference level recommended by the International Commission on Non-Ionizing Radiation Protection (ICNIRP) guidelines. For assessment of genotoxicity, we studied cellular proliferation, apoptosis and DNA damage by Cell Counting Kit-8 (CCK-8) assay, flow cytometry analysis, alkaline comet assay and phosphorylated histone H2AX (γH2AX) foci formation test. We did not detect any effect of a 90 kHz IFMF generated by WPT based on magnetic resonance on cell proliferation, apoptosis, comet assay, and γH2AX foci formation test. Our results indicated that exposure to 90 kHz IFMF generated by WPT based on magnetic resonance at 93.36 µT for 2 and 4 h does not cause detectable cellular genotoxicity. © 2014 Wiley Periodicals, Inc.

The organophosphate insecticide chlorpyrifos confers its genotoxic effects by inducing DNA damage and cell apoptosis.

PubMed

Li, Diqiu; Huang, Qingchun; Lu, Miaoqing; Zhang, Lei; Yang, Zhichuan; Zong, Mimi; Tao, Liming

2015-09-01

The organophosphate insecticide chlorpyrifos (CPF) is known to induce neurological effects, malformation and micronucleus formation, persistent developmental disorders, and maternal toxicity in rats and mice. The binding of chlorpyrifos with DNA to produce DNA adducts leads to an increasing social concern about the genotoxic risk of CPF in human, but CPF-induced cytotoxicity through DNA damage and cell apoptosis is not well understood. Here, we quantified the cytotoxicity and potential genotoxicity of CPF using the alkaline comet assay, γH2AX foci formation, and the DNA laddering assay in order to detect DNA damage and apoptosis in human HeLa and HEK293 cells in vitro. Drosophila S2 cells were used as a positive control. The alkaline comet assay showed that sublethal concentrations of CPF induced significant concentration-dependent increases in single-strand DNA breaks in the treated cells compared with the control. The percentage of γH2AX-positive HeLa cells revealed that CPF also causes DNA double-strand breaks in a time-dependent manner. Moreover, DNA fragmentation analysis demonstrated that exposure to CPF induced a significant concentration- and time-dependent increase in cell apoptosis. We conclude that CPF is a strongly genotoxic agent that induces DNA damage and cell apoptosis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Genotoxicity analysis of two halonitromethanes, a novel group of disinfection by-products (DBPs), in human cells treated in vitro

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

Liviac, Danae; Creus, Amadeu; Marcos, Ricard

Halonitromethanes (HNMs) constitute an emerging class of disinfection by-products (DBPs) produced when chlorine and/or ozone are used for water treatment. The HNMs are structurally similar to halomethanes, but have a nitro-group in place of hydrogen bonded to the central carbon atom. Since little information exists on the genotoxic potential of HNMs, a study has been carried out with two HNM compounds, namely trichloronitromethane (TCNM) and bromonitromethane (BNM) by using human cells. Primary damage induction has been measured with the Comet assay, which is used to determine both the repair kinetics of the induced damage and the proportion of induced oxidativemore » damage. In addition, the fixed DNA damage has been evaluated by using the micronucleus (MN) assay. The results obtained indicate that both compounds are genotoxic, inducing high levels of DNA breaks in the Comet assay, and that this DNA damage repairs well over time. In addition, oxidized bases constitute a high proportion of DNA-induced damage (50-75%). Contrarily, no positive effects were observed in the frequency of micronucleus, which measures both clastogenic and aneugenic effects, neither using TK6 cells nor peripheral blood lymphocytes. This lack of fixed genetic damage would minimize the potential mutagenic risk associated with HNMs exposure.« less

Houttuynia cordata Thunb extract modulates G0/G1 arrest and Fas/CD95-mediated death receptor apoptotic cell death in human lung cancer A549 cells

PubMed Central

2013-01-01

Background Houttuynia cordata Thunb (HCT) is commonly used in Taiwan and other Asian countries as an anti-inflammatory, antibacterial and antiviral herbal medicine. In this study, we investigated the anti-human lung cancer activity and growth inhibition mechanisms of HCT in human lung cancer A549 cells. Results In order to investigate effects of HCT on A549 cells, MTT assay was used to evaluate cell viability. Flow cytometry was employed for cell cycle analysis, DAPI staining, and the Comet assay was used for DNA fragmentation and DNA condensation. Western blot analysis was used to analyze cell cycle and apoptotic related protein levels. HCT induced morphological changes including cell shrinkage and rounding. HCT increased the G0/G1 and Sub-G1 cell (apoptosis) populations and HCT increased DNA fragmentation and DNA condensation as revealed by DAPI staining and the Comet assay. HCT induced activation of caspase-8 and caspase-3. Fas/CD95 protein levels were increased in HCT-treated A549 cells. The G0/G1 phase and apoptotic related protein levels of cyclin D1, cyclin A, CDK 4 and CDK 2 were decreased, and p27, caspase-8 and caspase-3 were increased in A549 cells after HCT treatment. Conclusions The results demonstrated that HCT-induced G0/G1 phase arrest and Fas/CD95-dependent apoptotic cell death in A549 cells PMID:23506616

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

PubMed

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

2013-01-01

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

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

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

PubMed

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

2013-11-01

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

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

PubMed Central

Pfuhler, Stefan

2013-01-01

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

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

PubMed

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

2010-06-01

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

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.

Genotoxicity studies on DNA-interactive telomerase inhibitors with application as anti-cancer agents.

PubMed

Harrington, Dean J; Cemeli, Eduardo; Carder, Joanna; Fearnley, Jamie; Estdale, Sian; Perry, Philip J; Jenkins, Terence C; Anderson, Diana

2003-01-01

Telomerase-targeted strategies have aroused recent interest in anti-cancer chemotherapy, because DNA-binding drugs can interact with high-order tetraplex rather than double-stranded (duplex) DNA targets in tumour cells. However, the protracted cell-drug exposure times necessary for clinical application require that telomerase inhibitory efficacy must be accompanied by both low inherent cytotoxicity and the absence of mutagenicity/genotoxicity. For the first time, the genotoxicity of a number of structurally diverse DNA-interactive telomerase inhibitors is examined in the Ames test using six Salmonella typhimurium bacterial strains (TA1535, TA1537, TA1538, TA98, TA100, and TA102). DNA damage induced by each agent was also assessed using the Comet assay with human lymphocytes. The two assay procedures revealed markedly different genotoxicity profiles that are likely to reflect differences in metabolism and/or DNA repair between bacterial and mammalian cells. The mutational spectrum for a biologically active fluorenone derivative, shown to be mutagenic in the TA100 strain, was characterised using a novel and rapid assay method based upon PCR amplification of a fragment of the hisG46 allele, followed by RFLP analysis. Preliminary analysis indicates that the majority (84%) of mutations induced by this compound are C --> A transversions at position 2 of the missense proline codon of the hisG46 allele. However, despite its genotoxic bacterial profile, this fluorenone agent gave a negative response in the Comet assay, and demonstrates how unwanted systemic effects (e.g., cytotoxicity and genotoxicity) can be prevented or ameliorated through suitable molecular fine-tuning of a candidate drug in targeted human tumour cells. Copyright 2003 Wiley-Liss, Inc.

Identification of irradiated refrigerated pork with the DNA comet assay

NASA Astrophysics Data System (ADS)

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

2004-09-01

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

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

PubMed

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

2009-05-17

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

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

PubMed

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

2009-02-15

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

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

PubMed

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

2002-01-01

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

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.

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

PubMed

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

2016-01-01

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

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

PubMed Central

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

2016-01-01

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

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

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

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

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

Evaluation of cytotoxicity, genotoxicity and teratogenicity of marine sediments from Qingdao coastal areas using in vitro fish cell assay, comet assay and zebrafish embryo test.

PubMed

Yang, Fan; Zhang, Qianqian; Guo, Huarong; Zhang, Shicui

2010-10-01

Marine sediments are often a final sink for numerous anthropogenic contaminants and may impose serious effects on benthic organisms and ecosystem. An in vitro cell assay using a cell line derived from flounder gill (FG) cells, an in vitro comet assay in FG cells, and an in vitro zebrafish embryo assay were used to evaluate the in vitro cytotoxicity (measured by MTT reduction), genotoxicity and teratogenicity of crude sediment extracts of Li Cang (LC), Zhan Qiao (ZQ) and Olympic Sailing Center (OSC) from Qingdao coastal area. Sediments from the three sites displayed different cytotoxicity, genotoxicity and teratogenicity potencies; however, all three assays yielded similar LOECs (lowest observed effect concentration) for each site, suggesting that the assays were equally sensitive to and suitable for initial screening of the LOECs of marine sediments. The cytotoxicity, genotoxicity and teratogenicity for these three sampling sites were in the same order of LC>ZQ>OSC, indicating different degrees of contamination. Interestingly, trials with the three sediment extracts at the doses inducing a similar cytotoxicity as evaluated with MTT reduction did not produce similar genotoxicity and teratogenicity, with the genotoxic and teratogenic activities of LC and ZQ extracts being markedly higher than those of OSC sediments. These findings indicate that cytotoxicity does not form a fully equivalent toxicity index with that of genotoxicity and teratogenicity. Therefore, in order to assess the true toxic potential of marine sediments, all three assays should be performed. Analysis of 16 EPA (US Environmental Protection Agency) priority PAHs in these three sediment samples showed a clear correlation between PAH concentrations and sediment toxicities, with a higher PAH content corresponding to higher toxicity although PAHs are surely not the only cause. Copyright © 2010 Elsevier Ltd. All rights reserved.

Pharmaceutical wastewater being composite mixture of environmental pollutants may be associated with mutagenicity and genotoxicity.

PubMed

Sharif, Ali; Ashraf, Muhammad; Anjum, Aftab Ahmed; Javeed, Aqeel; Altaf, Imran; Akhtar, Muhammad Furqan; Abbas, Mateen; Akhtar, Bushra; Saleem, Ammara

2016-02-01

Pharmaceutical industries are amongst the foremost contributor to industrial waste. Ecological well-being is endangered owing to its facile discharge. In the present study, heavy metals and organic contaminants in waste water were characterized using atomic absorption spectrophotometer and GC-MS, respectively. Mutagenicity and genotoxic potential of pharmaceutical waste water were investigated through bacterial reverse mutation assay and in vitro comet assay, respectively. Ames test and comet assay of first sample were carried out at concentrations of 100, 50, 25, 12.5, 6.25 % v/v effluent with distilled water. Chromium (Cr), lead (Pb), arsenic (As), and cadmium (Cd) were found in high concentrations as compared to WHO- and EPA-recommended maximum limits. Arsenic was found to be the most abundant metal and its maximum concentration was 0.8 mg.L(-1). GC-MS revealed the presence of lignocaine, digitoxin, trimethoprim, caffeine, and vitamin E in waste water. Dose-dependent decrease in mutagenic index was observed in both strains. Substantial increase in mutagenicity was observed for TA-100, when assay was done by incorporating an enzyme activation system, whereas a slight increase was detected for TA-102. In vitro comet assay of waste water exhibited decrease in damage index and percentage fragmentation with the increase in dilution of waste water. Tail length also decreased with an increase in the dilution factor of waste water. These findings suggest that pharmaceutical waste water being a mix of different heavy metals and organic contaminants may have a potent mutagenic and genotoxic effect on exposed living organisms.

Assessment of status of three water bodies in Serbia based on tissue metal and metalloid concentration (ICP-OES) and genotoxicity (comet assay).

PubMed

Sunjog, Karolina; Kolarević, Stoimir; Kračun-Kolarević, Margareta; Višnjić-Jeftić, Željka; Skorić, Stefan; Gačić, Zoran; Lenhardt, Mirjana; Vasić, Nebojša; Vuković-Gačić, Branka

2016-06-01

Metals and metalloids are natural components of the biosphere, which are not produced per se by human beings, but whose form and distribution can be affected by human activities. Like all substances, they are a contaminant if present in excess compared to background levels and/or in a form that would not normally occur in the environment. Samples of liver, gills, gonads and muscle from European chub, Squalius cephalus, were analyzed for Al, As, B, Ba, Cr, Cu, Fe, Hg, Mn, Mo, Sr and Zn using inductively coupled plasma optical emission spectrometry (ICP-OES) to highlight the importance of tissue selection in monitoring research. The comet assay or single cell gel electrophoresis (SCGE) was selected as an in vivo genotoxicity assay, a rapid and sensitive method for measuring genotoxic effects in blood, liver and gills of the European chub. Microscopic images of comets were scored using Comet IV Computer Software (Perceptive Instruments, UK). The objective of our study was to investigate two reservoirs, Zlatar and Garasi, and one river, Pestan by: (i) determining and comparing metal and metalloid concentrations in sediment, water and tissues of European chub: liver, gills, muscle and gonads (ii) comparing these findings with genotoxicity of water expressed through DNA damage of fish tissues. A clear link between the level of metals in water, sediment and tissues and between metal and genotoxicity levels at examined sites was not found. This suggests that other xenobiotics (possibly the organic compounds), contribute to DNA damage. Copyright © 2016 Elsevier Ltd. All rights reserved.

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.

Biodosimetry of Persons Chronically Exposed to Low and Therapeutic Doses of Ionizing Radiation

PubMed Central

Zedginidze, Alla; Namchevadze, Ema; Ormocadze, George; Kapanadze, Archil; Nikuradze, Tamara; Lomidze, Darejan

2016-01-01

Dynamic changes of the chromosomal aberrations and the DNA damage were analyzed in individuals exposed to low and therapeutic doses of radiation. The investigation included 37 persons living in areas where the radioactive sources were discovered 10–12 years ago. It was established by biodosimetry methods that the examined persons had absorbed dose of 0.2–0.7 Gy or had increased number of chromosomal aberrations, though insufficient to determine a dose. Clinical examination, chromosomal analysis, and assay of DNA damage by the comet (single-cell gel electrophoresis) assay were carried out. There was no correlation between the doses received 10 years ago and the cytogenetic changes with clinical outcome. The effect of the local fractionated gamma-irradiation with doses of 40–70 Gy was studied in cancer patients with localized head and neck tumors. The study of chromosomal abnormalities, the DNA damages by the comet assay, and the micronuclei detection of the buccal cells revealed a statistically significant correlation between the initial cytogenetic indices in cancer patients and their dynamic changes during and after the radiation exposure. In addition, the correlation was detected between the initial cytogenetic parameters and the functional stage of red blood system. Our results allow us to conclude that there is a need for further research to estimate the individual radiation risk to optimize and individualize the subsequent medical management of radiotherapy. PMID:28217288

Biodosimetry of Persons Chronically Exposed to Low and Therapeutic Doses of Ionizing Radiation.

PubMed

Zedginidze, Alla; Namchevadze, Ema; Ormocadze, George; Kapanadze, Archil; Nikuradze, Tamara; Lomidze, Darejan

2016-01-01

Dynamic changes of the chromosomal aberrations and the DNA damage were analyzed in individuals exposed to low and therapeutic doses of radiation. The investigation included 37 persons living in areas where the radioactive sources were discovered 10-12 years ago. It was established by biodosimetry methods that the examined persons had absorbed dose of 0.2-0.7 Gy or had increased number of chromosomal aberrations, though insufficient to determine a dose. Clinical examination, chromosomal analysis, and assay of DNA damage by the comet (single-cell gel electrophoresis) assay were carried out. There was no correlation between the doses received 10 years ago and the cytogenetic changes with clinical outcome. The effect of the local fractionated gamma-irradiation with doses of 40-70 Gy was studied in cancer patients with localized head and neck tumors. The study of chromosomal abnormalities, the DNA damages by the comet assay, and the micronuclei detection of the buccal cells revealed a statistically significant correlation between the initial cytogenetic indices in cancer patients and their dynamic changes during and after the radiation exposure. In addition, the correlation was detected between the initial cytogenetic parameters and the functional stage of red blood system. Our results allow us to conclude that there is a need for further research to estimate the individual radiation risk to optimize and individualize the subsequent medical management of radiotherapy.

Radio-protective effect of cinnamic acid, a phenolic phytochemical, on genomic instability induced by X-rays in human blood lymphocytes in vitro.

PubMed

Cinkilic, Nilufer; Tüzün, Ece; Çetintaş, Sibel Kahraman; Vatan, Özgür; Yılmaz, Dilek; Çavaş, Tolga; Tunç, Sema; Özkan, Lütfi; Bilaloğlu, Rahmi

2014-08-01

The present study was designed to determine the protective activity of cinnamic acid against induction by X-rays of genomic instability in normal human blood lymphocytes. This radio-protective activity was assessed by use of the cytokinesis-block micronucleus test and the alkaline comet assay, with human blood lymphocytes isolated from two healthy donors. A Siemens Mevatron MD2 (Siemens AG, USA, 1994) linear accelerator was used for the irradiation with 1 or 2 Gy. Treatment of the lymphocytes with cinnamic acid prior to irradiation reduced the number of micronuclei when compared with that in control samples. Treatment with cinnamic acid without irradiation did not increase the number of micronuclei and did not show a cytostatic effect in the lymphocytes. The results of the alkaline comet assay revealed that cinnamic acid reduces the DNA damage induced by X-rays, showing a significant radio-protective effect. Cinnamic acid decreased the frequency of irradiation-induced micronuclei by 16-55% and reduced DNA breakage by 17-50%, as determined by the alkaline comet assay. Cinnamic acid may thus act as a radio-protective compound, and future studies may focus on elucidating the mechanism by which cinnamic acid offers radioprotection. Copyright © 2014 Elsevier B.V. All rights reserved.

Role of Macronutrients and Micronutrients in DNA Damage: Results From a Food Frequency Questionnaire.

PubMed

Ladeira, Carina; Carolino, Elisabete; Gomes, Manuel C; Brito, Miguel

2017-01-01

The links between diet and genomic instability have been under investigation for several decades, and evidence suggests a significant causal or preventive role for various dietary factors. This study investigates the influence of macronutrients (calories, protein, and glucides) and micronutrients, such as vitamins and minerals, as assessed by a food frequency questionnaire, on genotoxicity biomarkers measured by cytokinesis-blocked micronucleus assay and comet assay. The results found significant positive and negative correlations. Micronucleus frequency tends to increase with higher intake of caffeine, calcium, magnesium, zinc, and protein ( P < .05, Spearman correlation). Calorie and omega-6 intakes are negatively correlated with DNA damage measured by the comet assay. These results are somewhat controversial because some of the correlations found are contrary to dominant views in the literature; however, we suggest that unraveling the association between diet and genetic instability requires a much better understanding of the modulating role of macronutrients and micronutrients.

Absence of genotoxic effects of the chalcone (E)-1-(2-hydroxyphenyl)-3-(4-methylphenyl)-prop-2-en-1-one) and its potential chemoprevention against DNA damage using in vitro and in vivo assays

PubMed Central

2017-01-01

The chalcone (E)-1-(2-hydroxyphenyl)-3-(4-methylphenyl)-prop-2-en-1-one), or 2HMC, displays antileishmanial, antimalarial, and antioxidant activities. The aim of this study was to investigate the cytotoxic, genotoxic, mutagenic, and protective effects of 2HMC using the Ames mutagenicity test, the mouse bone marrow micronucleus test, and the comet assay in mice. In the assessment using the Ames test, 2HMC did not increase the number of His+ revertants in Salmonella typhimurium strains, demonstrating lack of mutagenicity. 2HMC showed no significant increase in micronucleated polychromatic erythrocyte frequency (MNPCE) in the micronucleus test, or in DNA strand breaks using the comet assay, evidencing absence of genotoxicity. Regarding cytotoxicity, 2HMC exhibited moderate cytotoxicity in mouse bone marrow cells by micronucleus test. 2HMC showed antimutagenic action in co-administration with the positive controls, sodium azide (SA) and 4-nitroquinoline-1-oxide (4NQO), in the Ames test. Co-administered and mainly pre-administered with cyclophosphamide (CPA), 2HMC caused a decrease in the frequency of MNPCE using the micronucleus test and in DNA strand breaks using the comet assay. Thus, 2HMC exhibited antimutagenic and antigenotoxic effects, displaying a DNA-protective effect against CPA, SA, and 4NQO carcinogens. In conclusion, 2HMC presented antimutagenic, antigenotoxic and moderate cytotoxic effects; therefore it is a promising molecule for cancer prevention. PMID:28207781

Absence of genotoxic effects of the chalcone (E)-1-(2-hydroxyphenyl)-3-(4-methylphenyl)-prop-2-en-1-one) and its potential chemoprevention against DNA damage using in vitro and in vivo assays.

PubMed

Lima, Débora Cristina da Silva; Vale, Camila Regina do; Véras, Jefferson Hollanda; Bernardes, Aline; Pérez, Caridad Noda; Chen-Chen, Lee

2017-01-01

The chalcone (E)-1-(2-hydroxyphenyl)-3-(4-methylphenyl)-prop-2-en-1-one), or 2HMC, displays antileishmanial, antimalarial, and antioxidant activities. The aim of this study was to investigate the cytotoxic, genotoxic, mutagenic, and protective effects of 2HMC using the Ames mutagenicity test, the mouse bone marrow micronucleus test, and the comet assay in mice. In the assessment using the Ames test, 2HMC did not increase the number of His+ revertants in Salmonella typhimurium strains, demonstrating lack of mutagenicity. 2HMC showed no significant increase in micronucleated polychromatic erythrocyte frequency (MNPCE) in the micronucleus test, or in DNA strand breaks using the comet assay, evidencing absence of genotoxicity. Regarding cytotoxicity, 2HMC exhibited moderate cytotoxicity in mouse bone marrow cells by micronucleus test. 2HMC showed antimutagenic action in co-administration with the positive controls, sodium azide (SA) and 4-nitroquinoline-1-oxide (4NQO), in the Ames test. Co-administered and mainly pre-administered with cyclophosphamide (CPA), 2HMC caused a decrease in the frequency of MNPCE using the micronucleus test and in DNA strand breaks using the comet assay. Thus, 2HMC exhibited antimutagenic and antigenotoxic effects, displaying a DNA-protective effect against CPA, SA, and 4NQO carcinogens. In conclusion, 2HMC presented antimutagenic, antigenotoxic and moderate cytotoxic effects; therefore it is a promising molecule for cancer prevention.

Impact of cadmium on forage kale (Brassica oleracea var. viridis cv "Prover") after 3-,10- and 56-day exposure to a Cd-spiked field soil.

PubMed

Bernard, Fabien; Dumez, Sylvain; Lemière, Sébastien; Platel, Anne; Nesslany, Fabrice; Deram, Annabelle; Vandenbulcke, Franck; Cuny, Damien

2018-03-15

Cadmium (Cd) is a highly toxic element for living organisms and is widespread in metal-contaminated soils. As organisms which can grow up on these polluted areas, plants have some protection mechanisms against Cd issues. Among the plant kingdom, the Brassicaceae family includes species which are known to be able to tolerate and accumulate Cd in their tissues. In this study, Brassica oleracea var. viridis cv "Prover" was exposed to a range of artificially Cd-contaminated soils (from 2.5 up to 20 mg kg -1 ) during 3, 10, and 56 days and the effects on life traits, photosynthesis activity, antioxidant enzymatic activities were studied. Metal accumulation was quantified, as well as DNA damage, by means of the comet assay and immunodetection of 8-OHdG levels. Globally, B. oleracea was relatively tolerant to those Cd exposures. However, comet assay and detection of 8-OHdG revealed some DNA damage but which are not significant. According to metal accumulation analysis, B. oleracea var. viridis cv Prover could be a good candidate for alternative growing in contaminated areas.

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

PubMed

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

2016-01-01

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

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

PubMed Central

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

2016-01-01

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

In vivo antigenotoxic activity of watercress juice (Nasturtium officinale) against induced DNA damage.

PubMed

Casanova, Natalia A; Ariagno, Julia I; López Nigro, Marcela M; Mendeluk, Gabriela R; de los A Gette, María; Petenatti, Elisa; Palaoro, Luis A; Carballo, Marta A

2013-09-01

The present study was carried out to investigate the genotoxicity as well as possible protective activity against damage induced by cyclophosphamide (CP) of the aqueous juice of watercress (Nasturtium officinale, W.T. Aiton) in vivo. Male and female Swiss mice 7-8 weeks old (N = 48) were treated by gavage with 1 g kg(-1) body weight and 0.5 g kg(-1) body weight of watercress juice during 15 consecutive days. Genotoxicity and its possible protective effect were tested by the comet assay in peripheral blood cells and the micronucleus test in bone marrow. In addition, biopsies of the bladder, epididymis and testicles of mice were performed to extend the experimental design. Watercress juice per se did not induce genetic damage according to the comet assay and micronucleus study, exhibiting a protective activity against CP (P < 0.05 and P < 0.001, respectively). The comparative analysis of bladder histological changes obtained in the watercress plus CP group against those treated with CP alone suggests a probable protective effect. Further studies are needed in order to establish the protective role of watercress juice against DNA damage. Copyright © 2012 John Wiley & Sons, Ltd.

Cytogenetic status of human lymphocytes after exposure to low concentrations of p,p'-DDT, and its metabolites (p,p'-DDE, and p,p'-DDD) in vitro.

PubMed

Gerić, Marko; Ceraj-Cerić, Nikolina; Gajski, Goran; Vasilić, Želimira; Capuder, Željka; Garaj-Vrhovac, Vera

2012-06-01

Despite that the use of DDT has been restricted for more than 40 years to malaria affected areas, low doses of this pesticide and its metabolites DDE and DDD can be found in the environment around the world. Although it has been shown that these pollutants induce cell and DNA damage, the mechanisms of their cytogenotoxic activity remains largely unknown. This study looks into their possible genotoxic effects, at doses that can be found in body fluids, on human lymphocytes using the cytokinesis-block micronucleus assay and the comet assay. After exposure for 1, 6, and 24 h compounds p,p'-DDT (0.1 μg mL(-1)), p,p'-DDE (4.1 μg mL(-1)), and p,p'-DDD (3.9 μg mL(-1)) showed increase in DNA damage. The most significant results were observed at exposure period of 24 h where number of micronucleated cells increased from control 2.5±0.71 to 23.5±3.54, 13.5±0.71, and 16.5±6.36 for DDT, DDE, and DDD, respectively. Similar effect was observed using comet test where the percentage of DNA in comets tail increased from control 1.81±0.16 to 17.24±0.55, 11.21±0.56 and 9.28±0.50 for each compound, respectively. At the same time Fpg-comet assay failed to report induction of oxidative DNA damage of these pollutants. Additionally, the type of cell death was determined using diffusion assay and necrosis dominated. Our findings suggest that even at low concentrations, these pesticides could induce cytogenetic damage to human peripheral blood lymphocytes and in that manner have the impact on human health as well. Copyright © 2012 Elsevier Ltd. All rights reserved.

Dragon's blood Croton palanostigma induces genotoxic effects in mice.

PubMed

Maistro, Edson Luis; Ganthous, Giulia; Machado, Marina da Silva; Zermiani, Tailyn; Andrade, Sérgio Faloni de; Rosa, Paulo Cesar Pires; Perazzo, Fabio Ferreira

2013-05-20

Dragon's blood is a dark-red sap produced by species from the genus Croton (Euphorbiaceae), which has been used as a famous traditional medicine since ancient times in many countries, with scarce data about its safe use in humans. In this research, we studied genotoxicity and clastogenicity of Croton palanostigma sap using the comet assay and micronucleus test in cells of mice submitted to acute treatment. HPLC analysis was performed to identify the main components of the sap. The sap was administered by oral gavage at doses of 300 mg/kg, 1,000 mg/kg and 2,000 mg/kg. For the analysis, the comet assay was performed on the leukocytes and liver cells collected 24h after treatment, and the micronucleus test (MN) on bone marrow cells. Cytotoxicity was assessed by scoring 200 consecutive polychromatic (PCE) and normochromatic (NCE) erythrocytes (PCE/NCE ratio). The alkaloid taspine was the main compound indentified in the crude sap of Croton palanostigma. The results of the genotoxicity assessment show that all sap doses tested produced genotoxic effects in leukocytes and liver cells and also produced clastogenic/aneugenic effects in bone marrow cells of mice at the two higher doses tested. The PCE/NCE ratio indicated no cytotoxicity. The data obtained suggest caution in the use of Croton palanostigma sap by humans considering its risk of carcinogenesis. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Tests for genotoxicity and mutagenicity of furan and its metabolite cis-2-butene-1,4-dial in L5178Y tk+/- mouse lymphoma cells.

PubMed

Kellert, Marco; Brink, Andreas; Richter, Ingrid; Schlatter, Josef; Lutz, Werner K

2008-12-08

Furan is found in various food items and is cytotoxic and carcinogenic in the liver of rats and mice. Metabolism of furan includes the formation of an unsaturated dialdehyde, cis-2-butene-1,4-dial (BDA). In view of the multifunctional electrophilic reactivity of BDA, adduct formation with protein and DNA may explain some of the toxic effects. Short-term tests for genotoxicity of furan in mammalian cells are inconclusive, little is known for BDA. We investigated BDA generated by hydrolysis of 2,5-diacetoxy-2,5-dihydrofuran for genotoxicity in L5178Y tk+/- mouse lymphoma cells using standard procedures for the comet assay, the micronucleus test, and the mouse lymphoma thymidine kinase gene mutation assay, using 4-h incubation periods. Cytotoxicity was remarkable: cell viability at concentrations>or=50 microM was reduced to <50%. In the dose range up to 25 microM, viability was >90%. Measures of comet-tail length and thymidine-kinase mutant frequency were increased 1.6- and 2.4-fold above control, respectively. Analysis of three fully independent replicates with a linear mixed-effects model showed a highly significant increase with concentration for both endpoints. Compared to methyl methanesulfonate used as a positive control, BDA was of similar potency with respect to genotoxicity, but it was much more cytotoxic. Furan added to cell cultures at doses that resulted in time-averaged effective concentrations of up to 3100 microM was neither cytotoxic nor genotoxic. A potential cross-linking activity of BDA was investigated by checking whether gamma radiation-induced DNA migration in the comet assay could be reduced by pre-treatment with BDA. In contrast to the effect of the positive control glutaraldehyde, BDA treatment did not reduce the comet tail length. On the contrary, an increase was observed at >or=100 microM BDA, which was attributable to early apoptotic cells. Although BDA was found to be a relatively potent genotoxic agent in terms of the concentration necessary to double the background measures, cytotoxicity strongly limited the concentration range that produced interpretable results. This may explain some of the inconclusive results and indicates that non-genotoxic effects must be taken into account in the discussion of the modes of toxic and carcinogenic action of furan.

Chicken Fetal Liver DNA Damage and Adduct Formation by Activation-Dependent DNA-Reactive Carcinogens and Related Compounds of Several Structural Classes

PubMed Central

Williams, Gary M.; Duan, Jian-Dong; Brunnemann, Klaus D.; Iatropoulos, Michael J.; Vock, Esther; Deschl, Ulrich

2014-01-01

The chicken egg genotoxicity assay (CEGA), which utilizes the liver of an intact and aseptic embryo-fetal test organism, was evaluated using four activation-dependent DNA-reactive carcinogens and four structurally related less potent carcinogens or non-carcinogens. In the assay, three daily doses of test substances were administered to eggs containing 9–11-day-old fetuses and the fetal livers were assessed for two endpoints, DNA breaks using the alkaline single cell gel electrophoresis (comet) assay and DNA adducts using the 32P-nucleotide postlabeling (NPL) assay. The effects of four carcinogens of different structures requiring distinct pathways of bioactivation, i.e., 2-acetylaminofluorene (AAF), aflatoxin B1 (AFB1), benzo[a]pyrene (B[a]P), and diethylnitrosamine (DEN), were compared with structurally related non-carcinogens fluorene (FLU) and benzo[e]pyrene (B[e]P) or weak carcinogens, aflatoxin B2 (AFB2) and N-nitrosodiethanolamine (NDELA). The four carcinogens all produced DNA breaks at microgram or low milligram total doses, whereas less potent carcinogens and non-carcinogens yielded borderline or negative results, respectively, at higher doses. AAF and B[a]P produced DNA adducts, whereas none was found with the related comparators FLU or B[e]P, consistent with comet results. DEN and NDELA were also negative for adducts, as expected in the case of DEN for an alkylating agent in the standard NPL assay. Also, AFB1 and AFB2 were negative in NPL, as expected, due to the nature of ring opened aflatoxin adducts, which are resistant to enzymatic digestion. Thus, the CEGA, using comet and NPL, is capable of detection of the genotoxicity of diverse DNA-reactive carcinogens, while not yielding false positives for non-carcinogens. PMID:24973097

Tungsten carbide-cobalt as a nanoparticulate reference positive control in in vitro genotoxicity assays.

PubMed

Moche, Hélène; Chevalier, Dany; Barois, Nicolas; Lorge, Elisabeth; Claude, Nancy; Nesslany, Fabrice

2014-01-01

With the increasing human exposure to nanoparticles (NP), the evaluation of their genotoxic potential is of significant importance. However, relevance for NP of the routinely used in vitro genotoxicity assays is often questioned, and a nanoparticulate reference positive control would therefore constitute an important step to a better testing of NP, ensuring that test systems are really appropriate. In this study, we investigated the possibility of using tungsten carbide-cobalt (WC-Co) NP as reference positive control in in vitro genotoxicity assays, including 2 regulatory assays, the mouse lymphoma assay and the micronucleus assay, and in the Comet assay, recommended for the toxicological evaluation of nanomedicines by the French Agency of Human Health Products (Afssaps). Through these assays, we were able to study different genetic endpoints in 2 cell types commonly used in regulatory genotoxicity assays: the L5178Y mouse lymphoma cell line and primary cultures of human lymphocytes. Our results showed that the use of WC-Co NP as positive control in in vitro genotoxicity assays was conceivable, but that different parameters have to be considered, such as cell type and treatment schedule. L5178Y mouse lymphoma cells did not provide satisfactory results in the 3 performed tests. However, human lymphocytes were more sensitive to genotoxic effects induced by WC-Co NP, particularly after a 24-h treatment in the in vitro micronucleus assay and after a 4-h treatment in the in vitro Comet assay. Under such conditions, WC-Co could be used as a nanoparticulate reference positive control in these assays.

Evaluating the potential genotoxicity of phthalates esters (PAEs) in perfumes using in vitro assays.

PubMed

Al-Saleh, Iman; Al-Rajudi, Tahreer; Al-Qudaihi, Ghofran; Manogaran, Pulicat

2017-10-01

We previously reported high levels of phthalate esters (PAEs) added as solvents or fixatives in 47 brands of perfumes. Diethyl phthalate was the most abundant compound (0.232-23,649 ppm), and 83.3% of the perfumes had levels >1 ppm, the threshold limit cited by a Greenpeace investigation. All samples had dimethyl phthalate levels higher than its threshold limit of 0.1 ppm, and 88, 38, and 7% of the perfumes had benzyl butyl phthalate, di(2-ethylhexyl) phthalate, and dibutyl phthalate levels, respectively, above their threshold limits. The role of PAEs as endocrine disruptors has been well documented, but their effect on genotoxic behavior has received little attention. We used in vitro single-cell gel electrophoresis (comet) and micronucleus (MN) assays with human lymphoblastoid TK6 cells to evaluate the genotoxic potency of 42 of the same perfumes and to determine its association with PAEs. All perfumes induced more DNA damage than a negative control (NEG), ≥ 90% of the samples caused more damage than cells treated with the vehicles possibly used in perfume's preparations such as methanol (ME) and ethanol (ET), and 11.6% of the perfumes caused more DNA damage than a positive control (hydrogen peroxide). Chromosome breakage expressed as MN frequency was higher in cells treated with 71.4, 64.3, 57.1, and 4.8% of the perfumes than in NEG, cells treated with ME or ET, and another positive control (x-rays), respectively. The genotoxic responses in the comet and MN assays were not correlated. The comet assay indicated that the damage in TK6 cells treated with five PAEs at concentrations of 0.05 and 0.2 ppm either individually or as a mixture did not differ significantly from the damage in cells treated with the perfumes. Unlike the comet assay, the sensitivity of the MN assay to PAEs was weak at both low and high concentrations, and MN frequencies were generally low. This study demonstrates for the first time the possible contribution of PAEs in perfumes to DNA damage and suggests that their use as solvents or fixatives should be regulated. Other ingredients with mutagenic/genotoxic properties, however, may also have contributed to the DNA damage. Future studies should focus on applying a series of assays that use different cellular models with various endpoints to identify the spectrum of genotoxic mechanisms involved.

Summary of major conclusions from the 6th International Workshop on Genotoxicity Testing (IWGT), Foz do Iguacu, Brazil

EPA Science Inventory

The paper describes major conclusions of working groups convened in the following areas: comet assay; micronucleus test in the liver and organs other than bone marrow; pig-A assay; quantitative approaches to genotoxicity risk assessment; and approaches for identifying germ cell m...

Plant genotoxicity: a molecular cytogenetic approach in plant bioassays.

PubMed

Maluszynska, Jolanta; Juchimiuk, Jolanta

2005-06-01

It is important for the prevention of DNA changes caused by environment to understand the biological consequences of DNA damages and their molecular modes of action that lead to repair or alterations of the genetic material. Numerous genotoxicity assay systems have been developed to identify DNA reactive compounds. The available data show that plant bioassays are important tests in the detection of genotoxic contamination in the environment and the establishment of controlling systems. Plant system can detect a wide range of genetic damage, including gene mutations and chromosome aberrations. Recently introduced molecular cytogenetic methods allow analysis of genotoxicity, both at the chromosomal and DNA level. FISH gives a new possibility of the detection and analysis of chromosomal rearrangements in a great detail. DNA fragmentation can be estimated using the TUNEL test and the single cell gel electrophoresis (Comet assay).

Use of sensitive methods for detection of DNA damage on human lymphocytes exposed to p,p'-DDT: Comet assay and new criteria for scoring micronucleus test.

PubMed

Gajski, Goran; Ravlic, Sanda; Capuder, Zeljka; Garaj-Vrhovac, Vera

2007-08-01

Wide distribution, stability and long persistence in the environment of dichlorodiphenyltrichloroethane (DDT), probably the best-known and most useful insecticide in the world, imposes the need for further examination of the effect of this chemical on human health and especially on the human genome. In this study, peripheral blood human lymphocytes from a healthy donor were exposed to 0.025 mg/L concentration of p,p'-DDT at different time periods (1, 2, 24 and 48 h). For the assessment of genotoxic effect, the new criteria for scoring micronucleus test and alkaline comet assay were used. Both methods showed that p,p'-DDT induces DNA damage in low concentration used in this research. Results of micronucleus test showed a statistically significant (p < 0.05) genotoxic effect of p,p'-DDT on human lymphocytes compared with corresponding control and a different exposure time. A comet assay also showed increased DNA damage caused in p,p'-DDT-exposed human lymphocytes than in corresponding control cells for the tail length. Results obtained by measuring the level of DNA migration and incidence of micronuclei (MN), nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) indicate the sensitivity of these tests and their application in detection of primary genome damage after long-term exposure to establish the effect of p,p'-DDT on human genome.

Hot water extract of Chlorella vulgaris induced DNA damage and apoptosis

PubMed Central

Yusof, Yasmin Anum Mohd; Md. Saad, Suhana; Makpol, Suzana; Shamaan, Nor Aripin; Ngah, Wan Zurinah Wan

2010-01-01

OBJECTIVES: The aim of this study was to determine the antiproliferative and apoptotic effects of hot water extracts of Chlorella vulgaris on hepatoma cell line HepG2. INTRODUCTION: The search for food and spices that can induce apoptosis in cancer cells has been a major study interest in the last decade. Chlorella vulgaris, a unicellular green algae, has been reported to have antioxidant and anti‐cancer properties. However, its chemopreventive effects in inhibiting the growth of cancer cells have not been studied in great detail. METHODS: HepG2 liver cancer cells and WRL68 normal liver cells were treated with various concentrations (0‐4 mg/ml) of hot water extract of C. vulgaris after 24 hours incubation. Apoptosis rate was evaluated by TUNEL assay while DNA damage was assessed by Comet assay. Apoptosis proteins were evaluated by Western blot analysis. RESULTS: Chlorella vulgaris decreased the number of viable HepG2 cells in a dose dependent manner (p < 0.05), with an IC50 of 1.6 mg/ml. DNA damage as measured by Comet assay was increased in HepG2 cells at all concentrations of Chlorella vulgaris tested. Evaluation of apoptosis by TUNEL assay showed that Chlorella vulgaris induced a higher apoptotic rate (70%) in HepG2 cells compared to normal liver cells, WRL68 (15%). Western blot analysis showed increased expression of pro‐ apoptotic proteins P53, Bax and caspase‐3 in the HepG2 cells compared to normal liver cells WRL68, and decreased expression of the anti‐apoptotic protein Bcl‐2. CONCLUSIONS: Chlorella vulgaris may have anti‐cancer effects by inducing apoptosis signaling cascades via an increased expression of P53, Bax and caspase‐3 proteins and through a reduction of Bcl‐2 protein, which subsequently lead to increased DNA damage and apoptosis. PMID:21340229

Genetic damage induced by organic extract of coke oven emissions on human bronchial epithelial cells.

PubMed

Zhai, Qingfeng; Duan, Huawei; Wang, Yadong; Huang, Chuanfeng; Niu, Yong; Dai, Yufei; Bin, Ping; Liu, Qingjun; Chen, Wen; Ma, Junxiang; Zheng, Yuxin

2012-08-01

Coke oven emissions are known as human carcinogen, which is a complex mixture of polycyclic aromatic hydrocarbon. In this study, we aimed to clarify the mechanism of action of coke oven emissions induced carcinogenesis and to identify biomarkers of early biological effects in a human bronchial epithelial cell line with CYP1A1 activity (HBE-CYP1A1). Particulate matter was collected in the oven area on glass filter, extracted and analyzed by GC/MS. DNA breaks and oxidative damage were evaluated by alkaline and endonucleases (FPG, hOGG1 and ENDO III)-modified comet assays. Cytotoxicity and chromosomal damage were assessed by the cytokinesis-block micronucleus cytome (CBMN-Cyt) assay. The cells were treated with organic extract of coke oven emissions (OE-COE) representing 5, 10, 20, 40μg/mL extract for 24h. We found that there was a dose-effect relationship between the OE-COE and the direct DNA damage presented by tail length, tail intensity and Olive tail moment in the comet assay. The presence of lesion-specific endonucleases in the assays increased DNA migration after OE-COE treatment when compared to those without enzymes, which indicated that OE-COE produced oxidative damage at the level of pyrimidine and purine bases. The dose-dependent increase of micronuclei, nucleoplasmic bridges and nuclear buds in exposed cells was significant, indicating chromosomal and genomic damage induced by OE-COE. Based on the cytotoxic biomarkers in CBMN-Cyt assay, OE-COE may inhibit nuclear division, interfere with apoptosis, or induce cell necrosis. This study indicates that OE-COE exposure can induce DNA breaks/oxidative damage and genomic instability in HBE-CYP1A1 cells. The FPG-comet assay appears more specific for detecting oxidative DNA damage induced by complex mixtures of genotoxic substances. Copyright © 2012 Elsevier Ltd. All rights reserved.

The use of ex vivo human skin tissue for genotoxicity testing

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

Reus, Astrid A.; Usta, Mustafa; Krul, Cyrille A.M., E-mail: cyrille.krul@tno.nl

2012-06-01

As a result of the chemical legislation concerning the registration, evaluation, authorization and restriction of chemicals (REACH), and the Seventh Amendment to the Cosmetics Directive, which prohibits animal testing in Europe for cosmetics, alternative methods for safety evaluation of chemicals are urgently needed. Current in vitro genotoxicity assays are not sufficiently predictive for the in vivo situation, resulting in an unacceptably high number of misleading positives. For many chemicals and ingredients of personal care products the skin is the first site of contact, but there are no in vitro genotoxicity assays available in the skin for additional evaluation of positivemore » or equivocal responses observed in regulatory in vitro genotoxicity assays. In the present study ex vivo human skin tissue obtained from surgery was used for genotoxicity evaluation of chemicals by using the comet assay. Fresh ex vivo human skin tissue was cultured in an air–liquid interface and topically exposed to 20 chemicals, including true positive, misleading positive and true negative genotoxins. Based on the results obtained in the present study, the sensitivity, specificity and accuracy of the ex vivo skin comet assay to predict in vivo genotoxicity were 89%, 90% and 89%, respectively. Donor and experimental variability were mainly reflected in the magnitude of the response and not the difference between the presence and absence of a genotoxic response. The present study indicates that human skin obtained from surgery is a promising and robust model for safety evaluation of chemicals that are in direct contact with the skin. -- Highlights: ► We use human skin obtained from surgery for genotoxicity evaluation of chemicals. ► We use the comet assay as parameter for genotoxicity in ex vivo human skin. ► Sensitivity, specificity and accuracy to predict in vivo genotoxins are determined. ► Sensitivity, specificity and accuracy are 89%, 90% and 90%, respectively. ► The method is suitable for evaluation of chemicals that are in contact with skin.« less

Apigenin, a dietary flavonoid, induces apoptosis, DNA damage, and oxidative stress in human breast cancer MCF-7 and MDA MB-231 cells.

PubMed

Vrhovac Madunić, Ivana; Madunić, Josip; Antunović, Maja; Paradžik, Mladen; Garaj-Vrhovac, Vera; Breljak, Davorka; Marijanović, Inga; Gajski, Goran

2018-05-01

Apigenin is found in several dietary plant foods such as vegetables and fruits. To investigate potential anticancer properties of apigenin on human breast cancer, ER-positive MCF-7 and triple-negative MDA MB-231 cells were used. Moreover, toxicological safety of apigenin towards normal cells was evaluated in human lymphocytes. Cytotoxicity of apigenin towards cancer cells was evaluated by MTT assay whereas further genotoxic and oxidative stress parameters were measured by comet and lipid peroxidation assays, respectively. In order to examine the type of cell death induced by apigenin, several biomarkers were used. Toxicological safety towards normal cells was evaluated by cell viability and comet assays. After the treatment with apigenin, we observed changes in cell morphology in a dose- (10 to 100 μM) and time-dependent manner. Moreover, apigenin caused cell death in both cell lines leading to significant toxicity and dominantly to apoptosis. Furthermore, apigenin proved to be genotoxic towards the selected cancer cells with a potential to induce oxidative damage to lipids. Of great importance is that no significant cytogenotoxic effects were detected in normal cells. The observed cytogenotoxic and pro-cell death activities of apigenin coupled with its low toxicity towards normal cells indicate that this natural product could be used as a future anticancer modality. Therefore, further analysis to determine the exact mechanism of action and in vivo studies on animal models are warranted.

Genoprotective effects of green tea (Camellia sinensis) in human subjects: results of a controlled supplementation trial.

PubMed

Han, K C; Wong, W C; Benzie, Iris F F

2011-01-01

Green tea is rich in polyphenolic antioxidants and has widely reported but largely unsubstantiated health benefits. In the present study, genoprotective effects of two types of green tea were studied both in an in vitro and in a human supplementation trial. For the in vitro study, human lymphocytes were pre-incubated in tea (0·005-0·1 %, w/v), washed and subjected to oxidant challenge induced by H2O2. In a placebo-controlled, cross-over supplementation study, eighteen healthy volunteers took 2 x 150 ml/d of 1% (w/v) green tea ('Longjing' green tea or 'screw-shaped' green tea) or water (control) for 4 weeks (n 6). Subjects took all the three treatments in a random order, with 6 weeks' washout between each treatment. Fasting blood and urine were collected before and after each treatment. The comet assay was used to measure the resistance of lymphocytic DNA to H2O2-induced challenge. Basal oxidation-induced DNA damage was measured using the formamidopyrimidine glycosylase (Fpg) enzyme-assisted comet assay. Urine 7,8-dihydro-2-deoxyguanosine (8-oxodG, mol/mmol creatinine), a biomarker of whole-body oxidative stress, was measured by liquid chromatography with tandem MS. In vitro testing results of tea-treated cells showed increased (P < 0·05) resistance of DNA to the challenge. In the supplementation trial, a significant (P < 0·05) increase in resistance was also observed. Furthermore, the FPg comet data showed .20% decrease in DNA damage with tea supplementation: mean and standard deviation changes in %DNA in comet tail in the Fpg-assisted comet assay were: -5·96 (SD 3·83) % after Longjing tea; -6·22 (SD 3·34) % after screw-shaped tea; +0·91 (SD 5·79) % after water (P < 0·05). No significant changes in urine 8-oxodG were seen. The results indicate that green tea has significant genoprotective effects and provide evidence for green tea as a 'functional food'.

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.

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

Role of Macronutrients and Micronutrients in DNA Damage: Results From a Food Frequency Questionnaire

PubMed Central

Ladeira, Carina; Carolino, Elisabete; Gomes, Manuel C; Brito, Miguel

2017-01-01

The links between diet and genomic instability have been under investigation for several decades, and evidence suggests a significant causal or preventive role for various dietary factors. This study investigates the influence of macronutrients (calories, protein, and glucides) and micronutrients, such as vitamins and minerals, as assessed by a food frequency questionnaire, on genotoxicity biomarkers measured by cytokinesis-blocked micronucleus assay and comet assay. The results found significant positive and negative correlations. Micronucleus frequency tends to increase with higher intake of caffeine, calcium, magnesium, zinc, and protein (P < .05, Spearman correlation). Calorie and omega-6 intakes are negatively correlated with DNA damage measured by the comet assay. These results are somewhat controversial because some of the correlations found are contrary to dominant views in the literature; however, we suggest that unraveling the association between diet and genetic instability requires a much better understanding of the modulating role of macronutrients and micronutrients. PMID:28469462

Toxicity of tributyltin in the marine mollusc Mytilus edulis.

PubMed

Hagger, Josephine A; Depledge, Michael H; Galloway, Tamara S

2005-01-01

Our previous studies have demonstrated that tributyltin (TBT) is genotoxic to the early life stages of marine mussels and worms. Here, the toxicity of TBT to adult organisms was determined using a suite of biomarkers designed to detect cytotoxic, immunotoxic and genotoxic effects. Exposure of adult mussels, Mytilus edulis, to environmentally realistic concentrations of TBTO for 7 days resulted in a statistically significant decrease in cell viability at concentrations of 0.5 microg/l and above. TBT had no effect on phagocytic activity or antioxidant capacity (FRAP assay). There was a statistically significant increase in DNA damage detected using the comet and micronucleus assays between the controls and 0.5, 1 and 5 microg/l of TBTO (P > 0.0005). Furthermore there was a strong correlation between DNA strand breaks (comet assay) and formation of micronuclei (P = 0.0005; R2 = 61.5%). Possible mechanisms by which TBT could damage DNA either directly or indirectly are discussed including the possibility that TBT is genotoxic due to its ability to disrupt calcium homeostasis.

Radioprotective effects of honeybee venom (Apis mellifera) against 915-MHz microwave radiation-induced DNA damage in wistar rat lymphocytes: in vitro study.

PubMed

Gajski, Goran; Garaj-Vrhovac, Vera

2009-01-01

The aim of this study is to investigate the radioprotective effect of bee venom against DNA damage induced by 915-MHz microwave radiation (specific absorption rate of 0.6 W/kg) in Wistar rats. Whole blood lymphocytes of Wistar rats are treated with 1 microg/mL bee venom 4 hours prior to and immediately before irradiation. Standard and formamidopyrimidine-DNA glycosylase (Fpg)-modified comet assays are used to assess basal and oxidative DNA damage produced by reactive oxygen species. Bee venom shows a decrease in DNA damage compared with irradiated samples. Parameters of Fpg-modified comet assay are statistically different from controls, making this assay more sensitive and suggesting that oxidative stress is a possible mechanism of DNA damage induction. Bee venom is demonstrated to have a radioprotective effect against basal and oxidative DNA damage. Furthermore, bee venom is not genotoxic and does not produce oxidative damage in the low concentrations used in this study.

Cigarette smoke induced genotoxicity and respiratory tract pathology: evidence to support reduced exposure time and animal numbers in tobacco product testing

PubMed Central

Dalrymple, Annette; Ordoñez, Patricia; Thorne, David; Walker, David; Camacho, Oscar M.; Büttner, Ansgar; Dillon, Debbie; Meredith, Clive

2016-01-01

Abstract Many laboratories are working to develop in vitro models that will replace in vivo tests, but occasionally there remains a regulatory expectation of some in vivo testing. Historically, cigarettes have been tested in vivo for 90 days. Recently, methods to reduce and refine animal use have been explored. This study investigated the potential of reducing animal cigarette smoke (CS) exposure to 3 or 6 weeks, and the feasibility of separate lung lobes for histopathology or the Comet assay. Rats were exposed to sham air or CS (1 or 2 h) for 3 or 6 weeks. Respiratory tissues were processed for histopathological evaluation, and Alveolar type II cells (AEC II) isolated for the Comet assay. Blood was collected for Pig-a and micronucleus quantification. Histopathological analyses demonstrated exposure effects, which were generally dependent on CS dose (1 or 2 h, 5 days/week). Comet analysis identified that DNA damage increased in AEC II following 3 or 6 weeks CS exposure, and the level at 6 weeks was higher than 3 weeks. Pig-a mutation or micronucleus levels were not increased. In conclusion, this study showed that 3 weeks of CS exposure was sufficient to observe respiratory tract pathology and DNA damage in isolated AEC II. Differences between the 3 and 6 week data imply that DNA damage in the lung is cumulative. Reducing exposure time, plus analyzing separate lung lobes for DNA damage or histopathology, supports a strategy to reduce and refine animal use in tobacco product testing and is aligned to the 3Rs (replacement, reduction and refinement). PMID:27160659

Cigarette smoke induced genotoxicity and respiratory tract pathology: evidence to support reduced exposure time and animal numbers in tobacco product testing.

PubMed

Dalrymple, Annette; Ordoñez, Patricia; Thorne, David; Walker, David; Camacho, Oscar M; Büttner, Ansgar; Dillon, Debbie; Meredith, Clive

2016-06-01

Many laboratories are working to develop in vitro models that will replace in vivo tests, but occasionally there remains a regulatory expectation of some in vivo testing. Historically, cigarettes have been tested in vivo for 90 days. Recently, methods to reduce and refine animal use have been explored. This study investigated the potential of reducing animal cigarette smoke (CS) exposure to 3 or 6 weeks, and the feasibility of separate lung lobes for histopathology or the Comet assay. Rats were exposed to sham air or CS (1 or 2 h) for 3 or 6 weeks. Respiratory tissues were processed for histopathological evaluation, and Alveolar type II cells (AEC II) isolated for the Comet assay. Blood was collected for Pig-a and micronucleus quantification. Histopathological analyses demonstrated exposure effects, which were generally dependent on CS dose (1 or 2 h, 5 days/week). Comet analysis identified that DNA damage increased in AEC II following 3 or 6 weeks CS exposure, and the level at 6 weeks was higher than 3 weeks. Pig-a mutation or micronucleus levels were not increased. In conclusion, this study showed that 3 weeks of CS exposure was sufficient to observe respiratory tract pathology and DNA damage in isolated AEC II. Differences between the 3 and 6 week data imply that DNA damage in the lung is cumulative. Reducing exposure time, plus analyzing separate lung lobes for DNA damage or histopathology, supports a strategy to reduce and refine animal use in tobacco product testing and is aligned to the 3Rs (replacement, reduction and refinement).

Chemical form of selenium differentially influences DNA repair pathways following exposure to lead nitrate.

PubMed

McKelvey, Shauna M; Horgan, Karina A; Murphy, Richard A

2015-01-01

Lead, an environmental toxin is known to induce a broad range of physiological and biochemical dysfunctions in humans through a number of mechanisms including the deactivation of antioxidants thus leading to generation of reactive oxygen species (ROS) and subsequent DNA damage. Selenium on the other hand has been proven to play an important role in the protection of cells from free radical damage and oxidative stress, though its effects are thought to be form and dose dependent. As the liver is the primary organ required for metabolite detoxification, HepG2 cells were chosen to assess the protective effects of various selenium compounds following exposure to the genotoxic agent lead nitrate. Initially DNA damage was quantified using a comet assay, gene expression patterns associated with DNA damage and signalling were also examined using PCR arrays and the biological pathways which were most significantly affected by selenium were identified. Interestingly, the organic type selenium compounds (selenium yeast and selenomethionine) conferred protection against lead induced DNA damage in HepG2 cells; this is evident by reduction in the quantity of DNA present in the comet tail of cells cultured in their presence with lead. This trend also followed through the gene expression changes noted in DNA damage pathways analysed. These results were in contrast with those of inorganic sodium selenite which promoted lead induced DNA damage evident in both the comet assay results and the gene expression analysis. Over all this study provided valuable insights into the effects which various selenium compounds had on the DNA damage and signalling pathway indicating the potential for using organic forms of selenium such as selenium enriched yeast to protect against DNA damaging agents. Copyright © 2014 Elsevier GmbH. All rights reserved.

A battery of in vivo and in vitro tests useful for genotoxic pollutant detection in surface waters.

PubMed

Pellacani, Claudia; Buschini, Annamaria; Furlini, Mariangela; Poli, Paola; Rossi, Carlo

2006-04-20

Since the 1980s, stricter water quality regulations have been promulgated in many countries throughout the world. We discuss the application of a battery of both in vivo and in vitro genotoxicity tests on lake water as a tool for a more complete assessment of surface water quality. The lake water concentrated by adsorption on C18 silica cartridges were used for the following in vitro biological assays: gene conversion, point mutation, mitochondrial DNA mutability assays on the diploid Saccharomyces cerevisiae D7 strain, with or without endogenous P450 complex induction; DNA damage on fresh human leukocytes by the comet. Toxicity testing on yeast and human cells was also performed. In vivo genotoxicity was determined by the comet assay on two well-established bio-indicator organisms of water quality (Cyprinus carpio erythrocytes and Dreissena polymorpha haemocytes) exposed in situ. The in vivo experiments and the water samplings were carried out during different campaigns to detect seasonal variations of both the water contents and physiological state of the animals. Temperature and oxygen level seasonal variations and different pollutant contents in the lake water appeared to affect the DNA migration in carp and zebra mussel cells. Seasonal variability of lake water quality was also evident in the in vitro genotoxicity and cytotoxicity tests, with regards to water pollutant quantity and quality (direct-acting compounds or indirect-acting compounds on yeast cells). However, the measured biological effects did not appear clearly related to the physical-chemical characteristics of lake waters. Therefore, together with the conventional chemical analysis, mutagenicity/genotoxicity assays should be included as additional parameters in water quality monitoring programs: their use could permit the quantification of mutagenic hazard in surface waters.

A systematic review and meta-analysis to determine the effect of sperm DNA damage on in vitro fertilization and intracytoplasmic sperm injection outcome

PubMed Central

Simon, Luke; Zini, Armand; Dyachenko, Alina; Ciampi, Antonio; Carrell, Douglas T

2017-01-01

Sperm DNA damage is prevalent among infertile men and is known to influence natural reproduction. However, the impact of sperm DNA damage on assisted reproduction outcomes remains controversial. Here, we conducted a meta-analysis of studies on sperm DNA damage (assessed by SCSA, TUNEL, SCD, or Comet assay) and clinical pregnancy after IVF and/or ICSI treatment from MEDLINE, EMBASE, and PUBMED database searches for this analysis. We identified 41 articles (with a total of 56 studies) including 16 IVF studies, 24 ICSI studies, and 16 mixed (IVF + ICSI) studies. These studies measured DNA damage (by one of four assays: 23 SCSA, 18 TUNEL, 8 SCD, and 7 Comet) and included a total of 8068 treatment cycles (3734 IVF, 2282 ICSI, and 2052 mixed IVF + ICSI). The combined OR of 1.68 (95% CI: 1.49–1.89; P < 0.0001) indicates that sperm DNA damage affects clinical pregnancy following IVF and/or ICSI treatment. In addition, the combined OR estimates of IVF (16 estimates, OR = 1.65; 95% CI: 1.34–2.04; P < 0.0001), ICSI (24 estimates, OR = 1.31; 95% CI: 1.08–1.59; P = 0.0068), and mixed IVF + ICSI studies (16 estimates, OR = 2.37; 95% CI: 1.89–2.97; P < 0.0001) were also statistically significant. There is sufficient evidence in the existing literature suggesting that sperm DNA damage has a negative effect on clinical pregnancy following IVF and/or ICSI treatment. PMID:27345006

Detection of irradiated fresh fruits treated by e-beam or gamma rays

NASA Astrophysics Data System (ADS)

Marin-Huachaca, Nélida Simona; Lamy-Freund, Maria Tereza; Mancini-Filho, Jorge; Delincée, Henry; Villavicencio, Anna Lúcia C. H.

2002-03-01

Since about 1990, the amount of commercially irradiated food products available worldwide has increased. Commercial irradiation of foods has been allowed in Brazil since 1973 and now more than 20 different food products are approved. Among these products are a number of fresh fruits which may be irradiated for insect disinfestation, to delay ripening and to extend shelf-life. Today, there is a growing interest to apply radiation for the treatment of fruits instead of using fumigation or e.g. vapour-heat treatments, and an increased international trade in irradiated fruits is expected. To ensure free consumer choice, methods to identify irradiated foods are highly desirable. In this work, three detection methods for irradiated fruits have been employed: DNA Comet Assay, the half-embryo test and ESR. Both electron-beam (e-beam) and gamma rays were applied in order to compare the response with these two different kinds of radiation. Fresh fruits such as oranges, lemons, apples, watermelons and tomatoes were irradiated with doses in the range 0, 0.50, 0.75, 1.0, 2.0 and 4.0kGy. For analysis, the seeds of the fruits were utilized. Both DNA Comet Assay and the half-embryo test enabled an easy identification of the radiation treatment. However, under our conditions, ESR measurements were not satisfactory.

Genotoxicity of Advanced Glycation End Products: Involvement of Oxidative Stress and of Angiotensin II Type 1 Receptors

NASA Astrophysics Data System (ADS)

Schupp, Nicole; Schinzel, Reinhard; Heidland, August; Stopper, Helga

2005-06-01

In patients with chronic renal failure, cancer incidence is increased. This may be related to an elevated level of genomic damage, which has been demonstrated by micronuclei formation as well as by comet assay analysis. Advanced glycation end products (AGEs) are markedly elevated in renal failure. In the comet assay, the model AGEs methylglyoxal- and carboxy(methyl)lysine-modified bovine serum albumin (BSA) induced significant DNA damage in colon, kidney, and liver cells. The addition of antioxidants prevented AGE-induced DNA damage, suggesting enhanced formation of reactive oxygen species (ROS). The coincubation with dimethylfumarate (DMF), an inhibitor of NF-κB translocation, reduced the genotoxic effect, thereby underscoring the key role of NF-κB in this process. One of the genes induced by NF-κB is angiotensinogen. The ensuing proteolytic activity yields angiotensin II, which evokes oxidative stress as well as proinflammatory responses. A modulator of the renin-angiotensin system (RAS), the angiotensin II (Ang II) receptor 1 antagonist, candesartan, yielded a reduction of the AGE-induced DNA damage, connecting the two signal pathways, RAS and AGE signaling. We were able to identify important participants in AGE-induced DNA damage: ROS, NF-κB, and Ang II, as well as modulators to prevent this DNA damage: antioxidants, DMF, and AT1 antagonists.

By-product identification and phytotoxicity of biodegraded Direct Yellow 4 dye.

PubMed

Nouren, Shazia; Bhatti, Haq Nawaz; Iqbal, Munawar; Bibi, Ismat; Kamal, Shagufta; Sadaf, Sana; Sultan, Misbah; Kausar, Abida; Safa, Yusra

2017-02-01

Citrus limon peroxidase mediated decolourization of Direct Yellow 4 (DY4) was investigated. The process variables (pH, temperature, incubation time, enzyme dose, H 2 O 2 amount, dye concentration, co-metal ions and surfactants) were optimized for maximum degradation of dye. Maximum dye decolourization of 89.47% was achieved at pH 5.0, temperature 50 °C, enzyme dose 24 U/mL, H 2 O 2 concentration 0.25 mM and DY4 concentration 18.75 mg/L and incubation time 10 min. The co-metal ions and surfactants did not affect the dye decolourization significantly. Response surface analysis revealed that predicted values were in agreement with experimentally determined responses. The degradation products were identified by UPLC/MS analysis and degradation pathway was proposed. Besides, phytotoxicity assay revealed a considerable detoxification in response of biodegradation of DY4 dye. C. limon showed promising efficiency for DY4 degradation and could possibly be used for the remediation of textile effluents. Copyright © 2016 Elsevier Ltd. All rights reserved.

DNA Damage in Euonymus japonicus Leaf Cells Caused by Roadside Pollution in Beijing

PubMed Central

Li, Tianxin; Zhang, Minjie; Gu, Ke; Herman, Uwizeyimana; Crittenden, John; Lu, Zhongming

2016-01-01

The inhalable particles from vehicle exhaust can cause DNA damage to exposed organisms. Research on DNA damage is primarily focused on the influence of specific pollutants on certain species or the effect of environmental pollution on human beings. To date, little research has quantitatively studied the relationship between roadside pollution and DNA damage. Based on an investigation of the roadside pollution in Beijing, Euonymus japonicus leaves of differing ages grown in heavily-polluted sections were chosen as biomonitors to detect DNA damage using the comet assay technique. The percentage of DNA in the tail and tail moment was chosen as the analysis index based on SPSS data analysis. The roadside samples showed significantly higher levels of DNA damage than non-roadside samples, which increased in older leaves, and the DNA damage to Euonymus japonicus leaf cells was positively correlated with haze-aggravated roadside pollution. The correlation between damage and the Air Quality Index (AQI) are 0.921 (one-year-old leaves), 0.894 (two-year-old leaves), and 0.878 (three-year-old leaves). Over time, the connection between DNA damage and AQI weakened, with the sensitivity coefficient for δyear 1 being larger than δyear 2 and δyear 3. These findings support the suitability and sensitivity of the comet assay for surveying plants for an estimation of DNA damage induced by environmental genotoxic agents. This study might be applied as a preliminary quantitative method for Chinese urban air pollution damage assessment caused by environmental stress. PMID:27455298

Compounds used to produce cloned animals are genotoxic and mutagenic in mammalian assays in vitro and in vivo

PubMed Central

Oliveira, R.J.; Mantovani, M.S.; da Silva, A.F.; Pesarini, J.R.; Mauro, M.O.; Ribeiro, L.R.

2014-01-01

The compounds 6-dimethylaminopurine and cycloheximide promote the successful production of cloned mammals and have been used in the development of embryos produced by somatic cell nuclear transfer. This study investigated the effects of 6-dimethylaminopurine and cycloheximide in vitro, using the thiazolyl blue tetrazolium bromide colorimetric assay to assess cytotoxicity, the trypan blue exclusion assay to assess cell viability, the comet assay to assess genotoxicity, and the micronucleus test with cytokinesis block to test mutagenicity. In addition, the comet assay and the micronucleus test were also performed on peripheral blood cells of 54 male Swiss mice, 35 g each, to assess the effects of the compounds in vivo. The results indicated that both 6-dimethylaminopurine and cycloheximide, at the concentrations and doses tested, were cytotoxic in vitro and genotoxic and mutagenic in vitro and in vivo, altered the nuclear division index in vitro, but did not diminish cell viability in vitro. Considering that alterations in DNA play important roles in mutagenesis, carcinogenesis, and morphofunctional teratogenesis and reduce embryonic viability, this study indicated that 6-dimethylaminopurine and cycloheximide utilized in the process of mammalian cloning may be responsible for the low embryo viability commonly seen in nuclear transfer after implantation in utero. PMID:24714812

Compounds used to produce cloned animals are genotoxic and mutagenic in mammalian assays in vitro and in vivo.

PubMed

Oliveira, R J; Mantovani, M S; Silva, A F da; Pesarini, J R; Mauro, M O; Ribeiro, L R

2014-04-01

The compounds 6-dimethylaminopurine and cycloheximide promote the successful production of cloned mammals and have been used in the development of embryos produced by somatic cell nuclear transfer. This study investigated the effects of 6-dimethylaminopurine and cycloheximide in vitro, using the thiazolyl blue tetrazolium bromide colorimetric assay to assess cytotoxicity, the trypan blue exclusion assay to assess cell viability, the comet assay to assess genotoxicity, and the micronucleus test with cytokinesis block to test mutagenicity. In addition, the comet assay and the micronucleus test were also performed on peripheral blood cells of 54 male Swiss mice, 35 g each, to assess the effects of the compounds in vivo. The results indicated that both 6-dimethylaminopurine and cycloheximide, at the concentrations and doses tested, were cytotoxic in vitro and genotoxic and mutagenic in vitro and in vivo, altered the nuclear division index in vitro, but did not diminish cell viability in vitro. Considering that alterations in DNA play important roles in mutagenesis, carcinogenesis, and morphofunctional teratogenesis and reduce embryonic viability, this study indicated that 6-dimethylaminopurine and cycloheximide utilized in the process of mammalian cloning may be responsible for the low embryo viability commonly seen in nuclear transfer after implantation in utero.

The JaCVAM international validation study on the in vivo comet assay: Selection of test chemicals.

PubMed

Morita, Takeshi; Uno, Yoshifumi; Honma, Masamitsu; Kojima, Hajime; Hayashi, Makoto; Tice, Raymond R; Corvi, Raffaella; Schechtman, Leonard

2015-07-01

The Japanese Center for the Validation of Alternative Methods (JaCVAM) sponsored an international prevalidation and validation study of the in vivo rat alkaline pH comet assay. The main objective of the study was to assess the sensitivity and specificity of the assay for correctly identifying genotoxic carcinogens, as compared with the traditional rat liver unscheduled DNA synthesis assay. Based on existing carcinogenicity and genotoxicity data and chemical class information, 90 chemicals were identified as primary candidates for use in the validation study. From these 90 chemicals, 46 secondary candidates and then 40 final chemicals were selected based on a sufficiency of carcinogenic and genotoxic data, differences in chemical class or genotoxic or carcinogenic mode of action (MOA), availability, price, and ease of handling. These 40 chemicals included 19 genotoxic carcinogens, 6 genotoxic non-carcinogens, 7 non-genotoxic carcinogens and 8 non-genotoxic non-carcinogens. "Genotoxicity" was defined as positive in the Ames mutagenicity test or in one of the standard in vivo genotoxicity tests (primarily the erythrocyte micronucleus assay). These chemicals covered various chemicals classes, MOAs, and genotoxicity profiles and were considered to be suitable for the purpose of the validation study. General principles of chemical selection for validation studies are discussed. Copyright © 2015 Elsevier B.V. All rights reserved.

Evaluation of genetic damage induced by glyphosate isopropylamine salt using Tradescantia bioassays.

PubMed

Alvarez-Moya, Carlos; Silva, Mónica Reynoso; Arámbula, Alma Rosa Villalobos; Sandoval, Alfonso Islas; Vasquez, Hugo Castañeda; González Montes, Rosa María

2011-01-01

Glyphosate is noted for being non-toxic in fishes, birds and mammals (including humans). Nevertheless, the degree of genotoxicity is seriously controversial. In this work, various concentrations of a glyphosate isopropylamine salt were tested using two methods of genotoxicity assaying, viz., the pink mutation assay with Tradescantia (4430) and the comet assay with nuclei from staminal cells of the same plant. Staminal nuclei were studied in two different forms, namely nuclei from exposed plants, and nuclei exposed directly. Using the pink mutation assay, isopropylamine induced a total or partial loss of color in staminal cells, a fundamental criterion utilized in this test. Consequently, its use is not recommended when studying genotoxicity with agents that produce pallid staminal cells. The comet assay system detected statistically significant (p < 0.01) genotoxic activity by isopropylamine, when compared to the negative control in both the nuclei of treated plants and directly treated nuclei, but only the treated nuclei showed a dose-dependent increase. Average migration in the nuclei of treated plants increased, when compared to that in treated nuclei. This was probably due, either to the permanence of isopropylamine in inflorescences, or to the presence of secondary metabolites. In conclusion, isopropylamine possesses strong genotoxic activity, but its detection can vary depending on the test systems used.

Evaluation of genetic damage induced by glyphosate isopropylamine salt using Tradescantia bioassays

PubMed Central

Alvarez-Moya, Carlos; Silva, Mónica Reynoso; Arámbula, Alma Rosa Villalobos; Sandoval, Alfonso Islas; Vasquez, Hugo Castañeda; González Montes, Rosa María

2011-01-01

Glyphosate is noted for being non-toxic in fishes, birds and mammals (including humans). Nevertheless, the degree of genotoxicity is seriously controversial. In this work, various concentrations of a glyphosate isopropylamine salt were tested using two methods of genotoxicity assaying, viz., the pink mutation assay with Tradescantia (4430) and the comet assay with nuclei from staminal cells of the same plant. Staminal nuclei were studied in two different forms, namely nuclei from exposed plants, and nuclei exposed directly. Using the pink mutation assay, isopropylamine induced a total or partial loss of color in staminal cells, a fundamental criterion utilized in this test. Consequently, its use is not recommended when studying genotoxicity with agents that produce pallid staminal cells. The comet assay system detected statistically significant (p < 0.01) genotoxic activity by isopropylamine, when compared to the negative control in both the nuclei of treated plants and directly treated nuclei, but only the treated nuclei showed a dose-dependent increase. Average migration in the nuclei of treated plants increased, when compared to that in treated nuclei. This was probably due, either to the permanence of isopropylamine in inflorescences, or to the presence of secondary metabolites. In conclusion, isopropylamine possesses strong genotoxic activity, but its detection can vary depending on the test systems used. PMID:21637555

Indoor and outdoor genotoxic load detected by the Comet assay in leaves of Nicotiana tabacum cultivars Bel B and Bel W3.

PubMed

Restivo, Francesco Maria; Laccone, Maria Concetta; Buschini, Annamaria; Rossi, Carlo; Poli, Paola

2002-03-01

Environmental pollution assessment and control are priority issues for both developed and developing countries of the world. The use of plant material for a more complete picture of environmental health appears to be particularly appealing. Here we validate a previous plant-adapted Comet assay on leaf tissues of Nicotiana tabacum cultivars Bel B and Bel W3. The effects of H(2)O(2) on DNA damage in Bel B and Bel W3 agree with the hypothesis that some component of the machinery that protects DNA integrity from oxidative stress may be impaired in cv. Bel W3. Exposure in the field on sunny summer days (peak ozone concentration >80 p.p.b.) showed significantly higher DNA damage in cv. Bel W3 if plants were collected and subjected to the Comet assay when the air ozone concentration was reaching its peak value, but not when plants were sampled early in the morning and hence after a period of low ozone concentration. The different results suggest that Bel W3 possesses a less efficient recovery apparatus that requires a longer period of activity to be effective and/or is less protected against reactive oxygen species production during exposure to ozone. However, it cannot be excluded that the increase in mean DNA damage is the result of the presence of a genotoxic agent(s) other than ozone. Interestingly, Bel W3 also appears to be more responsive, compared with Bel B, when exposed to ambient indoor pollutants. The use of cv. Bel W3 increases the sensitivity of the assay under both indoor and field conditions. However, different classes of mutagens should be tested to define the range of profitable utilization of this tobacco cultivar for environmental genotoxicity detection.

Comparison in vivo Study of Genotoxic Action of High- Versus Very Low Dose-Rate γ-Irradiation

PubMed Central

Osipov, A. N.; Klokov, D. Y.; Elakov, A. L.; Rozanova, O. M.; Zaichkina, S. I.; Aptikaeva, G. F.; Akhmadieva, A. Kh.

2004-01-01

The aim of the present study was to compare genotoxicity induced by high- versus very low dose-rate exposure of mice to γ-radiation within a dose range of 5 to 61 cGy using the single-cell gel electrophoresis (comet) assay and the micronucleus test. CBA/lac male mice were irradiated at a dose rate of 28.2 Gy/h (high dose rate) or 0.07 mGy/h (very low dose rate). The comet assay study on spleen lymphocytes showed that very low dose-rate irradiation resulted in a statistically significant increase in nucleoid relaxation (DNA breaks), starting from a dose of 20 cGy. Further prolongation of exposure time and, hence, increase of a total dose did not, however, lead to further increase in the extent of nucleoid relaxation. Doses of 20 and 61 cGy were equal in inducing DNA breaks in mouse spleen lymphocytes as assayed by the comet assay. Of note, the level of DNA damage by 20–61 cGy doses of chronic irradiation (0.07 mGy/h) was similar to that an induced by an acute (28.2 Gy/h) dose of 14 cGy. The bone marrow micronucleus test revealed that an increase in polychromatic erythrocytes with micronuclei over a background level was induced by very low-level γ-irradiation with a dose of 61 cGy only, with the extent of the cytogenetic effect being similar to that of 10 cGy high-dose-rate exposure. In summary, presented results support the hypothesis of the nonlinear threshold nature of mutagenic action of chronic low dose-rate irradiation. PMID:19330145

Protective effects of melatonin-loaded lipid-core nanocapsules on paraquat-induced cytotoxicity and genotoxicity in a pulmonary cell line.

PubMed

Charão, Mariele F; Baierle, Marília; Gauer, Bruna; Goethel, Gabriela; Fracasso, Rafael; Paese, Karina; Brucker, Natália; Moro, Angela M; Bubols, Guilherme B; Dias, Bruna B; Matte, Ursula S; Guterres, Silvia S; Pohlmann, Adriana R; Garcia, Solange C

2015-06-01

Many acute poisonings lack effective and specific antidotes. Due to both intentional and accidental exposures, paraquat (PQ) causes thousands of deaths annually, especially by pulmonary fibrosis. Melatonin (Mel), when incorporated into lipid-core nanocapsules (Mel-LNC), has enhanced antioxidant properties. The effects of such a formulation have not yet been studied with respect to mitigation of PQ- induced cytotoxicity and DNA damage. Here, we have tested whether Mel-LNC can ameliorate PQ-induced toxicity in the A549 alveolar epithelial cell line. Physicochemical characterization of the formulations was performed. Cellular uptake was measured using nanocapsules marked with rhodamine B. Cell viability was determined by the MTT assay and DNA damage was assessed by the comet assay. The enzyme-modified comet assay with endonuclease III (Endo III) and formamidopyrimidine glycosylase (FPG) were used to investigate oxidative DNA damage. Incubation with culture medium for 24h did not alter the granulometric profile of Mel-LNC formulations. Following treatment (3 and 24h), red fluorescence was detected around the cell nucleus, indicating internalization of the formulation. Melatonin solution (Mel), Mel-LNC, and LNC did not have significant effects on cell viability or DNA damage. Pre-treatment with Mel-LNC enhanced cell viability and showed a remarkable reduction in % DNA in tail compared to the PQ group; this was not observed in cells pre-treated with Mel. PQ induces oxidative DNA damage detected with the enzyme-modified comet assay. Mel-LNC reduced this damage more effectively than did Mel. In summary, Mel-LNC is better than Mel at protecting A549 cells from the cytotoxic and genotoxic effects of PQ. Copyright © 2015 Elsevier B.V. All rights reserved.

DNA damage and repair capacity in workers exposed to low concentrations of benzene.

PubMed

Lovreglio, Piero; Doria, Denise; Fracasso, Maria Enrica; Barbieri, Anna; Sabatini, Laura; Drago, Ignazio; Violante, Francesco S; Soleo, Leonardo

2016-03-01

DNA damage and cellular repair capacity were studied in 18 male fuel tanker drivers and 13 male filling-station attendants exposed to low and very low concentrations of benzene, respectively, and compared to 20 males with no occupational exposure (controls). Exposure to airborne benzene was measured using passive personal samplers, and internal doses were assayed through the biomarkers t,t-muconic acid, S-phenylmercapturic acid and urinary benzene. DNA damage was evaluated using tail intensity (TI) determined by the comet assay in peripheral lymphocytes. Urinary 7-hydro-8-oxo-2'-deoxyguanosine (8-oxodG) was measured as a biomarker of oxidative damage. DNA repair kinetics were assessed using the comet assay in lymphocytes sampled 20 and 60 min post H2O2 exposure. Benzene exposure differed significantly between the drivers (median 246.3 µg/m(3)), attendants (median 13.8 µg/m(3)), and controls (median 4.1 µg/m(3)). There were no differences in TI and 8-oxodG among the three groups, or between smokers and non-smokers. DNA repair kinetics were similar among the drivers, attendants and controls, although the comet assay on H2 O2 -damaged lymphocytes after 60 min revealed significantly lower levels of TI only in drivers. The DNA repair process in smokers was similar to that observed in drivers. In conclusion, this study found no relationship between low levels of benzene exposure and DNA damage, although there was evidence that exposure interferes with DNA repair kinetics. The biological impact of this finding on the onset of genotoxic effects in exposed workers has still to be ascertained. © 2015 Wiley Periodicals, Inc.

DNA damage, repair monitoring and epigenetic DNA methylation changes in seedlings of Chernobyl soybeans.

PubMed

Georgieva, Mariyana; Rashydov, Namik M; Hajduch, Martin

2017-02-01

This pilot study was carried out to assess the effect of radio-contaminated Chernobyl environment on plant genome integrity 27 years after the accident. For this purpose, nuclei were isolated from root tips of the soybean seedlings harvested from plants grown in the Chernobyl area for seven generations. Neutral, neutral-alkaline, and methylation-sensitive comet assays were performed to evaluate the induction and repair of primary DNA damage and the epigenetic contribution to stress adaptation mechanisms. An increased level of single and double strand breaks in the radio-contaminated Chernobyl seedlings at the stage of primary root development was detected in comparison to the controls. However, the kinetics of the recovery of DNA breaks of radio-contaminated Chernobyl samples revealed that lesions were efficiently repaired at the stage of cotyledon. Methylation-sensitive comet assay revealed comparable levels in the CCGG methylation pattern between control and radio-contaminated samples with a slight increase of approximately 10% in the latter ones. The obtained preliminary data allow us to speculate about the onset of mechanisms providing an adaptation potential to the accumulated internal irradiation after the Chernobyl accident. Despite the limitations of this study, we showed that comet assay is a sensitive and flexible technique which can be efficiently used for genotoxic screening of plant specimens in natural and human-made radio-contaminated areas, as well as for safety monitoring of agricultural products. Copyright © 2016 Elsevier B.V. All rights reserved.

Evaluation of DNA Damage in Common Carp (Cyprinus carpio L.) by Comet Assay for Determination of Possible Pollution in Lake Mogan (Ankara)

PubMed Central

Çok, İsmet; Ulutaş, Onur Kenan; Okuşluk, Öncü; Durmaz, Emre; Demir, Nilsun

2011-01-01

Contamination of the aquatic environment with various concentrations of pollutants results in unexpected threats to humans and wildlife. The consequences of exposure and metabolism of pollutants/xenobiotics, especially carcinogens and mutagens, can be suitably assessed by investigating severe events, such as DNA damage; for example, DNA adducts and DNA strand breaks. One of the commonly used techniques to detect DNA damage in aquatic organisms is single-cell gel electrophoresis (comet assay). This study was carried out using Cyprinus carpio in order to identify the possible pollution in Lake Mogan, near Ankara, Turkey, where the city's sewer system and pesticides used in agriculture are believed to be the common causes of pollution. From the comet assay, the tail length (μm), tail intensity (%), and tail moment values of fish caught from Lake Mogan were found to be 31.10 ± 10.39, 7.77 ± 4.51, 1.50 ± 1.48, respectively, whereas for clean reference sites they were found to be 22.80 ± 1.08, 3.47 ± 1.59, 0.40 ± 0.51, respectively. The values are statistically different from each other (p < 0.0001, p < 0.0001, and p < 0.0013, respectively). These results indicate that Lake Mogan may be polluted with substances that have genotoxic effects and constitute an early warning for the lake system. Further detailed research is needed to establish the source of the pollution and the chemicals responsible. PMID:21805014

Detection of in vivo DNA damage induced by ethanol in multiple organs of pregnant mice using the alkaline single cell gel electrophoresis (Comet) assay.

PubMed

Kido, Ryoko; Sato, Itaru; Tsuda, Shuji

2006-01-01

Ethanol is principal ingredient of alcohol beverage, but considered as human carcinogen, and has neurotoxicity. Alcohol consumption during pregnancy often causes fetal alcohol syndrome. The DNA damage is one of the important factors in carcinogenicity or teratogenicity. To detect the DNA damage induced by ethanol, we used an in vivo alkaline single cell gel electrophoresis (Comet) assay in pregnant mice organs and embryos. Pregnant ICR mice on Day 7 of gestation were treated with 2, 4 or 8 g/kg ethanol, and maternal organs/tissues and embryos were subjected to the Comet assay at 4, 8, 12 and 24 hr after ethanol treatment. Four and 8 g/kg ethanol induced DNA damage in brain, lung and embryos at 4 or 8 hr after the treatment. Two g/kg ethanol did not cause any DNA damage, and 8 g/kg ethanol only increased the duration of DNA damage without distinct increase in the degree of the damage. No significant DNA damage was observed in the liver. To detect the effect of acetaldehyde, disulfiram, acetaldehyde dehydrogenase inhibitor, was administered before 4 g/kg ethanol treatment. No significant increase of DNA damage was observed in the disulfiram pre-treated group. These data indicate that ethanol induces DNA damage, which might be related to ethanol toxicity. Since pre-treatment of disulfiram did not increase DNA damage, DNA damage observed in this study might not be the effect of acetaldehyde.

Evaluation of DNA damage induced by gamma radiation in gill and muscle tissues of Cyprinus carpio and their relative sensitivity.

PubMed

M K, Praveen Kumar; Shyama, Soorambail K; D'Costa, Avelyno; Kadam, Samit B; Sonaye, Bhagatsingh Harisingh; Chaubey, Ramesh Chandra

2017-10-01

The effect of radiation on the aquatic environment is of major concern in recent years. Limited data is available on the genotoxicity of gamma radiation on different tissues of aquatic organisms. Hence, the present investigation was carried out to study the DNA damage induced by gamma radiation in the gill and muscle tissues and their relative sensitivity using the comet assay in the freshwater teleost fish, common carp (Cyprinus carpio). The comet assay was optimized and validated in common carp using cyclophosphamide (CP), a reference genotoxic agent. The fish were exposed (acute) to various doses of gamma radiation (2, 4, 6, 8 and 10Gy) and samplings (gill and muscle tissue) were done at regular intervals (24, 48 and 72h) to assess the DNA damage. A significant increase in DNA damage was observed as indicated by an increase in % tail DNA for all doses of gamma radiation in both tissues. We also observed a dose-related increase and a time-dependent decrease of DNA damage. In comparison, DNA damage showed different sensitivity among the tissues at different doses. This shows that a particular dose may have different effects on different tissues which could be due to physiological factors of the particular tissue. Our study also suggests that the gills and muscle of fish are sensitive and reliable tissues for evaluating the genotoxic effects of reference and environmental agents, using the comet assay. Copyright © 2017. Published by Elsevier Inc.

Chicken fetal liver DNA damage and adduct formation by activation-dependent DNA-reactive carcinogens and related compounds of several structural classes.

PubMed

Williams, Gary M; Duan, Jian-Dong; Brunnemann, Klaus D; Iatropoulos, Michael J; Vock, Esther; Deschl, Ulrich

2014-09-01

The chicken egg genotoxicity assay (CEGA), which utilizes the liver of an intact and aseptic embryo-fetal test organism, was evaluated using four activation-dependent DNA-reactive carcinogens and four structurally related less potent carcinogens or non-carcinogens. In the assay, three daily doses of test substances were administered to eggs containing 9-11-day-old fetuses and the fetal livers were assessed for two endpoints, DNA breaks using the alkaline single cell gel electrophoresis (comet) assay and DNA adducts using the (32)P-nucleotide postlabeling (NPL) assay. The effects of four carcinogens of different structures requiring distinct pathways of bioactivation, i.e., 2-acetylaminofluorene (AAF), aflatoxin B1 (AFB1), benzo[a]pyrene (B[a]P), and diethylnitrosamine (DEN), were compared with structurally related non-carcinogens fluorene (FLU) and benzo[e]pyrene (B[e]P) or weak carcinogens, aflatoxin B2 (AFB2) and N-nitrosodiethanolamine (NDELA). The four carcinogens all produced DNA breaks at microgram or low milligram total doses, whereas less potent carcinogens and non-carcinogens yielded borderline or negative results, respectively, at higher doses. AAF and B[a]P produced DNA adducts, whereas none was found with the related comparators FLU or B[e]P, consistent with comet results. DEN and NDELA were also negative for adducts, as expected in the case of DEN for an alkylating agent in the standard NPL assay. Also, AFB1 and AFB2 were negative in NPL, as expected, due to the nature of ring opened aflatoxin adducts, which are resistant to enzymatic digestion. Thus, the CEGA, using comet and NPL, is capable of detection of the genotoxicity of diverse DNA-reactive carcinogens, while not yielding false positives for non-carcinogens. © The Author 2014. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Multiple-endpoints gene alteration-based (MEGA) assay: A toxicogenomics approach for water quality assessment of wastewater effluents.

PubMed

Fukushima, Toshikazu; Hara-Yamamura, Hiroe; Nakashima, Koji; Tan, Lea Chua; Okabe, Satoshi

2017-12-01

Wastewater effluents contain a significant number of toxic contaminants, which, even at low concentrations, display a wide variety of toxic actions. In this study, we developed a multiple-endpoints gene alteration-based (MEGA) assay, a real-time PCR-based transcriptomic analysis, to assess the water quality of wastewater effluents for human health risk assessment and management. Twenty-one genes from the human hepatoblastoma cell line (HepG2), covering the basic health-relevant stress responses such as response to xenobiotics, genotoxicity, and cytotoxicity, were selected and incorporated into the MEGA assay. The genes related to the p53-mediated DNA damage response and cytochrome P450 were selected as markers for genotoxicity and response to xenobiotics, respectively. Additionally, the genes that were dose-dependently regulated by exposure to the wastewater effluents were chosen as markers for cytotoxicity. The alterations in the expression of an individual gene, induced by exposure to the wastewater effluents, were evaluated by real-time PCR and the results were validated by genotoxicity (e.g., comet assay) and cell-based cytotoxicity tests. In summary, the MEGA assay is a real-time PCR-based assay that targets cellular responses to contaminants present in wastewater effluents at the transcriptional level; it is rapid, cost-effective, and high-throughput and can thus complement any chemical analysis for water quality assessment and management. Copyright © 2017 Elsevier Ltd. All rights reserved.

Evaluation of genetic damage in tobacco and arsenic exposed population of Southern Assam, India using buccal cytome assay and comet assay.

PubMed

Roy, Prasenjit; Mukherjee, Anita; Giri, Sarbani

2016-02-01

Ground water is the principal source of drinking water in Assam. Ground water contamination of arsenic in drinking water is a great concern for human health and considered as a human carcinogen. The present cytogenetic biomonitoring study was undertaken to investigate the genotoxic effects associated with people of southern Assam consuming arsenic contaminated water and chewing tobacco. Employing the buccal cytome assay, exfoliated cells were analyzed in 138 individuals of age range 22-42 years and divided into four groups. Group I (n=54) are participants residing in localities where ground water contains arsenic concentration below the permissible limit (<10μg/l) and without any tobacco chewing history. Group II (n=32) participants from the same area but they are tobacco chewers. Group III (n=24) participants from localities where significantly high arsenic contamination in ground water were observed. Whereas the Group IV (n=28) consists of participants from the arsenic contaminated area and also tobacco chewers. Body mass index (BMI) in all the groups are found to be nearly same and in normal range. Statistically significant (P<0.001) increase in genotoxic, cell death parameters and cell proliferation biomarkers were observed in the Group IV compared to other groups. In the comet assay, percent of tail DNA gradually increases among the groups and has statistical significance. Spearman correlation revealed strong positive correlation between the arsenic exposed peoples and the binucleated cells (r=0.4763; P<0.001). Amount of chewing tobacco had significant positive correlation with micronucleus frequency (r=0.268; P<0.05) and karyolitic cells (r=0.217; P<0.05) and also in the percentage of tail DNA (r=0.5532, P<0.001). A statistically significant increase in glucose content and decrease in hemoglobin content as well as acetylcholine esterase in the blood of exposed individuals was observed. Our preliminary study indicate that population exposed to arsenic through drinking water may become more susceptible towards chewing tobacco induced nuclear damage as evaluated by buccal cytome assay and comet assay. Copyright © 2015 Elsevier Inc. All rights reserved.

Mixture genotoxicity of 2,4-dichlorophenoxyacetic acid, acrylamide, and maleic hydrazide on human Caco-2 cells assessed with comet assay.

PubMed

Syberg, Kristian; Binderup, Mona-Lise; Cedergreen, Nina; Rank, Jette

2015-01-01

Assessment of genotoxic properties of chemicals is mainly conducted only for single chemicals, without taking mixture genotoxic effects into consideration. The current study assessed mixture effects of the three known genotoxic chemicals, 2,4-dichlorophenoxyacetic acid (2,4-D), acrylamide (AA), and maleic hydrazide (MH), in an experiment with a fixed ratio design setup. The genotoxic effects were assessed with the single-cell gel electrophoresis assay (comet assay) for both single chemicals and the ternary mixture. The concentration ranges used were 0-1.4, 0-20, and 0-37.7 mM for 2,4-D, AA, and MH, respectively. Mixture toxicity was tested with a fixed ratio design at a 10:23:77% ratio for 2.4-D:AA:MH. Results indicated that the three chemicals yielded a synergistic mixture effect. It is not clear which mechanisms are responsible for this interaction. A few possible interactions are discussed, but further investigations including in vivo studies are needed to clarify how important these more-than-additive effects are for risk assessment.

Transcriptome changes induced in vitro by alcohol-containing mouthwashes in normal and dysplastic oral keratinocytes.

PubMed

Fox, Simon A; Currie, Sean S; Dalley, Andrew J; Farah, Camile S

2018-05-01

The role of alcohol-containing mouthwash as a risk factor for the development of oral cancer is a subject of conflicting epidemiological evidence in the literature despite alcohol being a recognised carcinogen. The aim of this study was to use in vitro models to investigate mechanistic and global gene expression effects of exposure to alcohol-containing mouthwash. Two brands of alcohol-containing mouthwash and their alcohol-free counterparts were used to treat two oral cell lines derived from normal (OKF6-TERT) and dysplastic (DOK) tissues. Genotoxicity was determined by Comet assay. RNA-seq was performed using the Ion Torrent platform. Bioinformatics analysis used R/Bioconductor packages with differential expression using DEseq2. Pathway enrichment analysis used EnrichR with the WikiPathways and Kegg databases. Both cell lines displayed dose-dependent DNA damage in response to acute exposure to ethanol and alcohol-containing mouthwashes as well as alcohol-free mouthwashes reconstituted with ethanol as shown by Comet assay. The transcriptomic effects of alcohol-containing mouthwash exposure were more complex with significant differential gene expression ranging from >2000 genes in dysplastic (DOK) cells to <100 genes in normal (OKF6-TERT) cells. Pathway enrichment analysis in DOK cells revealed alcohol-containing mouthwashes showed common features between the two brands used including DNA damage response as well as cancer-associated pathways. In OKF6-TERT cells, the most significantly enriched pathways involved inflammatory signalling. Alcohol-containing mouthwashes are genotoxic in vitro to normal and dysplastic oral keratinocytes and induce widespread changes in gene expression. Dysplastic cells are more susceptible to the transcriptomic effects of mouthwash. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

[Evaluation of cyto- and genotoxic action of ferronanomagnetic and constant magnetic field in in vivo system].

PubMed

Chekhun, V F; Lozovs'ka, Iu V; Luk'ianova, N Iu; Demash, D V; Todor, I M; Nalieskina, L A

2013-01-01

Cyto- and genotoxic effects of nanoparticles on the basis of FM, CMF or their combination have been studied in AKE cells, BM cells of erythroid line, and peripheral blood lymphocytes with the use of MN test and "DNA-comet" assay. It has been shown that expression of mentioned effects is related to FM concentration and duration of tested agent action. It has been also demonstrated that action of CMF alone in the studied cells did not cause any changes in cell architectonics or affect MN counts which are associated with DNA damage. When FM and CMF were used in combination there has been observed the phenomenon of induction of CMF action with FM nanoparticles. The obtained results allow recommend MN test and "DNA-comet" assay as the markers of genome stability in the tests of genotoxic effects of nanomaterials for development of vector nanosystems.

Cytogenetic investigation of subjects professionally exposed to radiofrequency radiation.

PubMed

Maes, Annemarie; Van Gorp, Urbain; Verschaeve, Luc

2006-03-01

Nowadays, virtually everybody is exposed to radiofrequency radiation (RFR) from mobile phone base station antennas or other sources. At least according to some scientists, this exposure can have detrimental health effects. We investigated cytogenetic effects in peripheral blood lymphocytes from subjects who were professionally exposed to mobile phone electromagnetic fields in an attempt to demonstrate possible RFR-induced genetic effects. These subjects can be considered well suited for this purpose as their RFR exposure is 'normal' though rather high, and definitely higher than that of the 'general population'. The alkaline comet assay, sister chromatid exchange (SCE) and chromosome aberration tests revealed no evidence of RFR-induced genetic effects. Blood cells were also exposed to the well known chemical mutagen mitomycin C in order to investigate possible combined effects of RFR and the chemical. No cooperative action was found between the electromagnetic field exposure and the mutagen using either the comet assay or SCE test.

Genotoxicity assessment of propyl thiosulfinate oxide, an organosulfur compound from Allium extract, intended to food active packaging.

PubMed

Mellado-García, P; Maisanaba, S; Puerto, M; Llana-Ruiz-Cabello, M; Prieto, A I; Marcos, R; Pichardo, S; Cameán, A M

2015-12-01

Essential oils from onion (Allium cepa L.), garlic (Allium sativum L.), and their main components, such as propyl thiosulfinate oxide (PTSO) are being intended for active packaging with the purpose of maintaining and extending food product quality and shelf life. The present work aims to assess for the first time the potential mutagenicity/genotoxicity of PTSO (0-50 µM) using the following battery of genotoxicity tests: (1) the bacterial reverse-mutation assay in Salmonella typhimurium (Ames test, OECD 471); (2) the micronucleus test (OECD 487) (MN) and (3) the mouse lymphoma thymidine-kinase assay (OECD 476) (MLA) on L5178YTk(+/-), cells; and (4) the comet assay (with and without Endo III and FPG enzymes) on Caco-2 cells. The results revealed that PTSO was not mutagenic in the Ames test, however it was mutagenic in the MLA assay after 24 h of treatment (2.5-20 µM). The parent compound did not induce MN on mammalian cells; however, its metabolites (in the presence S9) produced positive results (from 15 µM). Data from the comet assay indicated that PTSO did not induce DNA breaks or oxidative DNA damage. Further in vivo genotoxicity tests are needed to confirm its safety before it is used as active additive in food packaging. Copyright © 2015 Elsevier Ltd. All rights reserved.

Genotoxicity evaluation of the naturally-derived food colorant, gardenia blue, and its precursor, genipin.

PubMed

Hobbs, Cheryl A; Koyanagi, Mihoko; Swartz, Carol; Davis, Jeffrey; Maronpot, Robert; Recio, Leslie; Hayashi, Shim-Mo

2018-06-04

Gardenia blue is widely used in Eastern Asia as a natural food colorant. To evaluate the genotoxic potential of gardenia blue, as well as genipin, the natural starting material from which it is produced, a GLP-compliant test battery was conducted according to OECD guidelines. No evidence of mutagenicity of gardenia blue was detected in a 5-strain bacterial reverse mutation assay, with or without metabolic activation; an equivocal response for genipin occurred in S. typhimurium TA97a without metabolic activation. In in vitro micronucleus and chromosome aberration assays, genipin tested positive under some test conditions; however, gardenia blue tested negative in both assays. In combined micronucleus/comet assays conducted in male and female B6C3F1 mice, exposure to genipin at doses reaching maximal toxicity (74 and 222 mg/kg bw/day for males and females, respectively) or gardenia blue tested up to the limit dose (2000 mg/kg bw/day) did not induce micronuclei in peripheral blood or DNA damage in several examined tissues. Modified ("reverse") comet assays showed no evidence of DNA crosslinking potential of either genipin, known to form crosslinks with other macromolecules, or gardenia blue. Our results indicate that consumption of gardenia blue in food products does not pose a significant genotoxic concern for humans. Copyright © 2018. Published by Elsevier Ltd.

Aspects of nitrogen dioxide toxicity in environmental urban concentrations in human nasal epithelium

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

Koehler, C.; Ginzkey, C.; Friehs, G.

Cytotoxicity and genotoxicity of nitrogen dioxide (NO{sub 2}) as part of urban exhaust pollution are widely discussed as potential hazards to human health. This study focuses on toxic effects of NO{sub 2} in realistic environmental concentrations with respect to the current limit values in a human target tissue of volatile xenobiotics, the epithelium of the upper aerodigestive tract. Nasal epithelial cells of 10 patients were cultured as an air-liquid interface and exposed to 0.01 ppm NO{sub 2}, 0.1 ppm NO{sub 2}, 1 ppm NO{sub 2}, 10 ppm NO{sub 2} and synthetic air for half an hour. After exposure, genotoxicity wasmore » evaluated by the alkaline single-cell microgel electophoresis (Comet) assay and by induction of micronuclei in the micronucleus test. Depression of proliferation and cytotoxic effects were determined using the micronucleus assay and trypan blue exclusion assay, respectively. The experiments revealed genotoxic effects by DNA fragmentation starting at 0.01 ppm NO{sub 2} in the Comet assay, but no micronucleus inductions, no changes in proliferation, no signs of necrosis or apoptosis in the micronucleus assay, nor did the trypan blue exclusion assay show any changes in viability. The present data reveal a possible genotoxicity of NO{sub 2} in urban concentrations in a screening test. However, permanent DNA damage as indicated by the induction of micronuclei was not observed. Further research should elucidate the effects of prolonged exposure.« less

Antiproliferative Activity and Apoptosis Inducing Mechanism of Anthocephalus cadamba on Dalton’s Lymphoma Ascites Cells

PubMed Central

Dolai, Narayan; Islam, Aminul; Haldar, Pallab Kanti

2016-01-01

The purpose of this investigation was to evaluate the antiproliferative and apoptogenic mechanistic studies of methanol extract of Anthocephalus cadamba (MEAC) on Dalton’s lymphoma ascites (DLA) cells treated mice. Determination of antiproliferative activity was performed by using different DLA cells (2×106 cells, i.p.) inoculated mice groups (n = 12). Groups were treated for 14 consecutive days with MEAC at the doses of 200 and 400 mg/Kg b.w. respectively. The mechanism of antiproliferation activity of MEAC was investigated through morphological studies by acridine orange (AO)/ethidium bromide (EB) double staining method. Comet assay was estimated to check the DNA damage induced apoptosis property. Furthermore, flow cytometry (FACS) was used to quantitatively detect the apoptotic rate by double labeling techniques using Annexin-V FITC/propidium iodide staining analysis and apoptotic proteins expression done by western blotting assay method. MEAC exhibited significant (p<0.01) decrease the tumor volume, viable cell count, tumor weight and elevated the life span of DLA tumor bearing mice. Analysis of AO/EB staining and flow cytometry showed that MEAC possessed apoptosis induced antitumor activity on DLA cells in a dose dependant manner. Dose dependent induction of DNA damage on DLA cells were observed after MEAC treatment, which was evident from the appearance of comet tail length. Pro-apoptotic gene, Bax was up-regulated and down-regulation of the Bcl-2/Bax ratio, suggesting that Bcl-2 family involved in the control of apoptosis. Experimental results revealed that MEAC possess potent antitumor activity via induction of cancer cell apoptosis mechanism. PMID:27980586

Exposure to 1800 MHz radiofrequency electromagnetic radiation induces oxidative DNA base damage in a mouse spermatocyte-derived cell line.

PubMed

Liu, Chuan; Duan, Weixia; Xu, Shangcheng; Chen, Chunhai; He, Mindi; Zhang, Lei; Yu, Zhengping; Zhou, Zhou

2013-03-27

Whether exposure to radiofrequency electromagnetic radiation (RF-EMR) emitted from mobile phones can induce DNA damage in male germ cells remains unclear. In this study, we conducted a 24h intermittent exposure (5 min on and 10 min off) of a mouse spermatocyte-derived GC-2 cell line to 1800 MHz Global System for Mobile Communication (GSM) signals in GSM-Talk mode at specific absorption rates (SAR) of 1 W/kg, 2 W/kg or 4 W/kg. Subsequently, through the use of formamidopyrimidine DNA glycosylase (FPG) in a modified comet assay, we determined that the extent of DNA migration was significantly increased at a SAR of 4 W/kg. Flow cytometry analysis demonstrated that levels of the DNA adduct 8-oxoguanine (8-oxoG) were also increased at a SAR of 4 W/kg. These increases were concomitant with similar increases in the generation of reactive oxygen species (ROS); these phenomena were mitigated by co-treatment with the antioxidant α-tocopherol. However, no detectable DNA strand breakage was observed by the alkaline comet assay. Taking together, these findings may imply the novel possibility that RF-EMR with insufficient energy for the direct induction of DNA strand breaks may produce genotoxicity through oxidative DNA base damage in male germ cells. Crown Copyright © 2013. Published by Elsevier Ireland Ltd. All rights reserved.

Cytotoxicity, genotoxicity and gene expression changes elicited by exposure of human hepatic cells to Ginkgo biloba leaf extract.

PubMed

Grollino, Maria Giuseppa; Raschellà, Giuseppe; Cordelli, Eugenia; Villani, Paola; Pieraccioli, Marco; Paximadas, Irene; Malandrino, Salvatore; Bonassi, Stefano; Pacchierotti, Francesca

2017-11-01

The use of Ginkgo biloba leaf extract as nutraceutical is becoming increasingly common. As a consequence, the definition of a reliable toxicological profile is a priority for its safe utilization. Recently, contrasting data have been reported on the carcinogenic potential of Ginkgo biloba extract in rodent liver. We measured viability, Reactive Oxygen Species (ROS), apoptosis, colony-forming efficiency, genotoxicity by comet assay, and gene expression changes associated with hepato-carcinogenicity in human cells of hepatic origin (HepG2 and THLE-2) treated with different concentrations (0.0005-1.2 mg/mL) of Ginkgoselect ® Plus. Our analyses highlighted a decrease of cell viability, not due to apoptosis, after treatment with high doses of the extract, which was likely due to ROS generation by a chemical reaction between extract polyphenols and some components of the culture medium. Comet assay did not detect genotoxic effect at any extract concentration. Finally, the array analysis detected a slight decrease in the expression of only one gene (IGFBP3) in Ginkgo-treated THLE-2 cells as opposed to changes in 28 genes in Aflatoxin B1 treated-cells. In conclusion, our results did not detect any significant genotoxic or biologically relevant cytotoxic effects and gross changes in gene expression using the Ginkgo extract in the hepatic cells tested. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Mechanisms of sulfur mustard analog 2-chloroethyl ethyl sulfide-induced DNA damage in skin epidermal cells and fibroblasts.

PubMed

Inturi, Swetha; Tewari-Singh, Neera; Gu, Mallikarjuna; Shrotriya, Sangeeta; Gomez, Joe; Agarwal, Chapla; White, Carl W; Agarwal, Rajesh

2011-12-15

Employing mouse skin epidermal JB6 cells and dermal fibroblasts, here we examined the mechanisms of DNA damage by 2-chloroethyl ethyl sulfide (CEES), a monofunctional analog of sulfur mustard (SM). CEES exposure caused H2A.X and p53 phosphorylation as well as p53 accumulation in both cell types, starting at 1h, that was sustained for 24h, indicating a DNA-damaging effect of CEES, which was also confirmed and quantified by alkaline comet assay. CEES exposure also induced oxidative stress and oxidative DNA damage in both cell types, measured by an increase in mitochondrial and cellular reactive oxygen species and 8-hydroxydeoxyguanosine levels, respectively. In the studies distinguishing between oxidative and direct DNA damage, 1h pretreatment with glutathione (GSH) or the antioxidant Trolox showed a decrease in CEES-induced oxidative stress and oxidative DNA damage. However, only GSH pretreatment decreased CEES-induced total DNA damage measured by comet assay, H2A.X and p53 phosphorylation, and total p53 levels. This was possibly due to the formation of GSH-CEES conjugates detected by LC-MS analysis. Together, our results show that CEES causes both direct and oxidative DNA damage, suggesting that to rescue SM-caused skin injuries, pleiotropic agents (or cocktails) are needed that could target multiple pathways of mustard skin toxicities. Copyright © 2011 Elsevier Inc. All rights reserved.

Lack of genotoxicity in Astyanax bimaculatus and Oreochromis niloticus of 17α-methyltestosterone used in fish hatcheries to produce male monosex populations.

PubMed

Rivero-Wendt, C L G; Miranda-Vilela, A L; Ferreira, M F N; Amorim, F S; da Silva, V A G; Louvandini, H; Grisolia, C K

2013-10-24

17α-Methyltestosterone (MT) is widely used in fish hatcheries of many countries to produce male monosex populations. Its genotoxic risk to fish species is not well known and studies in other in vivo models are still inconclusive. MT was tested for genotoxicity in the fish species Oreochromis niloticus (tilapia), a target species, and Astyanax bimaculatus (lambari), a native non-target species. Genotoxicity was evaluated by the micronucleus test (MN), nuclear abnormalities (NA), and comet assay using peripheral erythrocytes of both species after a 96-h exposure to MT at concentrations of 0.01, 0.1, and 1.0 mg/L in the water. At the lowest exposure level of 0.01 mg/L, MT induced MN in both species and NA only in O. niloticus. These effects were not observed in the comet assay. Chromatographic analysis of water samples collected from aquariums at the beginning and end of each experiment showed that MT was consumed during the 96-h exposure. At the highest level of exposure (1.0 mg/L), 81.69% of the hormone was consumed during the exposure period. The chromatogram showed that at the lowest concentration level of 0.01 mg/L, 99.56% MT was consumed by the end of the exposure period. Thus, exposure to MT did not cause genotoxicity in either fish species.

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.

Evaluation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) adduct levels and DNA strand breaks in human peripheral blood lymphocytes exposed in vitro to polycyclic aromatic hydrocarbons with or without animal metabolic activation.

PubMed

Isabel, Rodríguez-Romero María; Sandra, Gómez-Arroyo; Rafael, Villalobos-Pietrini; Carmen, Martínez-Valenzuela; Josefina, Cortés-Eslava; del Carmen, Calderón-Ezquerro María; Rocío, García-Martínez; Francisco, Arenas-Huertero; Elena, Calderón-Segura María

2012-04-01

The polycyclic aromatic hydrocarbons (PAHs) dibenzo(a,h)anthracene, benzo(ghi)perylene, benzo(b)fluoranthene and benzo(a)pyrene have been identified in urban air from Mexico City and some of them are classified as human carcinogens. In the present study, human peripheral blood lymphocytes were exposed in vitro to different concentrations of PAHs with (+S9) or without (-S9) metabolic activation. The genotoxic and cytotoxic effects of each PAH were examined with an alkaline comet assay and trypan blue dye exclusion, and oxidative DNA damage was determined via the detection of 8-hydroxy-2'-deoxyguanosine (8-OhdG) adduct levels by enzyme-linked immunosorbent assay (ELISA). The DNA damage was evaluated with two genotoxicity parameters: the frequency of comets and the comet tail length. Concentrations of 20, 40, 80, 160 and 320 µM DB(a,h)A-S9; 20, 40, 80, 160 and 240 µM B(ghi)P-S9; 20, 30, 40, 60 and 80 µM B(b)F-S9; and 80 µM B(a)P-S9 for 24 h induced a small but significant increase in the means of comet frequency, in the tail length and in the 8-oHDg levels in relation to the control (0.5% DMSO-S9). However, all PAHs+S9 produced a more significant increase in DNA strand breaks and the level of 8-OHdG compared with the control (0.5% DMSO+S9), with a concentration-effect relationship. The viability of lymphocytes exposed to all PAHs-S9 and PAHs+S9 was not modified compared with the control. The results of this study demonstrate that the comet and ELISA are rapid, suitable and sensitive methods to detect in vitro PAH-induced DNA damage in human peripheral lymphocytes.

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

Primary DNA damage in chrome-plating workers.

PubMed

Gambelunghe, A; Piccinini, R; Ambrogi, M; Villarini, M; Moretti, M; Marchetti, C; Abbritti, G; Muzi, G

2003-06-30

In order to evaluate the primary DNA damage due to occupational exposure to chromium (VI), DNA strand-breaks and apoptosis in peripheral lymphocytes were measured in a group of 19 chrome-plating workers. DNA strand-breaks was assessed by alkaline (pH>13) single-cell microgel electrophoresis ('comet') assay, while apoptosis was measured by flow-cytometry after propidium iodide staining of the cells. Concentrations of chromium in urine, erythrocytes and lymphocytes were investigated as biological indicators of exposure. A group of 18 hospital workers (control group I) and another 20 university personnel (control group II) without exposure to chromium were also studied as controls. The results of the study show that chrome-plating workers have higher levels of chromium in urine, erythrocytes and lymphocytes than unexposed workers. Comet tail moment values, assumed as index of DNA damage, are increased in chromium-exposed workers and results are significantly correlated to chromium lymphocyte concentrations. No difference emerged in the percentage of apoptotic nuclei in exposed and unexposed workers. The study confirms that measurements of chromium in erythrocytes and lymphocytes may provide useful information about recent and past exposure to hexavalent chromium at the workplace. The increase in DNA strand-breaks measured by comet assay suggests this test is valid for the biological monitoring of workers exposed to genotoxic compounds such as chromium (VI).

High doses of alcohol during pregnancy cause DNA damages in osteoblasts of newborns rats.

PubMed

Carvalho, Isabel Chaves Silva; Dutra, Tamires Pereira; Andrade, Dennia Perez De; Balducci, Ivan; Pacheco-Soares, Cristina; Rocha, Rosilene Fernandes da

2016-02-01

Alcohol exerts teratogenic effects and its consumption during pregnancy can cause deficit of bone development. The aim of the current study was to evaluate the genotoxic effects of prenatal exposure to ethanol on newborn rat osteoblasts. Wistar rats were initially divided into two groups: Ethanol group which received Ethanol 20% V/V in liquid diet and solid diet ad libitum, and Control group, which received solid diet and water ad libitum. Each group received a specific diet for 8 weeks before breeding and throughout three weeks of gestation and the treatment was finished on the day the pups were killed. On the fifth day of life, the pups from each group were killed for removal of the calvaria and isolation of osteogenic cells by sequential enzymatic digestion. The cells were cultured for a maximum period of 14 days. The detection of genotoxic effects of alcohol was investigated by the comet and the micronucleus assay. Micronucleus and comet assay showed significant increases in DNA damage at 7 days in Ethanol group (p = 0.0302, p = 0.0446, respectively). However, at 14 days both assay showed no significant difference between the groups (p = 0.6194, p = 0.8326, respectively). Our results showed that prenatal exposure to ethanol induced DNA damage in osteoblasts, as shown by micronucleus formation and higher percentage of DNA in the comet tail. It can be concluded that prenatal exposure to ethanol damages osteoblast DNA in newborns exposed to high doses of ethanol during pregnancy, suggesting that prenatal ethanol consumption has a direct effect on fetal osteoblasts. © 2015 Wiley Periodicals, Inc.

DNA damage and micronuclei in parthenogenetic and bisexual Darevskia rock lizards from the areas with different levels of soil pollution.

PubMed

Simonyan, Anna; Hovhannisyan, Galina; Sargsyan, Anzhela; Arakelyan, Marine; Minasyan, Seyran; Aroutiounian, Rouben

2018-06-15

Natural species are widely used as indicator organisms to estimate of the impact of environmental pollution. Here we present the results of first study of a reliability of parthenogenetic Darevskia аrmeniaca and bisexual Darevskia raddei rock lizards as sentinels for monitoring of environmental genotoxicity. The comet assay and micronucleus test were applied to the lizards sampled in six areas in Armenia and Artsakh with different levels of soil contamination. The results obtained showed a clear relationship between the pollution level of lizards' habitats and the frequency of DNA damage in the comet assay. Low baseline frequency of micronuclei in D. аrmeniaca and D. raddei, however, makes this parameter ineffective for environmental genotoxicity evaluation. The parthenogenetic lizards D. аrmeniaca showed higher sensitivity toward genotoxic pollutions compared with bisexual D. raddei living in the same environment. The correlations between soil content of heavy metals Cr, Cu, Zn, Mo, Pb and DNA damage in D. аrmeniaca and between Cu, As, Mo, Pb and DNA damage in D. raddei were revealed. Overall, the lizards D. raddei and D. аrmeniaca appeared to be sensitive species in detecting soil pollution in natural environment. The application of the comet assay in Darevskia lizard species can be considered as a more appropriate method than a micronucleus test. The use of parthenogenetic lizards D. аrmeniaca as bioindicator will permit to assess the environmental genotoxicity independent of the genetic polymorphism of bisexual species. Copyright © 2018. Published by Elsevier Inc.

Effect of pollution on DNA damage and essential fatty acid profile in Cirrhinus mrigala from River Chenab

NASA Astrophysics Data System (ADS)

Hussain, Bilal; Sultana, Tayyaba; Sultana, Salma; Al-Ghanim, K. A.; Mahboob, Shahid

2017-05-01

The objective of this study was to evaluate the effect of anthropogenic pollution on DNA damage and the fatty acid profile of the bottom dweller fish ( Cirrhinus mrigala), collected from the River Chenab, in order to assess the effect of the toxicants on the quality of the fish meat. The levels of Cd, Hg, Cu, Mn, Zn, Pb, Cr and Sn and of phenols from this river were significantly higher than the permissible limits set by the USEPA. Comet assays showed DNA damage in Cirrhinus mrigala collected from three different sampling sites in the polluted area of the river. Significant differences were observed for DNA damage through comet assay in fish collected from polluted compared to control sites. No significant differences were observed for DNA damage between farmed and fish collected from upstream. The micronucleus assay showed similar trends. Fish from the highly polluted sites showed less number of fatty acids and more saturated fatty acids in their meat compared to fish from less polluted areas. Several fatty acids were missing in fish with higher levels of DNA in comet tail and micronucleus induction. Long-chain polyunsaturated fatty acids, eicosapentaenoic acid (20:5n-3) was found missing in the fish from polluted environment while it was found in considerable amount in farmed fish 7.8±0.4%. Docosahexaenoic acid (22:6n-3) also showed significant differences as 0.1±0.0 and 7.0±0.1% respectively, in wild polluted and farmed fishes.

Evaluation of the genotoxic potential of 3-monochloropropane-1,2-diol (3-MCPD) and its metabolites, glycidol and beta-chlorolactic acid, using the single cell gel/comet assay.

PubMed

El Ramy, R; Ould Elhkim, M; Lezmi, S; Poul, J M

2007-01-01

3-monochloropropane-1,2-diol (3-MCPD) is a member of a group of chemicals known as chloropropanols. It is found in many foods and food ingredients as a result of food processing. 3-MCPD is regarded as a rat carcinogen known to induce Leydig-cell and mammary gland tumours in males and kidney tumours in both genders. The aim of our study was to clarify the possible involvement of genotoxic mechanisms in 3-MCPD induced carcinogenicity at the target organ level. For that purpose, we evaluated DNA damages in selected target (kidneys and testes) and non-target (blood leukocytes, liver and bone marrow) male rat organs by the in vivo alkaline single cell gel electrophoresis (comet) assay, 3 and 24 h after 3-MCPD oral administration to Sprague-Dawley and Fisher 344 adult rats. 3-MCPD may be metabolised to a genotoxic intermediate, glycidol, whereas the predominant urinary metabolite in rats following 3-MCPD administration is beta-chlorolactic acid. Therefore, we also studied the DNA damaging effects of 3-MCPD and its metabolites, glycidol and beta-chlorolactic acid, in the in vitro comet assay on CHO cells. Our results show the absence of genotoxic potential of 3-MCPD in vivo in the target as well as in the non-target organs. Glycidol, the epoxide metabolite, induced DNA damages in CHO cells. beta-Chlorolactic acid, the main metabolite of 3-MCPD in rats, was shown to be devoid of DNA-damaging effects in vitro in mammalian cells.

The Imagery of Sound

NASA Technical Reports Server (NTRS)

2000-01-01

Automated Analysis Corporation's COMET is a suite of acoustic analysis software for advanced noise prediction. It analyzes the origin, radiation, and scattering of noise, and supplies information on how to achieve noise reduction and improve sound characteristics. COMET's Structural Acoustic Foam Engineering (SAFE) module extends the sound field analysis capability of foam and other materials. SAFE shows how noise travels while airborne, how it travels within a structure, and how these media interact to affect other aspects of the transmission of noise. The COMET software reduces design time and expense while optimizing a final product's acoustical performance. COMET was developed through SBIR funding and Langley Research Center for Automated Analysis Corporation.

Evaluation of the genotoxicity of waters impacted by domestic and industrial effluents of a highly industrialized region of São Paulo State, Brazil, by the comet assay in HTC cells.

PubMed

Manzano, Bárbara Cassu; Roberto, Matheus Mantuanelli; Hoshina, Márcia Miyuki; Menegário, Amauri Antônio; Marin-Morales, Maria Aparecida

2015-01-01

The problems that most affect the quality of the waters of rivers and lakes are associated with the discharges performed in these environments, mainly industrial and domestic effluents inappropriately treated or untreated. The comet assay is a sensitive tool and is recommended for studies of environmental biomonitoring, which aim to determine the genotoxicity potential of water pollutants. This study aimed to assess the genotoxic potential of the Ribeirão Tatu waters, region of Limeira, São Paulo (SP), by the comet assay with mammalian cells (hepatoma tissue culture (HTC)). Water samples were collected along the Ribeirão Tatu at three distinct periods: November 2008, February 2009 and August 2009, and five collection sites were established: P1, source of the stream; P2, site located downstream the urban perimeter of the municipality of Cordeirópolis and after receiving the pollution load of this city; P3, collection site located upstream the urban perimeter of the city of Limeira; P4, urban area of Limeira; and P5, rural area of Limeira, downstream the discharges of the city sewage. The results showed that for the November 2008 collection, there was no water sample-induced genotoxicity; for the February 2009 collection, the sites P1 and P2 were statistically significant in relation to the negative control (NC), and for the August 2009 collection, the site P5 was statistically significant. These results could be explained by the content of different metals during the different seasons that are under the influence of domestic, industrial and agricultural effluents and also due to the seasonality, since the water samples collected in the period of heavy rain (February 2009) presented a higher genotoxicity possibly due to the entrainment of contaminants into the bed of the stream promoted by the outflow of rainwaters. The comet assay showed to be a useful and sensitive tool in the evaluation of hydric resources impacted by pollutants of diverse origins, and a constant monitoring should be done in order to verify the influence of different factors (season, amount of contaminants) in the water quality.

Assessment of the in vitro and in vivo genotoxicity of extracts and indole monoterpene alkaloid from the roots of Galianthe thalictroides (Rubiaceae).

PubMed

Fernandes, L M; Garcez, W S; Mantovani, M S; Figueiredo, P O; Fernandes, C A; Garcez, F R; Guterres, Z R

2013-09-01

Roots of Galianthe thalictroides K. Schum. (Rubiaceae) are used in folk medicine in the State of Mato Grosso do Sul, Brazil, for treating and preventing cancer. To gain information about the genotoxicity of extracts (aqueous and EtOH), the CHCl₃ phase resulting from partition of the EtOH extract and the indole monoterpene alkaloid 1 obtained from this plant. The genotoxicity of 1 and extracts was evaluated in vivo through the Drosophila melanogaster wing Somatic Mutation and Recombination Test - SMART, while in vitro cytotoxic (MTT) and Comet assays were performed only with alkaloid 1. The results obtained with the SMART test indicated that the aqueous extract had no genotoxic activity. The EtOH extract was not genotoxic to ST descendants but genotoxic to HB ones. The CHCl₃ phase was genotoxic and cytotoxic. Alkaloid 1 showed significant mutational events with SMART, in the cytotoxicity assay (MTT), it showed a high cytotoxicity for human hepatoma cells (HepG2), whereas for the Comet assay, not showing genotoxic activity. The ethanol extract was shown to be genotoxic to HB descendants in the SMART assay, while the results obtained in this test for the monoterpene indole alkaloid 1 isolated from this extract. Copyright © 2013 Elsevier Ltd. All rights reserved.

17β-Estradiol induces cyto-genotoxicity on blood cells of common carp (Cyprinus carpio).

PubMed

Orozco-Hernández, Luis; Gutiérrez-Gómez, Adriana Andrea; SanJuan-Reyes, Nely; Islas-Flores, Hariz; García-Medina, Sandra; Galar-Martínez, Marcela; Dublán-García, Octavio; Natividad, Reyna; Gómez-Oliván, Leobardo Manuel

2018-01-01

17β-Estradiol, a natural hormone present at high concentrations in aquatic ecosystems, affects and modifies endocrine function in animals. In recent years research workers have expressed concern over its potential effects on aquatic organisms; however, little is known about its capacity to induce genetic damage or the pro-apoptotic effects of such damage on fish. Therefore, this study aimed to evaluate 17β-estradiol-induced cyto-genotoxicity in blood cells of the common carp Cyprinus carpio exposed to different concentrations (1 ng, 1 μg and 1 mg L -1 ). Peripheral blood samples were collected and evaluated by comet assay, micronucleus test, determination of caspase-3 activity and TUNEL assay at 12, 24, 48, 72 and 96 h of exposure. Increases in frequency of micronuclei, TUNEL-positive cells and caspase-3 activity were observed, particularly at the highest concentration. In contrast, the comet assay detected significant increases at 24 and 96 h with the 1 μg and 1 ng L -1 concentrations respectively. The set of assays used in the present study constitutes a reliable early warning biomarker for evaluating the toxicity induced by this type of emerging contaminants on aquatic species. Copyright © 2017 Elsevier Ltd. All rights reserved.

Matrix metalloproteases inhibition and biocompatibility of gold and platinum nanoparticles.

PubMed

Hashimoto, Masanori; Kawai, Koji; Kawakami, Hayato; Imazato, Satoshi

2016-01-01

Matrix metalloprotease (MMP) inhibitors improve the longevity of dental adhesives/tooth bonds; however, biocompatibility is required for their clinical use. This study evaluated the inhibition of MMPs and toxicity of two gold (AuNPs) and platinum nanoparticles (PtNPs) as possible compounds for use in dental adhesives. The MMP assay for studying the interaction of MMPs and nanoparticles (NPs) was evaluated by an MMP assay kit and gelatin zymography. Cultured L929 fibroblast cells or RAW264 macrophages were exposed to NPs. The cellular responses to NPs were examined using cytotoxic (cell viability) and genotoxic assays (comet assay), and transmission electron microscopic (TEM) analysis. The mechanical properties (elastic modulus) of the experimental resin loaded with NPs were examined using thermomechanical analysis. All NPs inhibited MMP activity at relatively low concentrations. The NPs inhibit MMPs by chelating with the Zn(2+) bound in the active sites of MMPs. No cytotoxic and genotoxic effects were found in AuNPs, whereas the PtNPs possessed both adverse effects. In TEM analysis, the NPs were localized mainly in lysosomes without penetration into nuclei. The mechanical properties of the resins increased when AuNPs were added in resins, but not by PtNPs. AuNPs are attractive candidates to inhibit MMPs and improve the mechanical properties of resins without cytotoxic/genotoxic effects to cells, and therefore should be suitable for applications in adhesive resin systems. © 2015 Wiley Periodicals, Inc.

Analysis of the Cytotoxic Potential of Anisomelic Acid Isolated from Anisomeles malabarica

PubMed Central

Preethy, Christo Paul; Alshatwi, Ali Abdullah; Gunasekaran, Muthukumaran; Akbarsha, Mohammad Abdulkadher

2013-01-01

Anisomelic acid (AA), one of the major compounds in Anisomeles malabarica, was tested for its cytotoxicity and apoptosis-inducing potential in breast and cervical cancer cells. The MTT assay for cell viability indicated that AA is cytotoxic to all of the four cell lines tested in a dose- and duration-dependent manner. Acridine Orange & Ethidium Bromide (AO & EB) and Hoechst 33258 staining of AA-treated cells revealed typical apoptotic morphology such as condensed chromatin and formation of apoptotic bodies. The comet assay revealed DNA strand break(s), indicating that AA induces DNA damage which culminates in apoptosis. Thus, the study revealed the anti-proliferative and apoptosis-inducing properties of AA in both breast and cervical cancer cells. Therefore, anisomelic acid offers potential for application in breast and cervical cancer therapy. PMID:23833721

Apoptosis in liver cancer (HepG2) cells induced by functionalized gold nanoparticles.

PubMed

Ashokkumar, Thirunavukkarasu; Prabhu, Durai; Geetha, Ravi; Govindaraju, Kasivelu; Manikandan, Ramar; Arulvasu, Chinnasamy; Singaravelu, Ganesan

2014-11-01

An ethnopharmacological approach for biosynthesis of gold nanoparticles is being demonstrated using seed coat of Cajanus cajan. Medicinal value of capping molecule investigated for anticancer activity and results disclose its greater potential. The active principle of the seed coat [3-butoxy-2-hydroxypropyl 2-(2,4-dihydroxyphenyl) acetate] is elucidated. Rapid one-step synthesis yields highly stable, monodisperse (spherical) gold nanoparticles in the size ranging from 9 to 41 nm. Anticancer activity has been studied using liver cancer cells and cytotoxic mechanism has been evaluated using MTT, Annexin-V/PI Double-Staining Assay, Cell cycle, Comet assay and Flow cytometric analysis for apoptosis. The present investigation will open up a new possibility of functionalizing gold nanoparticles for apoptosis studies in liver cancer cells. Copyright © 2014 Elsevier B.V. All rights reserved.

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 activity to other biological responses, could provide further understanding of adverse effects in aquatic environments. Copyright © 2013 Elsevier B.V. All rights reserved.

CORSAIR (COmet Rendezvous, Sample Acquisition, Investigation, and Return): A New Frontiers Mission Concept to Collect Samples from a Comet and Return Them to Earth for Study

NASA Astrophysics Data System (ADS)

Sandford, S. A.; Chabot, N. L.; Dello Russo, N.; Leary, J. C.; Reynolds, E. L.; Weaver, H. A.; Wooden, D. H.

2017-07-01

CORSAIR (COmet Rendezvous, Sample Acquisition, Investigation, and Return) is a mission concept submitted in response to NASA's New Frontiers 4 call. CORSAIR's proposed mission is to return comet nucleus samples to Earth for detailed analysis.

Effects of ozone exposure on human epithelial adenocarcinoma and normal fibroblasts cells.

PubMed

Poma, Anna; Colafarina, Sabrina; Aruffo, Eleonora; Zarivi, Osvaldo; Bonfigli, Antonella; Di Bucchianico, Sebastiano; Di Carlo, Piero

2017-01-01

Previous studies show variable ozone cytotoxicity and genotoxicity in cell cultures, laboratory animals and humans directly exposed to tropospheric ozone. The aim of this study was therefore to investigate and compare the cyto and genotoxic effects of ozone using adenocarcinoma human alveolar basal epithelial cells A549 and normal human fibroblasts Hs27. A cell culture chamber with controlled atmosphere (a simulation reactor) was built to inject a flow of 120 ppb of ozone, which is two times the threshold value for the protection of human health, fixed by the EU legislation. Cell proliferation was evaluated by a luminescent cell viability assay while we assessed the genotoxic potential of ozone by the induction of micronuclei as well as evaluating DNA strand breaks by the induction of micronuclei evaluated by means of the cytokinesis-block micronucleus (CBMN) assay as well as evaluating DNA strand breaks by Alkaline Comet Assay (CA) or Comet Assay. A549 cells viability decreases significantly at 24 hours treatment with 120 ppb of O3 while at 48 hours and 72 hours O3 treated cells viability doesn't differ in respect to the control. However a significative decrease of A549 viability is shown at 72 hours vs. 48 hours in both treated and not-treated cells. The viability trend in the Hs27 cells did not show any significant changes in treated samples compared to the control in all conditions. The two genotoxicity biomarkers, the micronucleus and the comet tests, showed in both the cell types exposed to ozone, a significant increase in the number of micronuclei and in the tail DNA % in respect to the control even if at different times/cell type. Moreover, we found that O3 provokes genotoxic effects more evident in A549 cancer cells than in normal fibroblasts Hs27 ones. We applied a cell growth simulation model referred to ozone treated or not cell lines to confirm that the ozone exposure causes a slackening in the cells replication.

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.

DNA DAMAGE AND EXTERNAL LESIONS IN BROWN BULLHEAD FROM CONTAMINATED HABITATS

EPA Science Inventory

The single cell gel electrophoresis ("Comet") assay was used to compare levels of DNA damage in brown bullheads (Ameiurus nebulosus) collected from three known contaminated locations, the Cuyahoga River, Ashtabula River, and Ashumet Pond (Cape Cod), with brown bullheads collected...

The effects of urbanization on Lepomis macrochirus using the comet assay

EPA Science Inventory

Urbanization has been linked to increased concentrations of polycyclic aromatic hydrocarbons in natural waterways. This study was designed to examine the impact of urbanization and a wastewater treatment plant by investigating the impact on field-collected bluegill (Lepomis macr...

DNA damage evaluation of hydroxyapatite on fibroblast cell L929 using the single cell gel electrophoresis assay.

PubMed

Rajab, N F; Yaakob, T A; Ong, B Y; Hamid, M; Ali, A M; Annuar, B O; Inayat-Hussain, S H

2004-05-01

Hydroxyapatite is the main component of the bone which is a potential biomaterial substance that can be applied in orthopaedics. In this study, the biocompatibility of this biomaterial was assessed using an in vitro technique. The cytotoxicity and genotoxicity effect of HA2 and HA3 against L929 fibroblast cell was evaluated using the MTT Assay and Alkaline Comet Assay respectively. Both HA2 and HA3 compound showed low cytotoxicity effect as determined using MTT Assay. Cells viability following 72 hours incubation at maximum concentration of both HA2 and HA3 (200 mg/ml) were 75.3 +/- 8.8% and 86.7 +/- 13.1% respectively. However, the cytotoxicity effect of ZnSO4.7H2O as a positive control showed an IC50 values of 46 mg/ml (160 microM). On the other hand, both HA2 and HA3 compound showed a slight genotoxicity effect as determined using the Alkaline Comet Assay following incubation at the concentration 200 mg/ml for 72 hours. This assay has been widely used in genetic toxicology to detect DNA strand breaks and alkali-labile site. The percentage of the cells with DNA damage for both substance was 27.7 +/- 1.3% and 15.6 +/- 1.0% for HA2 and HA3 respectively. Incubation of the cells for 24 hours with 38 microg/ml (IC25) of positive control showed an increase in percentage of cells with DNA damage (67.5 +/- 0.7%). In conclusion, our study indicated that both hydroxyapatite compounds showed a good biocompatibility in fibroblast cells.

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

NASA Astrophysics Data System (ADS)

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

2014-02-01

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

Genotoxic effect of Physalis angulata L. (Solanaceae) extract on human lymphocytes treated in vitro.

PubMed

Alves dos Santos, Raquel; Cabral, Teresinha Rosa; Cabral, Isabel Rosa; Antunes, Lusânia Maria; Pontes Andrade, Cristiane; Cerqueira dos Santos Cardoso, Plínio; de Oliveira Bahia, Marcelo; Pessoa, Claudia; Martins do Nascimento, José Luis; Rodríguez Burbano, Rommel; Takahashi, Catarina Satie

2008-08-01

Physalis angulata L (Solanaceae) is a medicinal plant from North of Brazil, whose different extracts and infusions are commonly used in the popular medicine for the treatment of malaria, asthma, hepatitis, dermatitis and rheumatism. However, the genotoxic effects of P. angulata on human cells is not well known. The main purpose of the present study was to evaluate the in vitro genotoxic effects of aqueous extract of P. angulata using the comet assay and the micronucleus assay in human lymphocytes provided from 6 healthy donors. Treatments with P. angulata extracts were performed in vitro in order to access the extent of DNA damage. The comet assay has shown that treatments with P. angulata at 0.5, 1.0, 2.0, 3.0 and 6.0 microg/mL in culture medium were genotoxic. Lymphocytes treated with P. angulata at the concentrations of 3.0 and 6.0 microg/mL in culture medium showed a statistically significant increase in the frequency of micronucleus (p<0.05), however, the cytokinesis blocked proliferation index (CBPI) was not decreased after P. angulata treatment. In conclusion, the present work demonstrated the genotoxic effects of P. angulata extract on human lymphocytes in vitro.

Seahorse (Hippocampus reidi) as a bioindicator of crude oil exposure.

PubMed

Delunardo, Frederico Augusto Cariello; de Carvalho, Luciano Rodrigues; da Silva, Bruno Ferreira; Galão, Michel; Val, Adalberto Luís; Chippari-Gomes, Adriana R

2015-07-01

This study explored the suitability of the seahorse Hippocampus reidi (Ginsburg, 1933) for assessing biomarkers of genotoxic effects and its use as a sentinel organism to detect the effects of acute exposure to petroleum hydrocarbons. Fish were exposed to three concentrations of crude oil (10, 20 and 30 g/kg) for 96 h, and the activity of phase II biotransformation enzyme glutathione S-transferase (GST) was measured. In addition, we performed genotoxicity assays, such as comet assay, micronucleus (MN) test and nuclear abnormalities (NA) induction, on the erythrocytes of the fish species. Our results revealed that the inhibition of hepatic GST activity in H. reidi was dependent on increasing crude oil concentrations. In contrast, an increase in the damage index (DI) and MN frequency were observed with increased crude oil concentrations. These results indicate that the alkaline comet assay and micronucleus test were suitable and useful in the evaluation of the genotoxicity of crude oil, which could improve determinations of the impact of oil spills on fish populations. In addition, H. reidi is a promising "sentinel organism" to detect the genotoxic impact of petroleum hydrocarbons. Copyright © 2015 Elsevier Inc. All rights reserved.

Evaluation of genotoxic potential of avarol, avarone, and its methoxy and methylamino derivatives in prokaryotic and eukaryotic test models.

PubMed

Kolarević, Stoimir; Milovanović, Dragana; Kračun-Kolarević, Margareta; Kostić, Jovana; Sunjog, Karolina; Martinović, Rajko; Đorđević, Jelena; Novaković, Irena; Sladić, Dušan; Vuković-Gačić, Branka

2018-01-04

In this study, mutagenic and genotoxic potential of anti-tumor compounds avarol, avarone, and its derivatives 3'-methoxyavarone, 4'-(methylamino)avarone and 3'-(methylamino)avarone was evaluated and compared to cytostatics commonly used in chemotherapy (5-fluorouracil, etoposid, and cisplatin). Mutagenic potential of selected hydroquinone and quinones was assessed in prokaryotic model by the SOS/umuC assay in Salmonella typhimurium TA1535/pSK1002. Genotoxic potential was also assessed in eukaryotic models using comet assay in human fetal lung cell line (MRC-5), human adenocarcinoma epithelial cell line (A549), and in human peripheral blood cells (HPBC). The results indicated that avarol and avarone do not exert mutagenic/genotoxic potential. Among the studied avarone derivatives, mutagenic potential was detected by SOS/umuC test for 3'-(methylamino)avarone, but only after metabolic activation. The results of comet assay indicated that 3'-methoxyavarone and 3'-(methylamino)avarone have a significant impact on the level of DNA damage in the MRC-5 cell line. Genotoxic potential was not observed in A549 cells or HPBC probably due to a different uptake rate for the compounds and lower in metabolism rate within these cells.

First cytotoxic, genotoxic, and antigenotoxic assessment of Euterpe oleracea fruit oil (açaí) in cultured human cells.

PubMed

Marques, E S; Tsuboy, M S F; Carvalho, J C T; Rosa, P C P; Perazzo, F F; Gaivão, I O M; Maistro, E L

2017-08-17

Euterpe oleracea Mart., popularly known as "açaí", is a tropical fruit from the Amazon region where it has considerable economic importance. Açaí has been used as food and for several medicinal purposes. Despite the widespread use of this fruit, there is a lack of data regarding the safety of using this fruit oil exclusively. Therefore, we evaluated the in vitro cytotoxic, genotoxic, and antigenotoxic effects of E. oleracea fruit oil (EOO) in cultured human lymphocytes (non-metabolizing cells) and HepG2 cell line (human hepatoma) (metabolizing cells) by using MTT, comet, and micronucleus assays. A wide range of EOO concentrations was tested with a preliminary MTT assay, which allowed selecting five concentrations for comet and micronucleus assays: 2.5, 10, 100, 500, and 1000 µg/mL. The results showed that none of the EOO tested concentrations presented cytotoxic effects. The genotoxic assessment revealed an absence of significant DNA and chromosome damage in human lymphocytes and HepG2 cells but did not show chemoprotection against the DNA damage induced by methyl methanesulfonate and benzo[a]pyrene, used as DNA-damaging agents.

Sleep duration is associated with sperm chromatin integrity among young men in Chongqing, China.

PubMed

Wang, Xiaogang; Chen, Qing; Zou, Peng; Liu, Taixiu; Mo, Min; Yang, Huan; Zhou, Niya; Sun, Lei; Chen, Hongqiang; Ling, Xi; Peng, Kaige; Ao, Lin; Yang, Huifang; Cao, Jia; Cui, Zhihong

2017-10-09

This study explores whether sleep duration is associated with sperm chromatin integrity. To do so, we conducted a three-phase panel study of 796 male volunteers from colleges in Chongqing (China) from 2013 to 2015. Sleep duration was measured using a modified Munich Chronotype Questionnaire. Sperm DNA integrity was examined via Sperm Chromatin Structure Assay and Comet assay. Setting 7-7.5 h day -1 of sleep duration as a reference, either longer or shorter sleep duration was associated negatively with high DNA stainability (HDS) (P = 0.009), which reflected the immaturity of sperm chromatin. The volunteers with > 9.0 h day -1 sleep and those with ≤ 6.5 h day -1 sleep had 40.7 and 30.3% lower HDS than did volunteers with 7-7.5 h day -1 sleep. No association was found between sleep duration and DNA fragmentation index or Comet assay parameters. This study suggests that sleep duration is associated with sperm chromatin integrity. Further studies are required to validate these findings and investigate the mechanism underlying this association. © 2017 European Sleep Research Society.

Evaluation the urban atmospheric conditions in different cities using comet and micronuclei assay in Tradescantia pallida.

PubMed

Sposito, Juliana Caroline Vivian; Crispim, Bruno do Amaral; Romãn, Amanda Izadora; Mussury, Rosilda Mara; Pereira, Joelson Gonçalves; Seno, Leonardo Oliveira; Grisolia, Alexeia Barufatti

2017-05-01

In the present study, genotoxicity and mutagenicity were investigated in Tradescantia pallida exposed to vehicular traffic at different sites in a high-altitude tropical climate. During March, May, July, September, and November 2014, a comet assay and micronucleus bioassays were conducted on young inflorescences and leaves of T. pallida collected from twelve towns in the southern region of Mato Grosso do Sul with different amounts of vehicular traffic. Weather parameters (temperature, relative humidity and rainfall) were measured and vehicles were counted to determine traffic levels in each town. A higher frequency of genotoxic and mutagenic damage was observed in the municipality of Dourados. The highest frequency of genetic damage was observed in September and November according to both assays. Relative humidity and rainfall were inversely proportional to the frequency of genetic damage in T. pallida during the collection period. Based on these results, we conclude that the bioassays are efficient for assessing the effects of vehicular traffic in these towns with respect to weather conditions over time. These bioassays can be applied to identify risk areas, which are determined by climatic conditions and air pollutants released. Copyright © 2017 Elsevier Ltd. All rights reserved.

Disappearance of 19P/Borrelly's Silicate Feature in 2001 Apparition Is Attributed to Increase in Grain Size

NASA Technical Reports Server (NTRS)

Wooden, D. H.; Woodward, C. E.; Harker, D. E.

2002-01-01

We report on observations and analysis of HIFOGS 10 microns spectrophotometry of short period comet 19P/Borrelly on 2003 October 13, 15 UT at the NASA IRTF. 19P/Borrelly is one of two short period comets, comet 4PIFaye being the other, to have a silicate feature detected. During Borrelly s perihelion passage in 1994 December, a silicate feature was present with a flux-to-continuum ratio of 0.25. Two apparitions later in 2003 October, the silicate feature is absent. Thermal emission modeling using amorphous olivine and amorphous carbon shows that a slight increase in grain size accounts for the disappearance of the silicate feature. Analysis of 19P/Borrelly suggests grain size, and not the absence of olivine minerals, may be responsible for the absence of silicate features in most short period comets. 19P/Borrelly is one of the more active short period comets. However, short period comets as a family are less active than long period comets. Short period comets probably originated in the Kuiper Belt and suffered collisions while in residence in the outer solar system. Upon evolution into orbits that take them through the inner solar system, the surfaces of short period comets are exposed to sunlight through their many perihelion passages. This is in contrast to long period comets which probably originated near Jupiter and were expelled to the Oort cloud where they have existed and been exposed to cosmic ray processing. By studying the grain properties in short period comets and comparing to long period comets, we compare the effects on the grain populations of different parent body evolution histories. Upcoming opportunities to study short and long period comets will be advertised.

Genotoxic risk identification of soil contamination at a major industrialized city in northeast China by a combination of in vitro and in vivo bioassays.

PubMed

Xiao, Rui-Yang; Wang, Zijian; Wang, Chun-Xia; Yu, Guo; Zhu, Yong-Guan

2006-10-01

The present study evaluated the genotoxicity of field soils in the Tianjin area, one of the most industrialized contaminated areas in northeast China. The genotoxicity of organic extracts of 41 soils was assayed by an in vitro SOS/ umu bioassay with Salmonella typhimurium TA 1535/pSK 1002. From the 41 soil samples, 11 samples were selected to confirm the genotoxic effect by in vivo single-cell gel electrophoresis (comet assay) using earthworms (Eisenia fetida). The results obtained demonstrated that, in the in vitro assay, genotoxicity expressed as induction ratios (IR) ranged from 1.00 to 4.60, and in the in vivo assay, the genotoxicity expressed as tail moment (TM) varied from 14.6 to 57.8 microm. All samples with high genotoxicity assessed by the SOS/umu bioassay possessed significantly high genotoxic effects in the comet assay, and there was a correlation (R2 = 0.736, p < 0.05) between IR and TM in both bioassays. It is concluded that soils in the Tianjin area were seriously contaminated by organic genotoxicants and higher levels of genotoxic effects existed in soils in the urban area of Tianjin as well as in areas near the coastal towns in the northeast part of the city. It can be concluded that a combination of in vivo and in vitro bioassays as a powerful and efficient genotoxicity-assessing tool could facilitate the assessment of genotoxic risk at a regional scale.

Effect of drinking water disinfection by-products in human peripheral blood lymphocytes and sperm.

PubMed

Ali, Aftab; Kurzawa-Zegota, Malgorzata; Najafzadeh, Mojgan; Gopalan, Rajendran C; Plewa, Michael J; Anderson, Diana

2014-12-01

Drinking water disinfection by-products (DBPs) are generated by the chemical disinfection of water and may pose hazards to public health. Two major classes of DBPs are found in finished drinking water: haloacetic acids (HAAs) and trihalomethanes (THMs). HAAs are formed following disinfection with chlorine, which reacts with iodide and bromide in the water. Previously the HAAs were shown to be cytotoxic, genotoxic, mutagenic, teratogenic and carcinogenic. To determine the effect of HAAs in human somatic and germ cells and whether oxidative stress is involved in genotoxic action. In the present study both somatic and germ cells have been examined as peripheral blood lymphocytes and sperm. The effects of three HAA compounds: iodoacetic acid (IAA), bromoacetic acid (BAA) and chloroacetic acid (CAA) were investigated. After determining appropriate concentration responses, oxygen radical involvement with the antioxidants, butylated hydroxanisole (BHA) and the enzyme catalase, were investigated in the single cell gel electrophoresis (Comet) assay under alkaline conditions, >pH 13 and the micronucleus assay. In the Comet assay, BHA and catalase were able to reduce DNA damage in each cell type compared to HAA alone. In the micronucleus assay, micronuclei (MNi) were found in peripheral lymphocytes exposed to all three HAAs and catalase and BHA were in general, able to reduce MNi induction, suggesting oxygen radicals play a role in both assays. These observations are of concern to public health since both human somatic and germ cells show similar genotoxic responses. Copyright © 2014. Published by Elsevier B.V.

Infrared Imaging, Spectroscopic, and Photometric Studies of Comets

NASA Technical Reports Server (NTRS)

Gehrz, Robert D.

1997-01-01

We have continued our program of infrared (IR) photometric, imaging, spectroscopic, and polarimetric temporal observations of comets to study the properties of comet dust and comet nuclei. During the first two years we digitized our IR data base on P/Halley and other recent comets to facilitate further analysis and comparison with other data bases, and found compelling evidence for the emission of a burst of small grains from P/Halley's nucleus at perihelion. We reported imaging and photometric observations of Comets Austin 1990 V and Swift-Tuttle 1992. The Swift-Tuttle 1992t observations included IR photometry, several 7-14 micron long-slit spectra of the coma and a time-sequence of more than 150 10 micron broadband images of the coma. An analysis of near-IR images of the inner coma of P/Halley obtained on three consecutive nights in 1986 March showed sunwardjets. We completed our analysis of IR imaging spectrosco-photometric data on comets. We also obtained observations of Comets Hyakutake 1996 B2 and Hale/Bopp 1995 01. We obtained infrared imaging, photometric, spectroscopic and polarimetric temporal observations of bright comets using a network of five telescopes, with emphasis on simultaneous observations of comets at many wavelengths with different instruments. Our program offers several unique advantages: 1) rapid observational response to new comets with dedicated infrared telescopes; 2) observations within a few degrees of the sun when comets are near perihelion and 3) access to advanced infrared array imagers and spectrometers. In particular, reduction, analysis, publication and archiving of our Jupiter/sl-9 and Comet Hyakutake infrared data received special emphasis. Instrumentation development included installation of the latest version of the innovative FORTH telescope control and a data acquisition system that enables us to control three telescopes remotely by telephone from anywhere in the world for comet observations in broad daylight. We have acquired more than 3000 256x256 images totaling nearly two gigabytes of data detailing the near-IR development of the impact sites of the S-L9 fragments on Jupiter. These data were obtained using the University of Rochester Imaging IR Camera at the cassegrain focus of the 92" at WIRO. The WIRO data set covers 8 days and is, to our knowledge, one of the most extensive observational records of the S-L/Jupiter encounter obtained by any ground-based telescope. This program benefitted from the compilation during these last few months of an upgrade to the data acquisition program at WIRO with support of this NASA contract.

Sodium hypochlorite-, chlorine dioxide- and peracetic acid-induced genotoxicity detected by the Comet assay and Saccharomyces cerevisiae D7 tests.

PubMed

Buschini, Annamaria; Carboni, Pamela; Furlini, Mariangela; Poli, Paola; Rossi, Carlo

2004-03-01

Mutagenicity of drinking water is due not only to industrial, agricultural and urban pollution but also to chlorine disinfection by-products. Furthermore, residual disinfection is used to provide a partial safeguard against low level contamination and bacterial re-growth within the distribution system. The aims of this study were to further evaluate the genotoxic potential of the world wide used disinfectants sodium hypochlorite and chlorine dioxide in human leukocytes by the Comet assay and in Saccharomyces cerevisiae strain D7 (mitotic gene conversion, point mutation and mitochondrial DNA mutability, with and without endogenous metabolic activation) and to compare their effects with those of peracetic acid, proposed as an alternative disinfectant. All three disinfectants are weakly genotoxic in human leukocytes (lowest effective dose 0.2 p.p.m. for chlorine dioxide, 0.5 p.p.m. for sodium hypochlorite and peracetic acid). The results in S.cerevisiae show a genotoxic response on the end-points considered with an effect only at doses higher (5- to 10-fold) than the concentration normally used for water disinfection; sodium hypochlorite and peracetic acid are able to induce genotoxic effects without endogenous metabolic activation (in stationary phase cells) whereas chlorine dioxide is effective in growing cells. The Comet assay was more sensitive than the yeast tests, with effective doses in the range normally used for water disinfection processes. The biological effectiveness of the three disinfectants on S.cerevisiae proved to be strictly dependent on cell-specific physiological/biochemical conditions. All the compounds appear to act on the DNA and peracetic acid shows effectiveness similar to sodium hypochlorite and chlorine dioxide.

Comet assay and micronucleus test in circulating erythrocytes of Cyprinus carpio specimens exposed in situ to lake waters treated with disinfectants for potabilization.

PubMed

Buschini, A; Martino, A; Gustavino, B; Monfrinotti, M; Poli, P; Rossi, C; Santoro, M; Dörr, A J M; Rizzoni, M

2004-02-14

The detection of a possible genotoxic effect of surface water treated with disinfectants for potabilization is the aim of the present work. The Comet assay and the micronucleus test were applied in circulating erythrocytes of Cyprinus carpio. Young specimens (20-30 g) were exposed in experimental basins, built within the potabilization plant of Castiglione del Lago (Perugia, Italy). In this plant the water of the Trasimeno Lake is treated and disinfected for potabilization before it is distributed to the people in the net of drinkable water. A continuous flow of water at a constant rate was supplied to basins; the water was continuously treated at a constant concentration with one of the three tested disinfectants (sodium hypochlorite, peracetic acid and chloride dioxide), one control basin being supplied with untreated water. Three sampling campaigns were performed: October 2000, February 2001 and June 2001. Repeated blood samplings through intracardiac punctures allowed to follow the same fish populations after different exposure times: before introduction of the disinfectant, and 10 or 20 days afterwards. An additional blood sampling was performed 3 h after addition of the disinfectant in other, simultaneously exposed, fish populations. Genotoxic damage was shown in fish exposed to water disinfected with sodium hypochlorite and chloride dioxide. The Comet assay showed an immediate response, i.e. DNA damage that was induced directly in circulating erythrocytes, whereas micronuclei reached their highest frequencies at later sampling times, when a genotoxic damage in stem cells of the cephalic kidney is expressed in circulating erythrocytes. The quality of the untreated surface water seems to be the most important parameter for the long-term DNA damage in circulating erythrocytes.

Biomonitoring of genotoxicity of industrial fertilizer pollutants in Aiolopus thalassinus (Orthoptera: Acrididae) using alkaline comet assay.

PubMed

Abdelfattah, Eman A; Augustyniak, Maria; Yousef, Hesham A

2017-09-01

Phosphate fertilizer industry is considered as one of the main sources of environmental pollutants. Besides solid waste products, e.g. phosphates, sulphates, and heavy metals, also atmospheric pollutants, such as hydrofluoric acid fumes (HF), sulphur dioxide (SO 2 ), nitrogen oxides (NO 2 ), and particulate matter with diameter up to 10 μm (PM 10 ) can be dangerous. Genotoxic effect of these pollutants was monitored by assessing the DNA damage using alkaline comet assay on cells from brain, thoracic muscles and gut of Aiolopus thalassinus collected at three sites (A-C) located at 1, 3, and 6 km away from Abu-Zaabal Company for Fertilizers and Chemical Industries. Control site was established 32 km from the source of pollution, at the Cairo University Campus. The level of the DNA damage was significantly higher in insects from polluted sites comparing to that from the control site. A strong negative correlation between percentage of cells with visible DNA damage (% of severed cells) and the distance of the sites from Abu-Zaabal Company was found. The best parameter for monitoring of fertilizer pollutants is % of severed cells. Possible impact of Abu-Zaabal Company (extremely high concentration of phosphates and sulphates in all the polluted sites) on DNA integrity in A. thalassinus tissues was discussed. The potential use of the comet assay as a biomonitoring method of the environmental pollution caused by fertilizer industry was proposed. Specific pollution resulting from the activity of the fertilizer industry can cause comparable adverse effects in the organisms inhabiting areas up to 6 km from the source of contamination. Copyright © 2017 Elsevier Ltd. All rights reserved.

Airborne observations of methane in Comet Kohoutek

NASA Technical Reports Server (NTRS)

Roche, A. E.; Wells, W. C.

1974-01-01

The experiment is described for airborne observations of Comet Kohoutek using an infrared tilting-filter photometer. Preliminary analysis of the data established an upper limit to the Comet's fluorescence radiation in methane lines at 3.3 microns.

COMET-AR User's Manual: COmputational MEchanics Testbed with Adaptive Refinement

NASA Technical Reports Server (NTRS)

Moas, E. (Editor)

1997-01-01

The COMET-AR User's Manual provides a reference manual for the Computational Structural Mechanics Testbed with Adaptive Refinement (COMET-AR), a software system developed jointly by Lockheed Palo Alto Research Laboratory and NASA Langley Research Center under contract NAS1-18444. The COMET-AR system is an extended version of an earlier finite element based structural analysis system called COMET, also developed by Lockheed and NASA. The primary extensions are the adaptive mesh refinement capabilities and a new "object-like" database interface that makes COMET-AR easier to extend further. This User's Manual provides a detailed description of the user interface to COMET-AR from the viewpoint of a structural analyst.

Exposure to non-ionizing electromagnetic fields emitted from mobile phones induced DNA damage in human ear canal hair follicle cells.

PubMed

Akdag, Mehmet; Dasdag, Suleyman; Canturk, Fazile; Akdag, Mehmet Zulkuf

2018-01-01

The aim of this study was to investigate effect of radiofrequency radiation (RFR) emitted from mobile phones on DNA damage in follicle cells of hair in the ear canal. The study was carried out on 56 men (age range: 30-60 years old)in four treatment groups with n = 14 in each group. The groups were defined as follows: people who did not use a mobile phone (Control), people use mobile phones for 0-30 min/day (second group), people use mobile phones for 30-60 min/day (third group) and people use mobile phones for more than 60 min/day (fourth group). Ear canal hair follicle cells taken from the subjects were analyzed by the Comet Assay to determine DNA damages. The Comet Assay parameters measured were head length, tail length, comet length, percentage of head DNA, tail DNA percentage, tail moment, and Olive tail moment. Results of the study showed that DNA damage indicators were higher in the RFR exposure groups than in the control subjects. In addition, DNA damage increased with the daily duration of exposure. In conclusion, RFR emitted from mobile phones has a potential to produce DNA damage in follicle cells of hair in the ear canal. Therefore, mobile phone users have to pay more attention when using wireless phones.

Pharmacological and Genotoxic Properties of Polyphenolic Extracts of Cedrela odorata L. and Juglans regia L. Barks in Rodents

PubMed Central

Almonte-Flores, Dulce Carolina; Paniagua-Castro, Norma; Escalona-Cardoso, Gerardo; Rosales-Castro, Martha

2015-01-01

Evaluation of the phenolic compounds and antioxidant activity of Cedrela odorata L. and Juglans regia L. bark extracts was performed in vitro. Juglans regia showed greater extract concentration and higher antioxidant activity. Hypoglycemic activity in rats was assessed by generating a glucose tolerance curve and determining the area under the curve (AUC). Diabetes was later induced by an injection with streptozotocin (65 mg/kg of b.w.) and confirmed after 24 hours. The extract was administered (200 mg/kg b.w.) over 10 days, and blood glucose was monitored and compared with a control group. The glucose AUC showed a hypoglycemic effect of J. regia and C. odorata in normal rats. Both extracts reduced hepatic lipid peroxidation in diabetic rats. Polyphenolic extracts reduced cholesterol levels in a hypercholesterolemic mouse model and decreased hepatic lipid peroxidation. Polyphenolic extract doses of 100 and 200 mg/kg b.w. were administered alone or with cyclophosphamide (CPA) 50 mg/kg ip, which was used as a positive control. Analyses were performed using leukocytes in a comet assay after 4 and 24 h of treatment. Genotoxic effects were evaluated by the comet assay, which showed that while J. regia extract had no effect, C. odorata extract induced slight damage at 200 mg/kg, with the formation of type 0 and 1 comets. PMID:25945104

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

PubMed

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

2016-10-01

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

A study of ion composition and dynamics at Comet Halley

NASA Technical Reports Server (NTRS)

Shelley, E. G.; Fuselier, S. A.

1991-01-01

This report details the participation by Lockheed co-investigators in the reduction, analysis, and interpretation of data obtained by the Ion Mass Spectrometer onboard the Giotto mission to Comet Halley. The data analysis activities and much of the scientific collaboration was shared by this team. One objective of the effort under this contract was to use data obtained by the Giotto Ion Mass Spectrometer (IMS) during the encounter with comet Halley for the purpose of advancing our understanding of the chemistry and physics of the interaction of the solar wind with comets and obtaining new information on the comet's composition. An additional objective was to make this unique data set available in a format which can be easily used by the reset of the cometary science community for other analysis in the future. The IMS has two sensors: the High Intensity Spectrometer (HIS) and the High Energy Range Spectrometer (HERS).

DNA DAMAGE AND EXTERNAL LESIONS IN BROWN BULLHEADS FROM CONTAMINATED HABITATS

EPA Science Inventory

The Comet assay was used to compare levels of DNA damage in brown bullheads (Ameiurus nebulosus) collected from three known contaminated locations, the Cuyahoga River, Ashtabula River, and Ashumet Pond (Cape Cod), with brown bullheads collected from three paired reference sites, ...

The antileishmanial drug miltefosine (Impavido(®)) causes oxidation of DNA bases, apoptosis, and necrosis in mammalian cells.

PubMed

Castelo Branco, Patrícia Valéria; Soares, Rossy-Eric Pereira; de Jesus, Luís Cláudio Lima; Moreira, Vanessa Ribeiro; Alves, Hugo José; de Castro Belfort, Marta Regina; Silva, Vera Lucia Maciel; Ferreira Pereira, Silma Regina

2016-08-01

Miltefosine was developed to treat skin cancer; further studies showed that the drug also has activity against Leishmania. Miltefosine is the first oral agent for treating leishmaniasis. However, its mechanism of action is not completely understood. We have evaluated the induction of DNA damage by miltefosine. Cytotoxicity and genotoxicity (comet assay) tests were performed on human leukocytes exposed to the drug in vitro. Apoptosis and necrosis were also evaluated. In vivo tests were conducted in Swiss male mice (Mus musculus) treated orally with miltefosine. Oxidation of DNA bases in peripheral blood cells was measured using the comet assay followed by digestion with formamidopyrimidine glycosylase (FPG), which removes oxidized guanine bases. The micronucleus test was performed on bone marrow erythrocytes. Miltefosine caused DNA damage, apoptosis, and necrosis in vitro. Mice treated with miltefosine showed an increase in the DNA damage score, which was further increased following FPG digestion. The micronucleus test was also positive. Copyright © 2016 Elsevier B.V. All rights reserved.

Absence of mutagenicity effects of Psidium cattleyanum Sabine (Myrtaceae) extract on peripheral blood and bone marrow cells of mice.

PubMed

Costa, T D A; Vieira, S; Andrade, S F; Maistro, E L

2008-07-29

Cattley guava (Psidium cattleyanum Sabine) is a native fruit of Brazil that is popular both as a sweet food and for its reputed therapeutic properties. We examined whether it could damage DNA using the alkaline single-cell gel electrophoresis (comet assay) and the micronucleus test in leukocytes and in bone marrow cells of mice. P. cattleyanum leaf extract was tested at concentrations of 1000, 1500 and 2000 mg/kg. N-nitroso-N-ethylurea was used as a positive control. Peripheral blood leukocytes were collected 4 and 24 h after the treatments for the comet assay, and bone marrow cells were collected after 24 and 48 h for the micronucleus test. Unlike N-nitroso-N-ethylurea, P. cattleyanum extract failed to induce a significant increase in cell DNA damage, in micronucleated cell frequency, and in bone marrow toxicity. The lack of mutagenicity and cytotoxicity with high doses of this plant extract means that it can be safely used in traditional medicine.

Rapid detection of irradiated frozen hamburgers

NASA Astrophysics Data System (ADS)

Delincée, Henry

2002-03-01

DNA comet assay can be employed as a rapid and inexpensive screening test to check whether frozen ground beef patties (hamburgers) have been irradiated as a means to increase their safety by eliminating pathogenic bacteria, e.g. E. coli O157:H7. Such a detection procedure will provide an additional check on compliance with existing regulations, e.g. enforcement of labelling and rules in international trade. Frozen ready prepared hamburgers from the market place were `electron irradiated' with doses of 0, 1.3, 2.7, 4.5 and 7.2kGy covering the range of potential commercial irradiation. DNA fragmentation in the hamburgers was made visible within a few hours using the comet assay, and non-irradiated hamburgers could be easily discerned from the irradiated ones. Even after 9 months of frozen storage, irradiated hamburgers could be identified. Since DNA fragmentation may also occur with other food processes (e.g. temperature abuse), positive screening tests shall be confirmed using a validated method to specifically prove an irradiation treatment, e.g. EN 1784 or EN 1785.

Nandrolone decanoate induces genetic damage in multiple organs of rats.

PubMed

Pozzi, Renan; Fernandes, Kelly Rosseti; de Moura, Carolina Foot Gomes; Ferrari, Raquel Agnelli Mesquita; Fernandes, Kristianne Porta Santos; Renno, Ana Claudia Muniz; Ribeiro, Daniel Araki

2013-04-01

To evaluate the impact potential of nandrolone decanoate on DNA damage in multiple organs of Wistar rats by means of single-cell gel (comet) assay and micronucleus test. A total of 15 animals were distributed into three groups of five animals each as follows: control group = animal not exposed to nandrolone decanoate; experimental group = animals exposed to nandrolone decanoate for 24 h at 5 mg/kg subcutaneously; and experimental group = animals exposed to nandrolone decanoate for 24 h at 15 mg/kg subcutaneously. Significant statistical differences (p < 0.05) were noted in peripheral blood, liver, and heart cells exposed to nandrolone decanoate at the two doses evaluated. A clear dose-response relationship was observed between groups. Kidney cells showed genetic damage at only the highest dose (15 mg/kg) used. However, micronucleus data did not show remarkable differences among groups. In conclusion, the present study indicates that nandrolone decanoate induces genetic damage in rat blood, liver, heart, and kidney cells as shown by single-cell gel (comet) assay results.

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.

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.

Evaluation of Genetic Variations in Maize Seedlings Exposed to Electric Field Based on Protein and DNA Markers

PubMed Central

AL-Huqail, Asma A.; Abdelhaliem, Ekram

2015-01-01

The current study analyzed proteins and nuclear DNA of electric fields (ELF) exposed and nonexposed maize seedlings for different exposure periods using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), isozymes, random amplified polymorphic DNA (RAPD), and comet assay, respectively. SDS-PAGE analysis revealed total of 46 polypeptides bands with different molecular weights ranging from 186.20 to 36.00 KDa. It generated distinctive polymorphism value of 84.62%. Leucine-aminopeptidase, peroxidase, and catalase isozymes showed the highest values of polymorphism (100%) based on zymograms number, relative front (R f), and optical intensity while esterase isozyme generated polymorphism value of 83.33%. Amino acids were analyzed using high-performance liquid chromatography, which revealed the presence of 17 amino acids of variable contents ranging from 22.65% to 28.09%. RAPD revealed that 78 amplified DNA products had highly polymorphism value (95.08%) based on band numbers, with variable sizes ranging from 120 to 992 base pairs and band intensity. Comet assay recorded the highest extent of nuclear DNA damage as percentage of tailed DNA (2.38%) and tail moment unit (5.36) at ELF exposure of maize nuclei for 5 days. The current study concluded that the longer ELF exposing periods had genotoxic stress on macromolecules of maize cells and biomarkers used should be augmented for reliable estimates of genotoxicity after exposure of economic plants to ELF stressors. PMID:26180815

Genotoxic and biochemical changes in Baccharis trimera induced by coal contamination.

PubMed

Menezes, A P S; Da Silva, J; Rossato, R R; Santos, M S; Decker, N; Da Silva, F R; Cruz, C; Dihl, R R; Lehmann, M; Ferraz, A B F

2015-04-01

The processing and combustion of coal in thermal power plants release anthropogenic chemicals into the environment. Baccharis trimera is a common plant used in folk medicine that grows readily in soils degraded by coal mining activities. This shrub bioaccumulates metals released into the environment, and thus its consumption may be harmful to health. The purpose of this study was to investigate the phytochemical profile, antioxidant capacity (DPPH), genotoxic (comet assay) and mutagenic potential (CBMN-cyt) in V79 cells of B. trimera aqueous extracts in the coal-mining region of Candiota (Bt-AEC), and in Bagé, a city that does not experience the effects of exposure to coal (Bt-AEB, a reference site). In the comet assay, only Bt-AEC was genotoxic at the highest doses (0.8mg/mL and 1.6mg/mL), compared to the control. For extracts from both areas, mutagenic effects were observed at higher concentrations compared to the control. The cell damage parameters were significantly high in both extracts; however, more striking values were observed for Bt-AEC, up to the dose of 0.8mg/mL. In chemical analysis, no variation was observed in the contents of flavonoids and phenolic compounds, neither the antioxidant activity, which may suggest that DNA damage observed in V79 cells was induced by the presence of coal contaminants absorbed by the plant. Copyright © 2015 Elsevier Inc. All rights reserved.

Factors influencing heterogeneity of radiation-induced DNA-damage measured by the alkaline comet assay.

PubMed

Seidel, Clemens; Lautenschläger, Christine; Dunst, Jürgen; Müller, Arndt-Christian

2012-04-20

To investigate whether different conditions of DNA structure and radiation treatment could modify heterogeneity of response. Additionally to study variance as a potential parameter of heterogeneity for radiosensitivity testing. Two-hundred leukocytes per sample of healthy donors were split into four groups. I: Intact chromatin structure; II: Nucleoids of histone-depleted DNA; III: Nucleoids of histone-depleted DNA with 90 mM DMSO as antioxidant. Response to single (I-III) and twice (IV) irradiation with 4 Gy and repair kinetics were evaluated using %Tail-DNA. Heterogeneity of DNA damage was determined by calculation of variance of DNA-damage (V) and mean variance (Mvar), mutual comparisons were done by one-way analysis of variance (ANOVA). Heterogeneity of initial DNA-damage (I, 0 min repair) increased without histones (II). Absence of histones was balanced by addition of antioxidants (III). Repair reduced heterogeneity of all samples (with and without irradiation). However double irradiation plus repair led to a higher level of heterogeneity distinguishable from single irradiation and repair in intact cells. Increase of mean DNA damage was associated with a similarly elevated variance of DNA damage (r = +0.88). Heterogeneity of DNA-damage can be modified by histone level, antioxidant concentration, repair and radiation dose and was positively correlated with DNA damage. Experimental conditions might be optimized by reducing scatter of comet assay data by repair and antioxidants, potentially allowing better discrimination of small differences. Amount of heterogeneity measured by variance might be an additional useful parameter to characterize radiosensitivity.

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. Copyright © 2014 Elsevier Ltd. All rights reserved.

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.

Study of mass flow distribution and chemical composition of comets from solar induced X-ray fluorescence

NASA Technical Reports Server (NTRS)

Gorenstein, P.

1979-01-01

The expected performance of an X-ray detector as an instrument aboard a mission to a comet was evaluated. The functions of the detector are both nondispersive analysis of chemical composition and measurement of mass flow from the comet nucleus. Measurements are to be carried out at a distance from the comet. The approach distances considered are of the order of 1000 km and 100 km. A new type of X-ray detector, a proportional scintillation detector, is considered as an X-ray counter for nondispersive elemental analysis.

Extended atmospheres of outer planet satellites and comets

NASA Technical Reports Server (NTRS)

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

1985-01-01

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

On the problem of origin of periodic comets.

NASA Astrophysics Data System (ADS)

Guliev, A. S.

The problem of origin of periodic comets is viewed under various aspects. A steady growth of the fraction of these comets in the overall population of comets is emphasized. The number of discovered periodic comets with small eccentricities and with the Jacobi constant close to 3 is also growing eventually. Comparison of maximum magnitudes of the same comets in different apparitions at the same elongations as well as the analysis of exhausted comets indicate that the age of these objects does not exceed 1000 years. Capture is considered as an efficient mechanism for preserving equilibrium over reasonable time intervals. The analysis of the data given by Everhart and the calculations of the evolution of cometary orbits reveal small efficiency of capture. Comparison of the number of well established capture cases with the corresponding time interval shows that the age of the system of periodic comets must be 17000 years within the framework of this mechanism. This is most unlikely. Secular variations in the distributions of semimajor axes, inclinations, longitudes of perihelia, eccentricities of orbits of periodic comets are analysed. On the average, the eccentricities tend to increase, but this conflicts with the capture mechanism. A conclusion is made that the concept of capture in its classical and modern versions is unable to solve the problem of the origin of periodic comets on the whole. Other, more effective sources and mechanisms seem to be also in operation in enlarging the cometary system.

Analytical study of comet nucleus samples

NASA Technical Reports Server (NTRS)

Albee, A. L.

1989-01-01

Analytical procedures for studying and handling frozen (130 K) core samples of comet nuclei are discussed. These methods include neutron activation analysis, x ray fluorescent analysis and high resolution mass spectroscopy.

Sperm DNA damage has a negative association with live-birth rates after IVF.

PubMed

Simon, L; Proutski, I; Stevenson, M; Jennings, D; McManus, J; Lutton, D; Lewis, S E M

2013-01-01

Sperm DNA damage has a negative impact on pregnancy rates following assisted reproduction treatment (ART). The aim of the present study was to examine the relationship between sperm DNA fragmentation and live-birth rates after IVF and intracytoplasmic sperm injection (ICSI). The alkaline Comet assay was employed to measure sperm DNA fragmentation in native semen and in spermatozoa following density-gradient centrifugation in semen samples from 203 couples undergoing IVF and 136 couples undergoing ICSI. Men were divided into groups according to sperm DNA damage. Following IVF, couples with <25% sperm DNA fragmentation had a live-birth rate of 33%; in contrast, couples with >50% sperm DNA fragmentation had a much lower live-birth rate of 13%. Following ICSI, no significant differences in sperm DNA damage were found between any groups of patients. Sperm DNA damage was also associated with low live-birth rates following IVF in both men and couples with idiopathic infertility: 39% of couples and 41% of men with idiopathic infertility have high sperm DNA damage. Sperm DNA damage assessed by the Comet assay has a close inverse relationship with live-birth rates after IVF. Sperm DNA damage has a negative impact on assisted reproduction treatment outcome, in particular, on pregnancy rates. The aim of the present study was to examine the relationship between sperm DNA fragmentation and live-birth rates after IVF and intracytoplasmic sperm injection (ICSI). The alkaline Comet assay was employed to measure sperm DNA fragmentation in native semen and in spermatozoa following density-gradient centrifugation in semen samples from 203 couples undergoing IVF and 136 couples undergoing ICSI. Men were divided into groups according to sperm DNA damage and treatment outcome. Following IVF, couples with <25% sperm DNA fragmentation had a live birth rate of 33%. In contrast, couples with >50% sperm DNA fragmentation had a much lower live-birth rate of 13% following IVF. Following ICSI, there were no significant differences in levels of sperm DNA damage between any groups of patients. Sperm DNA damage was also associated with the very low live-birth rates following IVF in both men and couples with idiopathic infertility: 39% of couples and 41% of men have high level of sperm DNA damage. Sperm DNA damage assessed by the Comet assay has a close inverse relationship with live-birth rates after IVF. Copyright © 2012 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Histological changes in lung tissues related with sub-chronic exposure to ambient urban levels of PM2.5 in Córdoba, Argentina

NASA Astrophysics Data System (ADS)

Tavera Busso, Iván; Vera, Anahí; Mateos, Ana Carolina; Amarillo, Ana Carolina; Carreras, Hebe

2017-10-01

Concentration of fine particulate matter (PM2.5) is one of the most important environmental parameters to estimate health impacts attributable to air pollution. Despite the fact there are many studies regarding PM2.5 effects on human health, most of them were performed under conditions that do not simulate the natural particles interaction with the organism. In the present paper, we studied the effects of mammals' sub-chronic exposure to PM2.5 on the lower respiratory tract, addressing realistic exposure conditions to normal urban air. Thus, we exposed Wistar rats under controlled settings to the same normal urban air, with and without particles. Next, we analyzed chemical composition of PM2.5 and lungs samples, performed a histologic examination and run the comet assay to assess genotoxic effects. We found a strong agreement between lung tissues and PM2.5 elemental composition suggesting that metals found in lungs came from the particles inhaled. Histological analysis showed a mild to moderate infiltration, with a reduction of alveoli lumen and increment of alveolar macrophages and periodic acid-Schiff (PAS) (+) cells in treated animals. We also observed an increase in the number of nuclei with comets, mostly comets type 3, with a high DNA fragmentation as well. These results provide strong evidence that sub-chronic exposure to low particle levels, even below the 24 h WHO standard, can cause injuries in lungs tissues and DNA damage, as well.

Disappearance of 19P/Borrelly's Silicate Feature in 2001 Apparition Is Attributed to Increase in Grain Size

NASA Astrophysics Data System (ADS)

Wooden, D. H.; Woodward, C. E.; Harker, D. E.

2003-05-01

We report on observations and analysis of HIFOGS 10 \\micron \\ spectrophotometry of short period comet 19P/Borrelly on 2003 October 13, 15 UT at the NASA IRTF. 19P/Borrelly is one of two short period comets, comet 4P/Faye being the other, to have a silicate feature detected (Hanner et al. 1996, Icarus, 124, 344). During Borrelly.s perihelion passage in 1994 December, a silicate feature was present with a flux-to-continuum ratio of 0.25. Two apparitions later in 2003 October, the silicate feature is absent. Thermal emission modeling (cf. Harker et al. 2002, ApJ, 580, 579) using amorphous olivine and amorphous carbon shows that a slight increase in grain size accounts for the disappearance of the silicate feature. Analysis of 19P/Borrelly suggests grain size, and not the absence of olivine minerals, may be responsible for the absence of silicate features in most short period comets. 19P/Borrelly is one of the more active short period comets. However, short period comets as a family are less active than long period comets. Short period comets probably originated in the Kuiper Belt and suffered collisions while in residence in the outer solar system. Upon evolution into orbits that take them through the inner solar system, the surfaces of short period comets are exposed to sunlight through their many perihelion passages. This is in contrast to long period comets which probably originated near Jupiter and were expelled to the Oort cloud where they have existed and been exposed to cosmic ray processing. By studying the grain properties in short period comets and comparing to long period comets, we compare the effects on the grain populations of different parent body evolution histories. Upcoming opportunities to study short and long period comets will be advertised. This research is supported in part by an NSF Grant to the University of Minnesota.

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.

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

PubMed

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

2017-04-01

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

Dust environment and dynamical history of a sample of short-period comets . II. 81P/Wild 2 and 103P/Hartley 2

NASA Astrophysics Data System (ADS)

Pozuelos, F. J.; Moreno, F.; Aceituno, F.; Casanova, V.; Sota, A.; López-Moreno, J. J.; Castellano, J.; Reina, E.; Climent, A.; Fernández, A.; San Segundo, A.; Häusler, B.; González, C.; Rodriguez, D.; Bryssinck, E.; Cortés, E.; Rodriguez, F. A.; Baldris, F.; García, F.; Gómez, F.; Limón, F.; Tifner, F.; Muler, G.; Almendros, I.; de los Reyes, J. A.; Henríquez, J. A.; Moreno, J. A.; Báez, J.; Bel, J.; Camarasa, J.; Curto, J.; Hernández, J. F.; González, J. J.; Martín, J. J.; Salto, J. L.; Lopesino, J.; Bosch, J. M.; Ruiz, J. M.; Vidal, J. R.; Ruiz, J.; Sánchez, J.; Temprano, J.; Aymamí, J. M.; Lahuerta, L.; Montoro, L.; Campas, M.; García, M. A.; Canales, O.; Benavides, R.; Dymock, R.; García, R.; Ligustri, R.; Naves, R.; Lahuerta, S.; Pastor, S.

2014-11-01

Aims: This paper is a continuation of the first paper in this series, where we presented an extended study of the dust environment of a sample of short-period comets and their dynamical history. On this occasion, we focus on comets 81P/Wild 2 and 103P/Hartley 2, which are of special interest as targets of the spacecraft missions Stardust and EPOXI. Methods: As in the previous study, we used two sets of observational data: a set of images, acquired at Sierra Nevada and Lulin observatories, and the Afρ data as a function of the heliocentric distance provided by the amateur astronomical association Cometas-Obs. The dust environment of comets (dust loss rate, ejection velocities, and size distribution of the particles) was derived from our Monte Carlo dust tail code. To determine their dynamical history we used the numerical integrator Mercury 6.2 to ascertain the time spent by these objects in the Jupiter family Comet region. Results: From the dust analysis, we conclude that both 81P/Wild 2 and 103P/Hartley 2 are dusty comets, with an annual dust production rate of 2.8 × 109 kg yr-1 and (0.4-1.5) × 109 kg yr-1, respectively. From the dynamical analysis, we determined their time spent in the Jupiter family Comet region as ~40 yr in the case of 81P/Wild 2 and ~1000 yr for comet 103P/Hartley 2. These results imply that 81P/Wild 2 is the youngest and the most active comet of the eleven short-period comets studied so far, which tends to favor the correlation between the time spent in JFCs region and the comet activity previously discussed.

Investigation on the toxic potential of Tribulus terrestris in vitro.

PubMed

Abudayyak, M; Jannuzzi, A T; Özhan, G; Alpertunga, B

2015-04-01

Tribulus terrestris L. (Zygophyllaceae) has been commonly used to energize, vitalize, and improve sexual function and physical performance in men. This study investigates the potential cytotoxic and genotoxic, and endocrine disrupting activities of T. terrestris in vitro. The whole T. terrestris plant was extracted with water, methanol, and chloroform. The genotoxic potential of T. terrestris extracts at 3-2400 µg/mL was assessed by Comet assay in a rat kidney cell line (NRK-52E) and by Ames assay in Salmonella typhimurium TA98 and TA100 strains. Endocrine disrupting effects of the extracts at concentrations of 0.22-25 000 µg/mL were assessed by YES/YAS assay in Saccharomyces cerevisiae. Cytotoxic activity of the extracts was determined by the MTT test in NRK-52E cells. The different exposure times were used for four tests (3-48 h). The methanol extract of T. terrestris IC50 value was 160 µg/mL. The other extracts did not show cytotoxic effects. In the Comet and Ames genotoxicity assays, none of the extracts possessed genotoxic activities at concentrations of 0-2400 µg/mL. Only the water extract of T. terrestris induced frame shift mutations after metabolic activation. The water extract also showed estrogenic activity by YES/YAS assay in S. cerevisiae at concentrations ≥27 µg/mL (≥2.6-fold), while the other T. terrestris extracts had anti-estrogenic properties. Tribulus terrestris had estrogenic and genotoxic activities. The study was useful in determining its toxicological effects and the precautions regarding consumption.

Sulforaphane suppresses the growth of glioblastoma cells, glioblastoma stem cell-like spheroids, and tumor xenografts through multiple cell signaling pathways.

PubMed

Bijangi-Vishehsaraei, Khadijeh; Reza Saadatzadeh, M; Wang, Haiyan; Nguyen, Angie; Kamocka, Malgorzata M; Cai, Wenjing; Cohen-Gadol, Aaron A; Halum, Stacey L; Sarkaria, Jann N; Pollok, Karen E; Safa, Ahmad R

2017-12-01

OBJECTIVE Defects in the apoptotic machinery and augmented survival signals contribute to drug resistance in glioblastoma (GBM). Moreover, another complexity related to GBM treatment is the concept that GBM development and recurrence may arise from the expression of GBM stem cells (GSCs). Therefore, the use of a multifaceted approach or multitargeted agents that affect specific tumor cell characteristics will likely be necessary to successfully eradicate GBM. The objective of this study was to investigate the usefulness of sulforaphane (SFN)-a constituent of cruciferous vegetables with a multitargeted effect-as a therapeutic agent for GBM. METHODS The inhibitory effects of SFN on established cell lines, early primary cultures, CD133-positive GSCs, GSC-derived spheroids, and GBM xenografts were evaluated using various methods, including GSC isolation and the sphere-forming assay, analysis of reactive oxygen species (ROS) and apoptosis, cell growth inhibition assay, comet assays for assessing SFN-triggered DNA damage, confocal microscopy, Western blot analysis, and the determination of in vivo efficacy as assessed in human GBM xenograft models. RESULTS SFN triggered the significant inhibition of cell survival and induced apoptotic cell death, which was associated with caspase 3 and caspase 7 activation. Moreover, SFN triggered the formation of mitochondrial ROS, and SFN-triggered cell death was ROS dependent. Comet assays revealed that SFN increased single- and double-strand DNA breaks in GBM. Compared with the vehicle control cells, a significantly higher amount of γ-H2AX foci correlated with an increase in DNA double-strand breaks in the SFN-treated samples. Furthermore, SFN robustly inhibited the growth of GBM cell-induced cell death in established cell cultures and early-passage primary cultures and, most importantly, was effective in eliminating GSCs, which play a major role in drug resistance and disease recurrence. In vivo studies revealed that SFN administration at 100 mg/kg for 5-day cycles repeated for 3 weeks significantly decreased the growth of ectopic xenografts that were established from the early passage of primary cultures of GBM10. CONCLUSIONS These results suggest that SFN is a potent anti-GBM agent that targets several apoptosis and cell survival pathways and further preclinical and clinical studies may prove that SFN alone or in combination with other therapies may be potentially useful for GBM therapy.

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

The Water Production Rate of Recent Comets (2013-2014) by SOHO/SWAN: 2P/Encke (2013), C/2013 R1 (Lovejoy), and C/2013 A1 (Siding Spring)

NASA Astrophysics Data System (ADS)

Combi, Michael R.; Mäkinen, J. T.; Bertaux, J. L.; Quémerais, Eric; Ferron, Stéphane

2014-11-01

The all-sky hydrogen Lyman-alpha camera, SWAN (Solar Wind Anisotropies), on the SOlar and Heliospheric Observatory (SOHO) satellite makes observations of the hydrogen comae of comets. Most water vapor produced by the comet is ultimately photodissociated into two H atoms and one O atom producing a huge atomic hydrogen coma that is routinely observed in the daily full-sky SWAN images in comets of sufficient brightness. Water production rates are calculated using our time-resolved model (Mäkinen & Combi, 2005, Icarus 177, 217), typically yielding about 1 observation every 2 days on the average. Here we describe the progress in analysis of observations of comets observed during 2013-2014 and those selected from the archive for analysis. These include comets 2P/Encke (2013), 45P/Honda Mrkos-Pajdusakova (2011), C/2013 R1 (Lovejoy), as well as C/2013 A1 (Siding Spring), for which results are expected. A status report on the entire SOHO/SWAN archive of water production rates in comets will be given. SOHO is an international cooperative mission between ESA and NASA. Support from grants NNX11AH50G from the NASA Planetary Astronomy Program and NNX13AQ66G from the NASA Planetary Mission Data Analysis Program are gratefully acknowledged.

Collisions with meteoroid streams as one possible mechanism for the formation of hyperbolic cometary orbits

NASA Astrophysics Data System (ADS)

Guliyev, Ayyub; Nabiyev, Shaig

2017-07-01

This paper presents the results of a statistical analysis of the dynamic parameters of 300 comets that have osculating hyperbolic orbits. It is shown that such comets differ from other comets by their large perihelion distances and by a predominance of retrograde motion. It is shown that the values of i, the inclination of the hyperbolic comets, are in comparative excess over the interval 90-120°. The dominance by q, the perihelion distance, renders it difficult to suggest that the excess hyperbolic velocity of these comets can be the result of physical processes that take place in their nuclei. Aspects of the following working hypothesis, that the hyperbolic excess of parameter e might be formed after comets pass through meteoroid streams, are also studied. To evaluate this hypothesis, the distribution of the orbits of hyperbolic comets relative to the plane of motion of 112 established meteoroid streams are analyzed. The number (N) of orbit nodes for hyperbolic comets with respect to the plane of each stream at various distances is calculated. To determine the degree of redundancy of N, a special computing algorithm was applied that provided the expected value nav as well as the standard deviation σ for the number of cometary nodes at the plane of each stream. A comparative analysis of the N and nav values that take σ into account suggests an excess in 40 stream cases. This implies that the passage of comets through meteoroid streams can lead to an acceleration of the comets' heliocentric velocity.

Chlorella vulgaris Induces Apoptosis of Human Non-Small Cell Lung Carcinoma (NSCLC) Cells.

PubMed

Zhang, Zhi-Dong; Liang, Kai; Li, Kun; Wang, Guo-Quan; Zhang, Ke-Wei; Cai, Lei; Zhai, Shui-Ting; Chou, Kuo-Chen

2017-01-01

Chlorella vulgaris (C. vulgaris), a unicellular green microalga, has been widely used as a food supplement and reported to have antioxidant and anticancer properties. The current study was designed to assess the cytotoxic, apoptotic, and DNA-damaging effects of C. vulgaris growth factor (CGF), hot water C. vulgaris extracts, inlung tumor A549 and NCI-H460 cell lines. A549 cells, NCI-H460 cells, and normal human fibroblasts were treated with CGF at various concentrations (0-300 μg/ml) for 24 hr. The comet assay and γH2AX assay showed DNA damage in A549 and NCI-H460 cells upon CGF exposure. Evaluation of apoptosis by the TUNEL assay and DNA fragmentation analysis by agarose gel electrophoresis showed that CGF induced apoptosis in A549 and NCI-H460 cells. Chlorella vulgaris hot water extract induced apoptosis and DNA damage in human lung carcinoma cells. CGF can thus be considered a potential cytotoxic or genotoxic drug for treatment of lung carcinoma. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Discovery of water ice nearly everywhere in the solar system

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

Zuppero, A.

1995-10-01

During the last decade we have discovered sources of accessible water in some form nearly everywhere in the solar system. Water ice has been found on the planet Mercury; probably on the Earth`s Moon; on Mars; on near Earth objects; on comets whose orbits frequently come close to that of Earth`s orbit; probably on Ceres, the largest inner asteroid; and on comets previously and incorrectly considered to be out of practical reach. The comets also provide massive quantities of hydrocarbons, similar to oil shale. The masses of either water or hydrocarbons are measured in units of cubic kilometers. The watermore » is key to space transportation because it can be used as a rocket propellant directly, and because thermal process alone can be used to convert it and hydrocarbons into hydrogen, the highest performing rocket propellant. This presentation outlines what is currently known about the locations of the water ice, and sketches the requirements and environments of missions to prospect for and assay the water sources.« less

Application of micronucleus test and comet assay to evaluate BTEX biodegradation.

PubMed

Mazzeo, Dânia Elisa Christofoletti; Matsumoto, Silvia Tamie; Levy, Carlos Emílio; de Angelis, Dejanira de Franceschi; Marin-Morales, Maria Aparecida

2013-01-01

The BTEX (benzene, toluene, ethylbenzene and xylene) mixture is an environmental pollutant that has a high potential to contaminate water resources, especially groundwater. The bioremediation process by microorganisms has often been used as a tool for removing BTEX from contaminated sites. The application of biological assays is useful in evaluating the efficiency of bioremediation processes, besides identifying the toxicity of the original contaminants. It also allows identifying the effects of possible metabolites formed during the biodegradation process on test organisms. In this study, we evaluated the genotoxic and mutagenic potential of five different BTEX concentrations in rat hepatoma tissue culture (HTC) cells, using comet and micronucleus assays, before and after biodegradation. A mutagenic effect was observed for the highest concentration tested and for its respective non-biodegraded concentration. Genotoxicity was significant for all non-biodegraded concentrations and not significant for the biodegraded ones. According to our results, we can state that BTEX is mutagenic at concentrations close to its water solubility, and genotoxic even at lower concentrations, differing from some described results reported for the mixture components, when tested individually. Our results suggest a synergistic effect for the mixture and that the biodegradation process is a safe and efficient methodology to be applied at BTEX-contaminated sites. Copyright © 2012 Elsevier Ltd. All rights reserved.

Formation and removal of genotoxic activity during UV/H(2)O(2)-GAC treatment of drinking water.

PubMed

Heringa, M B; Harmsen, D J H; Beerendonk, E F; Reus, A A; Krul, C A M; Metz, D H; Ijpelaar, G F

2011-01-01

The objective of this study was to determine the genotoxic activity of water after UV/H(2)O(2) oxidation and GAC filtration. Pre-treated surface water from three locations was treated with UV/H(2)O(2) with medium pressure (MP) lamps and passed through granulated activated carbon (GAC). Samples taken before and after each treatment step were extracted and concentrated by solid phase extraction (SPE) and analyzed for genotoxicity using the Comet assay with HepG2 cells and the Ames II assay. The Comet assay showed no genotoxic response in any of the samples. In the Ames II, no genotoxic response was obtained with the TAMix (a mix of six strains), but the TA98 strain showed an increase in genotoxic activity after MP-UV/H(2)O(2) for all three locations. GAC post treatment effectively reduced the activities to control levels at two of the three locations and to below the level of the pre-treated water at one site. The results indicate that UV/H(2)O(2) treatment may lead to the formation of genotoxic by-products, which can be removed by subsequent GAC filtration. Copyright © 2010 Elsevier Ltd. All rights reserved.

CoMET: Cost and Mass Evaluation Tool for Spacecraft and Mission Design

NASA Technical Reports Server (NTRS)

Bieber, Ben S.

2005-01-01

New technology in space exploration is often developed without a complete knowledge of its impact. While the immediate benefits of a new technology are obvious, it is harder to understand its indirect consequences, which ripple through the entire system. COMET is a technology evaluation tool designed to illuminate how specific technology choices affect a mission at each system level. COMET uses simplified models for mass, power, and cost to analyze performance parameters of technologies of interest. The sensitivity analysis that CoMET provides shows whether developing a certain technology will greatly benefit the project or not. CoMET is an ongoing project approaching a web-based implementation phase. This year, development focused on the models for planetary daughter craft, such as atmospheric probes, blimps and balloons, and landers. These models are developed through research into historical data, well established rules of thumb, and engineering judgment of experts at JPL. The model is validated by corroboration with JpL advanced mission studies. Other enhancements to COMET include adding launch vehicle analysis and integrating an updated cost model. When completed, COMET will allow technological development to be focused on areas that will most drastically improve spacecraft performance.

An alkaline comet assay study on the antimalarial drug atovaquone in human peripheral blood lymphocytes: a study based on clinically relevant concentrations.

PubMed

Dinter, Domagoj; Gajski, Goran; Garaj-Vrhovac, Vera

2013-01-01

Atovaquone, a hydroxynaphthoquinone, is an anti-parasite drug, selectively targeting the mitochondrial respiratory chain of malaria parasite. It is used for both the treatment and prevention of malaria, usually in a fixed combination with proguanil. Although atovaquone has not often been associated with severe adverse reactions in the recommended dosages and has a relatively favorable side effect profile, the present study was undertaken to evaluate its cytogenotoxic potential towards human peripheral blood lymphocytes. Two different concentrations of atovaquone found in plasma when used in fixed-dose combination with proguanile hydrochloride were used with and without S9 metabolic activation: 2950 ng ml(-1) used for prophylactic treatment and 11 800 ng ml(-1) used in treatment of malaria. The results showed that lymphocyte viability was not affected after the treatment, suggesting that atovaquone was not cytotoxic in the given concentrations. With the alkaline comet assay we demonstrated that in human peripheral blood lymphocytes no significant changes in comet parameters occurred after the treatment. There were no differences in tested parameters with the addition of S9 metabolic activation, indicating that atovaquone either has no metabolite or it is not toxic in the given concentrations. Since no effects were observed after the treatment, it is to be concluded that atovaquone is safe from the aspect of genototoxicity in the recommended dosages. Copyright © 2011 John Wiley & Sons, Ltd.

Antioxidant Activity of Orange Flesh and Peel Extracted with Various Solvents

PubMed Central

Park, Jae-Hee; Lee, Minhee; Park, Eunju

2014-01-01

The aim of this study was to investigate the antioxidant activity of orange (Citrus auranthium) flesh (OF) and peel (OP) extracted with acetone, ethanol, and methanol. Antioxidant potential was examined by measuring total phenolic content (TPC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity (RSA), total radical-trapping anti-oxidant potential (TRAP), oxygen radical absorbance capacity (ORAC), and cellular antioxidant activity (CAA). The comet assay was used to determine the protective effects of OF and OP against H2O2-induced DNA damage. TPC was highest in the acetone extracts of OF and OP. DPPH RSA was also higher in the acetone extracts than in the ethanol extracts. The DPPH RSA was highest in the acetone extracts of OF. The TRAP and ORAC values of the all extracts increased in a dose-dependent manner. In the TRAP assay, the acetone extracts of OF and OP had the lowest IC50 values. In the CAA assay, the methanol and acetone extracts of OP had the lowest IC50 values. All of the samples protected against H2O2-induced DNA damage in human leukocytes, as measured by the comet assay, but the acetone extracts of OP had the strongest effect. These results suggest that acetone is the best solvent for the extraction of antioxidant compounds from OF and OP. Furthermore, the high antioxidant activity of OP, which is a by-product of orange processing, suggests that it can be used in nutraceutical and functional foods. PMID:25580393

Lack of genotoxic effect of food dyes amaranth, sunset yellow and tartrazine and their metabolites in the gut micronucleus assay in mice.

PubMed

Poul, Martine; Jarry, Gérard; Elhkim, Mostafa Ould; Poul, Jean-Michel

2009-02-01

The food dyes amaranth, sunset yellow and tartrazine were administered twice, at 24h intervals, by oral gavage to mice and assessed in the in vivo gut micronucleus test for genotoxic effects (frequency of micronucleated cells) and toxicity (apoptotic and mitotic cells). The concentrations of each compound and their main metabolites (sulfanilic acid and naphthionic acid) were measured in faeces during a 24-h period after single oral administrations of the food dyes to mice. Parent dye compounds and their main aromatic amine metabolites were detected in significant amounts in the environment of colonic cells. Acute oral exposure to food dye additives amaranth, sunset yellow and tartrazine did not induce genotoxic effect in the micronucleus gut assay in mice at doses up to 2000 mg/kg b.w. Food dyes administration increased the mitotic cells at all dose levels when compared to controls. These results suggest that the transient DNA damages previously observed in the colon of mice treated by amaranth and tartrazine by the in vivo comet assay [Sasaki, Y.F., Kawaguchi, S., Kamaya, A., Ohshita, M., Kabasawa, K., Iwama, K., Taniguchi, K., Tsuda, S., 2002. The comet assay with 8 mouse organs: results with 39 currently used food additives. Mutat. Res. 519, 103-119] are unable to be fixed in stable genotoxic lesions and might be partly explained by local cytotoxicity of the dyes.

Effects of ozone exposure on human epithelial adenocarcinoma and normal fibroblasts cells

PubMed Central

Colafarina, Sabrina; Aruffo, Eleonora; Zarivi, Osvaldo; Bonfigli, Antonella; Di Bucchianico, Sebastiano; Di Carlo, Piero

2017-01-01

Previous studies show variable ozone cytotoxicity and genotoxicity in cell cultures, laboratory animals and humans directly exposed to tropospheric ozone. The aim of this study was therefore to investigate and compare the cyto and genotoxic effects of ozone using adenocarcinoma human alveolar basal epithelial cells A549 and normal human fibroblasts Hs27. A cell culture chamber with controlled atmosphere (a simulation reactor) was built to inject a flow of 120 ppb of ozone, which is two times the threshold value for the protection of human health, fixed by the EU legislation. Cell proliferation was evaluated by a luminescent cell viability assay while we assessed the genotoxic potential of ozone by the induction of micronuclei as well as evaluating DNA strand breaks by the induction of micronuclei evaluated by means of the cytokinesis-block micronucleus (CBMN) assay as well as evaluating DNA strand breaks by Alkaline Comet Assay (CA) or Comet Assay. A549 cells viability decreases significantly at 24 hours treatment with 120 ppb of O3 while at 48 hours and 72 hours O3 treated cells viability doesn’t differ in respect to the control. However a significative decrease of A549 viability is shown at 72 hours vs. 48 hours in both treated and not-treated cells. The viability trend in the Hs27 cells did not show any significant changes in treated samples compared to the control in all conditions. The two genotoxicity biomarkers, the micronucleus and the comet tests, showed in both the cell types exposed to ozone, a significant increase in the number of micronuclei and in the tail DNA % in respect to the control even if at different times/cell type. Moreover, we found that O3 provokes genotoxic effects more evident in A549 cancer cells than in normal fibroblasts Hs27 ones. We applied a cell growth simulation model referred to ozone treated or not cell lines to confirm that the ozone exposure causes a slackening in the cells replication. PMID:28886142

Exposure of composite tannery effluent on snail, Pila globosa: A comparative assessment of toxic impacts of the untreated and membrane treated effluents.

PubMed

Bhattacharya, Priyankari; Swarnakar, Snehasikta; Mukhopadhyay, Aniruddha; Ghosh, Sourja

2016-04-01

Effluent from tannery industries can significantly affect the aquatic environment due to the presence of a variety of recalcitrant components. The present study focuses on a comparative assessment of the toxic impacts of an untreated tannery effluent and membrane treated effluents using snail, Pila globosa as an aquatic model. Composite tannery effluent collected from a common effluent treatment plant was selected as the untreated effluent. To investigate the effect of treated effluents on the aquatic organism the effluent was treated by two ways, viz. a single stage microfiltration (MF) using ceramic membrane and a two-step process involving MF followed by reverse osmosis (RO). The whole body tissue, gonad and mantle of P. globosa were subjected to enzyme assays like superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GSH-GPx), glutathione S- transferase (GST), etc. for assessing toxic impact. Changes in the biochemical parameters like protein, carbohydrate and amino acid were observed including histological studies of gonad and mantle tissue upon treatment with tannery effluents. To examine potential DNA damage due to the exposure of the effluent, comet assay was conducted. The study revealed that with an exposure to the untreated effluent, activity of the antioxidant enzymes increased significantly while the protein and carbohydrate content reduced largely in the whole body tissue, gonad as well as mantle tissues of P. globosa. Histological study indicated considerable damage in the gonad and mantle tissues following exposure to the untreated effluent. Comet assay using hemolymph of P. globosa following exposure to tannery effluent, showed significant genotoxicity. Interestingly, compared to the untreated effluent, damaging effect was reduced in molluscs tissues when exposed to MF treated effluent and even lesser when exposed to MF+RO treated effluent. Apart from the reduced activities of oxidative stress enzymes, the protein, amino acid and carbohydrate content of molluscs exposed to both of the treated effluent were found close to that of control. Comet assay revealed no damage in the DNA for MF and MF+RO treated effluent indicating that the membrane based treatment procedure restores environmental condition to control level. Copyright © 2015 Elsevier Inc. All rights reserved.

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. (iv) To build a constructive criticism and summarize the needs for protocol improvements for future test applications within the field of ecotoxicology. The Comet Assay is still developing and its potential is yet underexploited in experimental models, mesocosmos or natural ecosystems. PMID:26089833

Organophosphorus pesticides enhance the genotoxicity of benzo(a)pyrene by modulating its metabolism.

PubMed

Hreljac, Irena; Filipic, Metka

2009-12-01

Organophosphorus compounds (OPs) are widely used as pesticides. They act primarily as neurotoxins, but there is increasing evidence for secondary mechanisms of their toxicity. We have shown that the model OPs, methyl parathion (PT) and methyl paraoxon (PO), are genotoxic. Benzo(a)pyrene (BaP) is a widespread environmental genotoxin found in cigarette smoke, polluted air and grilled food. As people are constantly exposed to low concentrations of BaP and also to OPs, the aim of this study was to determine possible synergistic effects of PT and PO on BaP-induced genotoxicity. In the bacterial reverse mutation assay, PT and PO increased the number of BaP-induced mutations. The comet assay with human hepatoma HepG2 cells showed that BaP-induced DNA strand breaks were increased by PT but slightly decreased by PO. Using the acellular comet assay with UVC-induced DNA strand breaks, we observed a decrease in DNA migration, indicating that OPs cause cross-linking, thus interfering with comet assay results. In HepG2 cells the two OPs induced micronuclei formation at very low doses (0.01 microg/ml) and together with BaP, a more than additive increase of micronuclei was observed, confirming their co-genotoxic effect. We demonstrated for the first time that PT and PO modulate the metabolic activation of BaP. Addition of PT or PO increased aldo-keto reductase (AKR1C1/2) levels in the presence of BaP, while cytochrome 1A (CYP1A) mRNA expression and activity were decreased. Further, specific inhibition of CYP1A had no effect on BaP or OP+BaP-induced micronuclei, whereas inhibition of AKR1C dramatically decreased the number of micronuclei induced by BaP or OP+BaP. Based on these results we propose that co-genotoxicity results from OPs mediated modulation of BaP metabolism, favouring the induction of AKR1C enzymes known to catalyse the formation of DNA reactive BaP o-quinones and the production of reactive oxygen species.

Vitamin C in synergism with cisplatin induces cell death in cervical cancer cells through altered redox cycling and p53 upregulation.

PubMed

Leekha, Ankita; Gurjar, Bahadur S; Tyagi, Aakriti; Rizvi, Moshahid A; Verma, Anita K

2016-12-01

Cervical cancer is the second most prevalent cancer in women worldwide. Survival of patients has been improved by cisplatin-based chemotherapy, but its effectiveness is limited due to its adverse effects on many tissues, especially nephrotoxicity. To optimize the efficacy of CDDP, we propose a combination therapy using natural products with minimal side effects. Vitamin C being a natural antioxidant is capable of selectively targeting cancer cells at pharmacological concentrations. Vitamin C synergistically enhances the activity of chemotherapeutic agents without increasing toxicity to normal cells. Therefore, we exploited co-therapy with cisplatin and vitamin C to kill cervical cancer cells. We elucidated the role of CDDP and VC on cervical cancer cell line (SiHa) by using cell growth assays, DNA fragmentation analysis, comet assay, in vitro morphological assessment of apoptosis (AO/EB and DAPI staining), ROS analysis by DCFDA, flow cytometry, biochemical assays (GST, GSH, NO, catalase, TPA) and Western blotting. Our results clearly demonstrated that CDDP and VC treatment exhibited ameliorative effect on induction of cell death by p53 overexpression and generation of hydrogen peroxide in SiHa cells, thereby reducing the dosage of CDDP required to induce cell death in cancer cells. These studies provide novel approaches to combat cisplatin resistance in cervical cancer.

Identification of mammalian cell genotoxins in respirable diesel exhaust particles by bioassay-directed chemical analysis.

PubMed

Oh, Seung-Min; Chung, Kyu-Hyuck

2006-03-01

A bioassay-directed chemical analysis which consists of mammalian cell bioassays (comet assay, CBMN assay and EROD-microbioassay) in conjunction with analytical measurements was performed to identify the most biologically active compounds of the diesel exhaust particulate matters (DEPs) on mutagenic activity. These bioassay systems were suitable to estimate the mammalian genotoxic potentials of pollutants present in low concentrations in limited environmental samples, as is the case with DEPEs. The results from mutagenic assay showed that the aromatic and slightly polar fraction of DEPs induced chromosomal damage and DNA breakage in a non-cytotoxic dose. It was also revealed that indirect-acting mutagens may mainly contribute to the mutagenic effect of aromatic fraction via the enzyme metabolism system. In the aromatic fraction, several indirect-acting mutagenic PAHs such as dibenzo(a,h)anthracene, chrysene, and 1,2-benzanthracene were detected by GC-MS and the complex mixture effect of this fraction was quantified in terms of its biological-TCDD equivalent concentration (bio-TEQ) which was 32.82 bio-TEQ ng/g-DEPs by EROD-microbioassay. Conclusively, we confirmed that indirect-acting mutagens contained in aromatic fraction may be important causatives of the genotoxicity of extracts of DEPs by integrating the results obtained from a mammalian cell bioassay-directed fractionation.

Mammalian cell line-based bioassays for toxicological evaluation of landfill leachate treated by Pseudomonas sp. ISTDF1.

PubMed

Ghosh, Pooja; Das, Mihir Tanay; Thakur, Indu Shekhar

2014-01-01

Landfill leachate has become a serious environmental concern because of the presence of many hazardous compounds which even at trace levels are a threat to human health and environment. Therefore, it is important to assess the toxicity of leachate generated and discharge it conforming to the safety standards. The present work examined the efficiency of an earlier reported Pseudomonas sp. strain ISTDF1 for detoxification of leachate collected from Okhla landfill site (New Delhi, India). GC-MS analysis performed after treatment showed the removal of compounds like alpha-limonene diepoxide, brominated dioxin-2-one, Bisphenol A, nitromusk, phthalate derivative, and nitrobenzene originally found in untreated leachate. ICP-AES analysis for heavy metals also showed reduction in concentrations of Zn, Cd, Cr, Fe, Ni, and Pb bringing them within the limit of safety discharge. Methyl tetrazolium (MTT) assay for cytotoxicity, alkaline comet assay for genotoxicity, and 7-ethoxyresorufin-O-deethylase (EROD) assay for dioxin-like behavior were carried out in human hepato-carcinoma cell line HepG2 to evaluate the toxic potential of treated and untreated leachates. The bacterium reduced toxicity as shown by 2.5-fold reduction of MTT EC50 value, 7-fold reduction in Olive Tail Moment, and 2.8-fold reduction in EROD induction after 240 h of bacterial treatment.

Factors influencing heterogeneity of radiation-induced DNA-damage measured by the alkaline comet assay

PubMed Central

2012-01-01

Background To investigate whether different conditions of DNA structure and radiation treatment could modify heterogeneity of response. Additionally to study variance as a potential parameter of heterogeneity for radiosensitivity testing. Methods Two-hundred leukocytes per sample of healthy donors were split into four groups. I: Intact chromatin structure; II: Nucleoids of histone-depleted DNA; III: Nucleoids of histone-depleted DNA with 90 mM DMSO as antioxidant. Response to single (I-III) and twice (IV) irradiation with 4 Gy and repair kinetics were evaluated using %Tail-DNA. Heterogeneity of DNA damage was determined by calculation of variance of DNA-damage (V) and mean variance (Mvar), mutual comparisons were done by one-way analysis of variance (ANOVA). Results Heterogeneity of initial DNA-damage (I, 0 min repair) increased without histones (II). Absence of histones was balanced by addition of antioxidants (III). Repair reduced heterogeneity of all samples (with and without irradiation). However double irradiation plus repair led to a higher level of heterogeneity distinguishable from single irradiation and repair in intact cells. Increase of mean DNA damage was associated with a similarly elevated variance of DNA damage (r = +0.88). Conclusions Heterogeneity of DNA-damage can be modified by histone level, antioxidant concentration, repair and radiation dose and was positively correlated with DNA damage. Experimental conditions might be optimized by reducing scatter of comet assay data by repair and antioxidants, potentially allowing better discrimination of small differences. Amount of heterogeneity measured by variance might be an additional useful parameter to characterize radiosensitivity. PMID:22520045

Effects of cold atmospheric plasma on mucosal tissue culture

NASA Astrophysics Data System (ADS)

Welz, Christian; Becker, Sven; Li, Yang-Fang; Shimizu, Tetsuji; Jeon, Jin; Schwenk-Zieger, Sabina; Thomas, Hubertus M.; Isbary, Georg; Morfill, Gregor E.; Harréus, Ulrich; Zimmermann, Julia L.

2013-01-01

Thermal plasmas have been commonly used in medical applications such as plasma ablation and blood coagulation. Newer developments show that plasmas can be generated with ion temperatures close to room temperature: these non-thermal or so-called cold atmospheric plasmas (CAPs) therefore open up a wide range of further biomedical applications. Based on the understanding of the bactericidal, virucidal and fungicidal properties of CAPs, information about the effects of CAP on mucosal cells and tissue is still lacking. Therefore this study focuses on the interaction of CAP with healthy head and neck mucosal cells on a molecular level. To analyse this interaction in detail, fresh tissue samples from healthy nasal and pharyngeal mucosa were harvested during surgery, assembled to a three-dimensional tissue culture model (mini organ cultures) and treated with CAP for different treatment times. Effects on the viability, necrosis induction and mutagenic activity were evaluated with the trypan blue exclusion test, Annexin-V/PI staining and alkaline microgel electrophoresis (comet assay). Trypan blue exclusion test revealed that the CAP treatment significantly decreases the cell viability for all tested treatment times (5, 10, 30, 60 and 120 s p < 0.05), but only a treatment time of 120 s showed a cytotoxic effect as the viability dropped below 90%. Annexin-V/PI staining revealed a significant increase in necrosis in CAP treated pharyngeal tissue cultures for treatment times of 60 and 120 s (p < 0.05). For nasal tissue this effect was already detected for a 30 s treatment (p < 0.05). Comet assay analysis showed no mutagenic effects after exposure to CAP.

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

PubMed

Holliday, Richard; Kist, Ralf; Bauld, Linda

2016-03-01

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

Comet composition and density analyzer

NASA Technical Reports Server (NTRS)

Clark, B. C.

1982-01-01

Distinctions between cometary material and other extraterrestrial materials (meteorite suites and stratospherically-captured cosmic dust) are addressed. The technique of X-ray fluorescence (XRF) for analysis of elemental composition is involved. Concomitant with these investigations, the problem of collecting representative samples of comet dust (for rendezvous missions) was solved, and several related techniques such as mineralogic analysis (X-ray diffraction), direct analysis of the nucleus without docking (electron macroprobe), dust flux rate measurement, and test sample preparation were evaluated. An explicit experiment concept based upon X-ray fluorescence analysis of biased and unbiased sample collections was scoped and proposed for a future rendezvous mission with a short-period comet.

Effect of Treatment Media on the Agglomeration of Titanium Dioxide Nanoparticles: Impact on Genotoxicity, Cellular Interaction, and Cell Cycle

EPA Science Inventory

ABSTRACT The widespread use of titanium dioxide (TiO2) nanoparticles in consumer products increases the probability of exposure to humans and the environment. Although TiO2 nanoparticles have been shown to induce DNA damage (comet assay) and chromosome damage (micronucleus ass...

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.

Assessment of in vitro cyto/genotoxicity of sequentially treated electroplating effluent on the human hepatocarcinoma HuH-7 cell line.

PubMed

Naik, Umesh Chandra; Das, Mihir Tanay; Sauran, Swati; Thakur, Indu Shekhar

2014-03-01

The present study compares in vitro toxicity of electroplating effluent after the batch treatment process with that obtained after the sequential treatment process. Activated charcoal prepared from sugarcane bagasse through chemical carbonization, and tolerant indigenous bacteria, Bacillus sp. strain IST105, were used individually and sequentially for the treatment of electroplating effluent. The sequential treatment involving activated charcoal followed by bacterial treatment removed 99% of Cr(VI) compared with the batch processes, which removed 40% (charcoal) and 75% (bacteria), respectively. Post-treatment in vitro cyto/genotoxicity was evaluated by the MTT test and the comet assay in human HuH-7 hepatocarcinoma cells. The sequentially treated sample showed an increase in LC50 value with a 6-fold decrease in comet-assay DNA migration compared with that of untreated samples. A significant decrease in DNA migration and an increase in LC50 value of treated effluent proved the higher effectiveness of the sequential treatment process over the individual batch processes. Copyright © 2014 Elsevier B.V. All rights reserved.

Genotoxicity of endosseous implants using two cellular lineages in vitro.

PubMed

Matsumoto, Mariza; Filho, Hugo Nary; Ferrari, Raquel; Fernandes, Kristianne; Renno, Ana Claudia; Ribeiro, Daniel

2014-02-01

The genotoxic potential of corrosion eluates obtained from a single dental implant using murine fibroblasts or osteoblasts cells in vitro by the single-cell gel (comet) assay was examined. A single commercially available dental implant (Biotechnology) was eluted in a solution consisting of equal amounts of acetic acid and sodium chloride (0.1 M) for 1, 3, 7, 14, and 21 days. Murine fibroblast or osteoblast cultures were then exposed to all corrosion eluates obtained from endosseous dental implants for 30 minutes at 37°C. The results suggest that none of the eluates produced genotoxic changes in murine fibroblasts regardless of the length of exposure to the eluate. Similarly, no genotoxicity was found in osteoblasts. The results suggest that the dental implant eluates tested in this study did not induce genetic damage as depicted by the single-cell gel (comet) assay. Because DNA damage is an important event during oncogenesis, this study represents a relevant contribution to estimate the real risks to the cellular system induced by the corrosion products of a dental implant.

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

Cytogenetic biomonitoring of peripheral blood and oral mucosa cells from car painters.

PubMed

Pereira da Silva, Victor Hugo; Gomes de Moura, Carolina Foot; Spadari-Bratfisch, Regina Célia; Araki Ribeiro, Daniel

2012-09-01

The aim of the present study was to comparatively evaluate genomic damage and cellular death in exfoliated oral mucosa cells and peripheral blood from car painters. A total of 24 car painters and 19 healthy controls (non-exposed individuals) were included in this setting. Individuals had epithelial cells from cheek mucosa (left and right side) mechanically exfoliated, placed in fixative and dropped in clean slides which were checked for the specific nuclear phenotypes. A total of 5 μL from peripheral blood was collected for the single cell gel (comet) assay. The results pointed out statistically significant differences (p < 0.05) of micronucleated oral mucosa cells from car painters. In addition, DNA damage was detected in peripheral blood cells by single cell gel (comet) assay. Nevertheless, exposure to car paints did not cause increases other nuclear alterations closely related to cytotoxicity such as karrhyorexis, pyknosis and karyolysis in buccal mucosa cells. In summary, the results of the present study suggest that car painters comprise a high risk group since paints can induce genotoxic and mutagenic effects in peripheral blood and oral mucosa cells, respectively.

Assessment of trophic transfer of benzo(a)pyrene genotoxicity from the post-larval pink shrimp F. brasiliensis to the juvenile Florida pompano T. carolinus.

PubMed

Rocha, Arthur José da Silva; Santos, Thaís Cruz Alves; Gomes, Vicente; Bícego, Márcia Caruso; Barbosa, Ana Cecília Rizzatti de Albergaria; Passos, Maria José de Arruda Campos Rocha; Hasue, Fabio Matsu; Van Ngan, Phan

2012-11-01

In the present study, the polycyclic aromatic hydrocarbon (PAH) genotoxicity was investigated in a one-step predator-prey relationship with the trophic-related marine species. Florida pompanos were fed for 5 and 10 days with pink shrimp post larvae previously exposed to benzo(a)pyrene (BaP) concentrations. Parent BaP body burden was measured in samples of Farfantepenaeus brasiliensis. BaP metabolites were determined in bile samples of Trachinotus carolinus and DNA damage was assessed through the comet and erythrocyte nuclear abnormalities (ENAs) assays in fish erythrocytes. BaP body burden increased significantly with the PAH concentration in pink shrimp PLs as well as the fish bile BaP metabolites. Both, comet and ENAs assays indicated significant increase on erythrocyte DNA damage of Florida pompanos fed with BaP-exposed pink shrimp on both feeding periods. The trophic route of BaP genotoxicity is discussed as well as the PAH biotransformation as the inducing mechanism for the DNA damages observed. Copyright © 2012 Elsevier B.V. All rights reserved.

Genotoxicity of corrosion eluates obtained from endosseous implants.

PubMed

Ribeiro, Daniel Araki; Matsumoto, Mariza Akemi; Padovan, Luís Eduardo Marques; Marques, Mariângela Esther Alencar; Salvadori, Daisy Maria Fávero

2007-03-01

Commercially pure titanium alloys are currently used as metallic biomaterials in implantology. Corrosion phenomena appear to play a decisive role in metallic implant long-term behavior. Thus, the goal of this study was to examine the genotoxic potential of corrosion eluates obtained from dental implants using Chinese ovary hamster cells in vitro by the single-cell gel (comet) assay. This technique detects deoxyribonucleic acid strand breaks in individual cells in alkaline conditions. The materials tested included 3 dental implants commercially available. Each of the tested materials was corroded in a solution consisting of equal amounts of acetic acid and sodium chloride (0.1 M) for 1, 3, 7, 14, and 21 days. The Chinese ovary hamster cultures were then exposed to all corrosion eluates obtained from endosseous dental implants for 30 minutes at 37 degrees C. None of the eluates was found to exhibit genotoxicity, regardless of the type of dental implant used. The results suggest that all dental implants tested in this study did not induce deoxyribonucleic acid breakage as depicted by the single-cell gel (comet) assay.

DNA damage in blood cells exposed to low-level lasers.

PubMed

Sergio, Luiz Philippe da Silva; Silva, Ana Paula Almeida da; Amorim, Philipi Freitas; Campos, Vera Maria Araújo; Magalhães, Luis Alexandre Gonçalves; de Paoli, Flavia; de Souza da Fonseca, Adenilson

2015-04-01

In regenerative medicine, there are increasing applications of low-level lasers in therapeutic protocols for treatment of diseases in soft and in bone tissues. However, there are doubts about effects on DNA, and an adequate dosimetry could improve the safety of clinical applications of these lasers. This work aimed to evaluate DNA damage in peripheral blood cells of Wistar rats induced by low-level red and infrared lasers at different fluences, powers, and emission modes according to therapeutic protocols. Peripheral blood samples were exposed to lasers and DNA damage was accessed by comet assay. In other experiments, DNA damage was accessed in blood cells by modified comet assay using formamidopyrimidine DNA glycosylase (Fpg) and endonuclease III enzymes. Data show that exposure to low-level red and infrared lasers induce DNA damage depending on fluence, power and emission mode, which are targeted by Fpg and endonuclease III. Oxidative DNA damage should be considered for therapeutic efficacy and patient safety in clinical applications based on low-level red and infrared lasers. © 2015 Wiley Periodicals, Inc.

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.

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.

A biological evaluation of DNA damage detected by comet assay in healthy populations residing in areas that differ in lung cancer incidence.

PubMed

Heepchantree, Worapa; Paratasilpin, Thipmani; Kangwanpong, Daoroong

2006-06-01

The comet assay was performed to evaluate the effect of environmental exposure between human populations residing in two areas that differ in lung cancer incidence, Saraphi (n = 91) and Chom Thong (n = 94). Three parameters, the tail length, tail intensity, and tail moment, were used to detect DNA damage in peripheral blood and stimulated lymphocytes with and without the DNA repair inhibitor, aphidicolin. Internal standards, cryopreserved isolated lymphocytes, and isolated lymphocytes irradiated with 2 Gy gamma rays, were used to correct the interexperimental variability. Results revealed a significant difference between two populations only when the tail length was used to measure DNA damage. The evaluation of various potential confounding factors, such as gender, pesticide exposure, smoking, alcohol drinking, and fermented tea leaf or betel nut chewing, indicated no significant influence in DNA damage. In conclusion, significant difference in DNA damage, detected only by tail length between the two populations residing in the areas with different incidence of lung cancer, may reflect a nonhazardous level of exposure to toxic substances.

Genotoxic and mutagenic effects of polluted surface water in the midwestern region of Brazil using animal and plant bioassays

PubMed Central

Dourado, Priscila Leocádia Rosa; da Rocha, Monyque Palagano; Roveda, Liriana Mara; Raposo, Jorge Luiz; Cândido, Liliam Sílvia; Cardoso, Claudia Andréa Lima; Morales, Maria Aparecida Marin; de Oliveira, Kelly Mari Pires; Grisolia, Alexeia Barufatti

2016-01-01

Abstract This study aimed to evaluate DNA damage in animal and plant cells exposed to water from the Água Boa stream (Dourados, Mato Grosso do Sul, Brazil) by using bioassays, and to identify the chemical compounds in the water to determine the water quality in the area. Through the cytotoxicity bioassay with Allium cepa, using micronucleus test, and comet assay, using Astyanax altiparanae fish, the results indicated that biological samples were genetically altered. Micronuclei were observed in erythrocytes of A. altiparanae after exposure to water from locations close to industrial waste discharge. The highest DNA damage observed with the comet assay in fish occurred with the exposure to water from locations where the presence of metals (Cu, Pb, Cd, Ni) was high, indicating the possibility of genotoxic effects of these compounds. Thus, these results reinforce the importance of conducting genotoxicity tests for developing management plans to improve water quality, and indicate the need for waste management before domestic and industrial effluents are released into the rivers and streams. PMID:27801481

Protective effects of acerola juice on genotoxicity induced by iron in vivo

PubMed Central

Horta, Roberta Nunes; Kahl, Vivian Francilia Silva; Sarmento, Merielen da Silva; Nunes, Marisa Fernanda Silva; Porto, Carem Rejane Maglione; de Andrade, Vanessa Moraes; Ferraz, Alexandre de Barros Falcão; Silva, Juliana Da

2016-01-01

Abstract Metal ions such as iron can induce DNA damage by inducing reactive oxygen species (ROS) and oxidative stress. Vitamin C is one of the most widely consumed antioxidants worldwide, present in many fruits and vegetables, especially inMalpighia glabra L., popularly known as acerola, native to Brazil. Acerola is considered a functional fruit due to its high antioxidant properties and phenolic contents, and therefore is consumed to prevent diseases or as adjuvant in treatment strategies. Here, the influence of ripe and unripe acerola juices on iron genotoxicity was analyzed in vivo using the comet assay and micronucleus test. The comet assay results showed that acerola juice exerted no genotoxic or antigenotoxic activity. Neither ripe nor unripe acerola juices were mutagenic to animals treated with juices, in micronucleus test. However, when compared to iron group, the pre-treatment with acerola juices exerted antimutagenic activity, decreasing significantly micronucleus mean values in bone marrow. Stage of ripeness did not influence the interaction of acerola compounds with DNA, and both ripe and unripe acerola juices exerted protective effect over DNA damage generated by iron. PMID:27007905

Comparison of Comet Enflow and VA One Acoustic-to-Structure Power Flow Predictions

NASA Technical Reports Server (NTRS)

Grosveld, Ferdinand W.; Schiller, Noah H.; Cabell, Randolph H.

2010-01-01

Comet Enflow is a commercially available, high frequency vibroacoustic analysis software based on the Energy Finite Element Analysis (EFEA). In this method the same finite element mesh used for structural and acoustic analysis can be employed for the high frequency solutions. Comet Enflow is being validated for a floor-equipped composite cylinder by comparing the EFEA vibroacoustic response predictions with Statistical Energy Analysis (SEA) results from the commercial software program VA One from ESI Group. Early in this program a number of discrepancies became apparent in the Enflow predicted response for the power flow from an acoustic space to a structural subsystem. The power flow anomalies were studied for a simple cubic, a rectangular and a cylindrical structural model connected to an acoustic cavity. The current investigation focuses on three specific discrepancies between the Comet Enflow and the VA One predictions: the Enflow power transmission coefficient relative to the VA One coupling loss factor; the importance of the accuracy of the acoustic modal density formulation used within Enflow; and the recommended use of fast solvers in Comet Enflow. The frequency region of interest for this study covers the one-third octave bands with center frequencies from 16 Hz to 4000 Hz.

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.

Ballistic intercept missions to Comet Encke

NASA Technical Reports Server (NTRS)

Mumma, M. (Compiler)

1975-01-01

The optimum ballistic intercept of a spacecraft with the comet Encke is determined. The following factors are considered in the analysis: energy requirements, encounter conditions, targeting error, comet activity, spacecraft engineering requirements and restraints, communications, and scientific return of the mission. A baseline model is formulated which includes the basic elements necessary to estimate the scientific return for the different missions considered. Tradeoffs which have major impact on the cost and/or scientific return of a ballistic mission to comet Encke are identified and discussed. Recommendations are included.

Extended atmospheres of outer planet satellites and comets

NASA Technical Reports Server (NTRS)

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

1984-01-01

The cometary hydrogen particle-trajectory model was used successfully to analyze observations of Comet P/Encke. The Pioneer Venus Orbiter Ultraviolet Spectrometer observed the comet at 1216A (hydrogen Lyman-alpha) on 15 April 1984, when the comet was .58 AU from the Sun and 1.02 AU from Venus. The analysis implies a production rate at .58 AU of 3.3 x 10 to the 28th power/sec of the water molecules which photodissociate to produce the observed hydrogen.

[Cytotoxicity and genotoxicity of fluorides in human mucosa and lymphocytes].

PubMed

Kleinsasser, N H; Weissacher, H; Wallner, B C; Kastenbauer, E R; Harréus, U A

2001-04-01

Fluorides are widely used in dental health products and drinking water, due to their beneficial effects in caries-prophylaxis and -treatment. Nevertheless, irritation of the gingiva and oropharyngeal mucosa as well as in gastric mucosa is observed since neither local nor systemic application is restricted to the teeth. These effects may partly be attributed to a known cytotoxicity of fluorides. Whether fluorides also have genotoxic effects on human mucosa or lymphocytes as a possible factor in tumor initiation was investigated in this study. Human oropharyngeal epithelial cells and peripheral lymphocytes were incubated after single cell preparation with the aminefluoride Olaflur at concentrations of 2 ppm, 21 ppm, 35 ppm, 71 ppm and 213 ppm. The extent of cytotoxicity was investigated using the trypan blue exclusion test. Following incubation, electrophoresis for migration of DNA fragments, fluorescence staining and digital image analysis according to a standard protocol of the single cell microgel electrophoresis assay (Comet assay) followed. DNA damage was characterized using the Olive Tail Moment (OTM). For fluoride concentrations of 2 ppm to 35 ppm, non vital cells of less than 10% could be shown. After incubation with 71 ppm and 213 ppm Olaflur, there were 15% and 43% of damaged cells, respectively. Weak genotoxic effects on mucosal cells as well as on lymphocytes could be demonstrated at all concentrations tested. In fluoride concentrations of 213 ppm genotoxicity increased to max. OTM-levels of 23. Beside the cytotoxic effect of fluorides, also a minor genotoxic impact on human mucosa and on peripheral lymphocytes could be demonstrated using the Comet assay. Further investigations are warranted to examine fluorides in a model allowing for repeated or long term incubations on structurally intact human mucosa in vitro. Such a model will help to distinguish between DNA damage that may be repaired successfully and other impairments that may show an additive character in repetitive or chronic exposure in vivo.

Lead induces DNA damage and alteration of ALAD and antioxidant genes mRNA expression in construction site workers.

PubMed

Akram, Zertashia; Riaz, Sadaf; Kayani, Mahmood Akhtar; Jahan, Sarwat; Ahmad, Malik Waqar; Ullah, Muhammad Abaid; Wazir, Hizbullah; Mahjabeen, Ishrat

2018-01-16

Oxidative stress and DNA damage are considered as possible mechanisms involved in lead toxicity. To test this hypothesis, DNA damage and expression variations of aminolevulinic acid dehydratase (ALAD), superoxide dismutase 2 (SOD2), and 8-oxoguanine DNA glycosylase 2a (OGG1-2a) genes was studied in a cohort of 100 exposed workers and 100 controls with comet assay and real-time polymerse chain reaction (PCR). Results indicated that increased number of comets was observed in exposed workers versus controls (p < 0.001). After qPCR analysis, significant down-regulation in ALAD (p < 0.0001), SOD2 (p < 0.0001), and OGG1-2a (p < 0.0001) level was observed in exposed workers versus controls. Additionally, a positive spearmen correlation was observed between ALAD versus SOD2 (r = 0.402**, p < 0.001), ALAD versus OGG1-2a (r = 0.235*, p < 0.05), and SOD2 versus OGG1-2a (r = 0.292*, p < 0.05). This study showed that lead exposure induces DNA damage, which is accompanied by an elevated intensity of oxidative stress and expression variation of lead-related gene.

Temporary satellite capture of comets by Jupiter

NASA Astrophysics Data System (ADS)

Emel'yanenko, N. Yu.

2012-05-01

This paper studies the dynamical evolution of 97 Jupiter-family comets over an 800-year time period. More than two hundred encounters with Jupiter are investigated, with the observed comets moving during a certain period of time in an elliptic jovicentric orbit. In most cases this is an ordinary temporary satellite capture of a comet in Everhart's sense, not associated with a transition of the small body into Jupiter's family of satellites. The phenomenon occurs outside the Hill sphere with comets with a high Tisserand constant relative to Jupiter; the comets' orbits have a small inclination to the ecliptic plane. An analysis of 236 encounters has allowed the determination within the planar pair two-body problem of a region of orbits in the plane ( a, e) whose semimajor axes and eccentricities contribute to the phenomenon under study. Comets with orbits belonging to this region experience a temporary satellite capture during some of their encounters; the jovicentric distance function has several minima; and the encounters are characterized by reversions of the line of apsides and some others features of their combination that are intrinsic to comets in this region. Therefore, this region is called a region of comets with specific features in their encounters with Jupiter. Twenty encounters (out of 236), whereby the comet enters an elliptic jovicentric orbit in the Hill sphere, are identified and investigated. The size and shape of the elliptic heliocentric orbits enabling this transition are determined. It is found that in 11 encounters the motion of small bodies in the Hill sphere has features the most important of which is multiple minima of the jovicentric distance function. The study of these 20 encounters has allowed the introduction of the concept of temporary gravitational capture of a small body into the Hill sphere. An analysis of variations in the Tisserand constant in these (20) encounters of the observable comets shows that their motion is unstable in Hill's sense.

Oxidative stress, activity behaviour and body mass in captive parrots

PubMed Central

Larcombe, S D; Tregaskes, C A; Coffey, J; Stevenson, A E; Alexander, L G

2015-01-01

Abstract Many parrot species are kept in captivity for conservation, but often show poor reproduction, health and survival. These traits are known to be influenced by oxidative stress, the imbalance between the production of reactive oxygen species (ROS) and ability of antioxidant defences to ameliorate ROS damage. In humans, oxidative stress is linked with obesity, lack of exercise and poor nutrition, all of which are common in captive animals. Here, we tested whether small parrots (budgerigars, Melopsittacus undulatus) maintained in typical pet cages and on ad libitum food varied in oxidative profile, behaviour and body mass. Importantly, as with many birds held in captivity, they did not have enough space to engage in extensive free flight. Four types of oxidative damage, single-stranded DNA breaks (low-pH comet assay), alkali-labile sites in DNA (high-pH comet assay), sensitivity of DNA to ROS (H2O2-treated comet assay) and malondialdehyde (a byproduct of lipid peroxidation), were uncorrelated with each other and with plasma concentrations of dietary antioxidants. Without strenuous exercise over 28 days in a relatively small cage, more naturally ‘active’ individuals had more single-stranded DNA breaks than sedentary birds. High body mass at the start or end of the experiment, coupled with substantial mass gain, were all associated with raised sensitivity of DNA to ROS. Thus, high body mass in these captive birds was associated with oxidative damage. These birds were not lacking dietary antioxidants, because final body mass was positively related to plasma levels of retinol, zeaxanthin and α-tocopherol. Individuals varied widely in activity levels, feeding behaviour, mass gain and oxidative profile despite standardized living conditions. DNA damage is often associated with poor immunocompetence, low fertility and faster ageing. Thus, we have candidate mechanisms for the limited lifespan and fecundity common to many birds kept for conservation purposes. PMID:27293729

Oxidative stress, activity behaviour and body mass in captive parrots.

PubMed

Larcombe, S D; Tregaskes, C A; Coffey, J; Stevenson, A E; Alexander, L G; Arnold, K E

2015-01-01

Many parrot species are kept in captivity for conservation, but often show poor reproduction, health and survival. These traits are known to be influenced by oxidative stress, the imbalance between the production of reactive oxygen species (ROS) and ability of antioxidant defences to ameliorate ROS damage. In humans, oxidative stress is linked with obesity, lack of exercise and poor nutrition, all of which are common in captive animals. Here, we tested whether small parrots (budgerigars, Melopsittacus undulatus) maintained in typical pet cages and on ad libitum food varied in oxidative profile, behaviour and body mass. Importantly, as with many birds held in captivity, they did not have enough space to engage in extensive free flight. Four types of oxidative damage, single-stranded DNA breaks (low-pH comet assay), alkali-labile sites in DNA (high-pH comet assay), sensitivity of DNA to ROS (H2O2-treated comet assay) and malondialdehyde (a byproduct of lipid peroxidation), were uncorrelated with each other and with plasma concentrations of dietary antioxidants. Without strenuous exercise over 28 days in a relatively small cage, more naturally 'active' individuals had more single-stranded DNA breaks than sedentary birds. High body mass at the start or end of the experiment, coupled with substantial mass gain, were all associated with raised sensitivity of DNA to ROS. Thus, high body mass in these captive birds was associated with oxidative damage. These birds were not lacking dietary antioxidants, because final body mass was positively related to plasma levels of retinol, zeaxanthin and α-tocopherol. Individuals varied widely in activity levels, feeding behaviour, mass gain and oxidative profile despite standardized living conditions. DNA damage is often associated with poor immunocompetence, low fertility and faster ageing. Thus, we have candidate mechanisms for the limited lifespan and fecundity common to many birds kept for conservation purposes.

Basal damage and oxidative DNA damage in children with chronic kidney disease measured by use of the comet assay.

PubMed

Aykanat, Banu; Demircigil, Gonca Cakmak; Fidan, Kibriya; Buyan, Necla; Gulleroglu, Kaan; Baskin, Esra; Bayrakci, Umut Selda; Sepici, Aylin; Buyukkaragoz, Bahar; Karakayali, Hamdi; Haberal, Mehmet; Burgaz, Sema

2011-10-09

One consequence of chronic kidney disease (CKD) is an elevated risk for cancer. There is sufficient evidence to conclude that there is an increased incidence of at least some cancers in kidney-dialysis patients. Cancer risk after kidney transplantation has mainly been attributed to immunosuppressive therapy. There are no data evaluating DNA damage in children with CKD, in dialysis patients, or following kidney transplantation. In this study, the comet assay and the enzyme-modified comet assay - with the use of endonuclease III (Endo III) and formamidopyrimidine glycosylase (FPG) enzymes - were conducted to investigate the basal damage and the oxidative DNA damage as a result of treatment in peripheral blood lymphocytes of children. Children at various stages of treatment for kidney disease, including pre-dialysis patients (PreD) (n=17), regular hemodialysis patients (HD) (n=15), and those that received kidney transplants (Tx) (n=17), comprised the study group. They were compared with age- and gender-matched healthy children (n=20) as a control group. Our results show that the %DNA intensity, a measure of basal damage, was significantly increased in children with CKD (mean ± SD) (5.22 ± 1.57) and also in each of the PreD, HD, and Tx groups [(4.92 ± 1.23), (4.91 ± 1.35), and (5.79 ± 1.94), respectively, vs the healthy children (2.74 ± 2.91) (p<0.001). Significant increases in oxidative DNA damage were only found in the FPG-sensitive sites for the PreD and Tx groups, compared with control and HD groups (p<0.05), suggesting that basal DNA damage was more evident for the PreD, HD, and Tx groups. The findings of the present study indicate a critical need for further research on genomic damage with different endpoints and also for preventive measures and improvements in treatment of pediatric patients, in order to improve their life expectancy. 2011 Elsevier B.V. All rights reserved.

Genotoxicity of 2-[2-(acetylamino)-4-[bis(2-hydroxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-6) and 4-amino-3,3'-dichloro-5,4'-dinitro-biphenyl (ADDB) in goldfish (Carassius auratus) using the micronucleus test and the comet assay.

PubMed

Masuda, Shuichi; Deguchi, Yuya; Masuda, Yumi; Watanabe, Tetsushi; Nukaya, Haruo; Terao, Yoshiyasu; Takamura, Takeji; Wakabayashi, Keiji; Kinae, Naohide

2004-05-09

2-[2-(Acetylamino)-4-[bis(2-hydroxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-6) and 4-amino-3,3'-dichloro-5,4'-dinitrobiphenyl (ADDB) are two compounds, which show strong mutagenicity toward bacteria, that have been identified as major mutagens in river water in Japan. In the present study, we examined the genotoxicity of PBTA-6 and ADDB in goldfish (Carassius auratus) by the micronucleus test and single-cell gel electrophoresis (comet assay). The frequencies of micronuclei in gill cells gradually increased until 96h after i.p. injection of PBTA-6 and ADDB at doses of 50mg/kg body weight, and then decreased 144h after injection. PBTA-6 induced micronuclei in gill cells dose-dependently at a dose range of 1-100mg/kg body weight, giving significantly high frequencies at doses of 50 and 100mg/kg body weight. On the other hand, no significant increase was observed in the peripheral erythrocytes of goldfish exposed to PBTA-6 or ADDB. In the comet assay, values of DNA tail moment and tail length in peripheral erythrocytes increased significantly until 6h after the i.p. injection of PBTA-6 (50mg/kg body weight), only to decrease by 9h after injection. Both the DNA tail moment and tail length were dose-dependently increased by injections of PBTA-6 at doses ranging from 1 to 50mg/kg. Significantly high values for tail moment and tail length were found in peripheral erythrocytes 3h after an i.p. injection of ADDB and persisted for up to 6h. These results show that both PBTA-6 and ADDB have genotoxic effects in goldfish.

Chemopreventive activity of compounds extracted from Casearia sylvestris (Salicaceae) Sw against DNA damage induced by particulate matter emitted by sugarcane burning near Araraquara, Brazil

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

Prieto, A.M.; Santos, A.G.; Csipak, A.R.

Ethanolic extract of Casearia sylvestris is thought to be antimutagenic. In this study, we attempted to determine whether this extract and casearin X (a clerodane diterpene from C. sylvestris) are protective against the harmful effects of airborne pollutants from sugarcane burning. To that end, we used the Tradescantia micronucleus test in meiotic pollen cells of Tradescantia pallida, the micronucleus test in mouse bone marrow cells, and the comet assay in mouse blood cells. The mutagenic compound was total suspended particulate (TSP) from air. For the Tradescantia micronucleus test, T. pallida cuttings were treated with the extract at 0.13, 0.25, ormore » 0.50 mg/ml. Subsequently, TSP was added at 0.3 mg/ml, and tetrads from the inflorescences were examined for micronuclei. For the micronucleus test in mouse bone marrow cells and the comet assay in mouse blood cells, Balb/c mice were treated for 15 days with the extract—3.9, 7.5, or 15.0 mg/kg body weight (BW)—or with casearin X—0.3, 0.25, or 1.2 mg/kg BW—after which they received TSP (3.75 mg/kg BW). In T. pallida and mouse bone marrow cells, the extract was antimutagenic at all concentrations tested. In mouse blood cells, the extract was antigenotoxic at all concentrations, whereas casearin X was not antimutagenic but was antigenotoxic at all concentrations. We conclude that C. sylvestris ethanolic extract and casearin X protect DNA from damage induced by airborne pollutants from sugarcane burning. -- Highlights: ► We assessed DNA protection of C. sylvestris ethanolic extract. ► We assessed DNA protection of casearin X. ► We used Tradescantia pallida micronucleus test as screening. ► We used comet assay and micronucleus test in mice. ► The compounds protected DNA against sugar cane burning pollutants.« less

Developmental toxicity of PAH mixtures in fish early life stages. Part I: adverse effects in rainbow trout.

PubMed

Le Bihanic, Florane; Morin, Bénédicte; Cousin, Xavier; Le Menach, Karyn; Budzinski, Hélène; Cachot, Jérôme

2014-12-01

A new gravel-contact assay using rainbow trout, Oncorhynchus mykiss, embryos was developed to assess the toxicity of polycyclic aromatic hydrocarbons (PAHs) and other hydrophobic compounds. Environmentally realistic exposure conditions were mimicked with a direct exposure of eyed rainbow trout embryos incubated onto chemical-spiked gravels until hatching at 10 °C. Several endpoints were recorded including survival, hatching delay, hatching success, biometry, developmental abnormalities, and DNA damage (comet and micronucleus assays). This bioassay was firstly tested with two model PAHs, fluoranthene and benzo[a]pyrene. Then, the method was applied to compare the toxicity of three PAH complex mixtures characterized by different PAH compositions: a pyrolytic extract from a PAH-contaminated sediment (Seine estuary, France) and two petrogenic extracts from Arabian Light and Erika oils, at two environmental concentrations, 3 and 10 μg g(-1) sum of PAHs. The degree and spectrum of toxicity were different according to the extract considered. Acute effects including embryo mortality and decreased hatching success were observed only for Erika oil extract. Arabian Light and pyrolytic extracts induced mainly sublethal effects including reduced larvae size and hemorrhages. Arabian Light and Erika extracts both induced repairable DNA damage as revealed by the comet assay versus the micronucleus assay. The concentration and proportion of methylphenanthrenes and methylanthracenes appeared to drive the toxicity of the three PAH fractions tested, featuring a toxic gradient as follows: pyrolytic < Arabian Light < Erika. The minimal concentration causing developmental defects was as low as 0.7 μg g(-1) sum of PAHs, indicating the high sensitivity of the assay and validating its use for toxicity assessment of particle-bound pollutants.

Equol as a potent radiosensitizer in estrogen receptor-positive and -negative human breast cancer cell lines.

PubMed

Taghizadeh, Bita; Ghavami, Laleh; Nikoofar, Alireza; Goliaei, Bahram

2015-07-01

Breast cancer is the most common cause of cancer death among women worldwide, and diet plays an important role in its prevention and progression. Radiotherapy has a limited but important role in the management of nearly every stage of breast cancer. We studied whether equol, the major metabolite of the soybean isoflavone daidzein, could enhance radiosensitivity in two human breast cancer cell lines (T47D and MDA-MB-231). MTT assay was used to examine equol's effect on cell viability. Sensitivity of cells to equol, radiation and a combination of both was determined by colonogenic assays. Induction of apoptosis by equol, radiation and the combination of both was also determined by acridine orange/ethidium bromide double staining fluorescence microscopy. DNA strand breaks were assessed by Comet assay. MTT assay showed that equol (0.1-350 μM) inhibited MDA-MB-231 and T47D cell growth in a time- and dose-dependent manner. Treatment of cells with equol for 72 h (MDA-MB-231) and 24 h (T47D) was found to inhibit cell growth with IC50 values of 252 μM and 228 μM, respectively. Furthermore, pretreatment of cells with 50 μM equol for 72 h (MDA-MB-231) and 24 h (T47D) sensitized the cells to irradiation. Equol was also found to enhance radiation-induced apoptosis. Comet assay results showed that the radiosensitizing effect of equol was accompanied by increased radiation-induced DNA damages. These results suggest for the first time that equol can be considered as a radiosensitizing agent and its effects may be due to increasing cell death following irradiation, increasing the remaining radiation-induced DNA damage and thus reducing the surviving fraction of irradiated cells.

Collisional quenching of OH radio emission from comet Hale-Bopp

NASA Technical Reports Server (NTRS)

Schloerb, F. P.; Devries, C. H.; Lovell, A. J.; Irvine, W. M.; Senay, M.; Wootten, H. A.; Ferris, J. P. (Principal Investigator)

1997-01-01

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

Genotoxicity of nanomaterials: DNA damage and micronuclei induced by carbon nanotubes and graphite nanofibres in human bronchial epithelial cells in vitro.

PubMed

Lindberg, Hanna K; Falck, Ghita C-M; Suhonen, Satu; Vippola, Minnamari; Vanhala, Esa; Catalán, Julia; Savolainen, Kai; Norppa, Hannu

2009-05-08

Despite the increasing industrial use of different nanomaterials, data on their genotoxicity are scant. In the present study, we examined the potential genotoxic effects of carbon nanotubes (CNTs; >50% single-walled, approximately 40% other CNTs; 1.1 nm x 0.5-100 microm; Sigma-Aldrich) and graphite nanofibres (GNFs; 95%; outer diameter 80-200 nm, inner diameter 30-50 nm, length 5-20 microm; Sigma-Aldrich) in vitro. Genotoxicity was assessed by the single cell gel electrophoresis (comet) assay and the micronucleus assay (cytokinesis-block method) in human bronchial epithelial BEAS 2B cells cultured for 24h, 48h, or 72h with various doses (1-100 microg/cm(2), corresponding to 3.8-380 microg/ml) of the carbon nanomaterials. In the comet assay, CNTs induced a dose-dependent increase in DNA damage at all treatment times, with a statistically significant effect starting at the lowest dose tested. GNFs increased DNA damage at all doses in the 24-h treatment, at two doses (40 and 100 microg/cm(2)) in the 48-h treatment (dose-dependent effect) and at four doses (lowest 10 microg/cm(2)) in the 72-h treatment. In the micronucleus assay, no increase in micronucleated cells was observed with either of the nanomaterials after the 24-h treatment or with CNTs after the 72-h treatment. The 48-h treatment caused a significant increase in micronucleated cells at three doses (lowest 10 microg/cm(2)) of CNTs and at two doses (5 and 10 microg/cm(2)) of GNFs. The 72-h treatment with GNFs increased micronucleated cells at four doses (lowest 10 microg/cm(2)). No dose-dependent effects were seen in the micronucleus assay. The presence of carbon nanomaterial on the microscopic slides disturbed the micronucleus analysis and made it impossible at levels higher than 20 microg/cm(2) of GNFs in the 24-h and 48-h treatments. In conclusion, our results suggest that both CNTs and GNFs are genotoxic in human bronchial epithelial BEAS 2B cells in vitro. This activity may be due to the fibrous nature of these carbon nanomaterials with a possible contribution by catalyst metals present in the materials-Co and Mo in CNTs (<5wt.%) and Fe (<3wt.%) in GNFs.

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.

Water production rates of recent comets (2016-2017) by SOHO/SWAN: 2P/Encke, 41P/Tuttle-Giacobini-Kresak, 45P/ Honda-Mrkos-Pajdusakova, and C/2015 ER61 (PanSTARRS)

NASA Astrophysics Data System (ADS)

Combi, Michael R.; Mäkinen, Terhi; Bertaux, Jean-Loup; Quémerais, Eric; Ferron, Stephane

2017-10-01

The all-sky hydrogen Lyman-alpha camera, SWAN (Solar Wind Anisotropies), on the SOlar and Heliospheric Observatory (SOHO) satellite makes observations of the hydrogen coma of comets. Most water molecules produced by comets are ultimately photodissociated into two H atoms and one O atom producing a huge atomic hydrogen coma that is routinely observed in the daily full-sky SWAN images in comets of sufficient brightness. Water production rates are calculated using our time-resolved model (Mäkinen & Combi, 2005, Icarus 177, 217), typically yielding about 1 observation every 2 days on the average for each comet over the brightest part of its apparition. Here we describe the progress in analysis of observations of comets observed in 2016 and 2017. These include comets 2P/Encke, 41P/Tuttle-Giacobini-Sresak, 45P/ Honda-Mrkos-Pajdusakova, and C/2015 ER61 (PanSTARRS). A status update on the entire SOHO/SWAN archive of water production rates in comets will also be given.SOHO is an international cooperative mission between ESA and NASA. Support from grants NNX15AJ81G from the NASA Solar System Observations Planetary Astronomy Program and a previous grant NNX13AQ66G from the NASA Planetary Mission Data Analysis Program are gratefully acknowledged, as is support from CNRS, CNES, and the Finnish Meteorological Institute (FMI).

A tail like no other. The RPC-MAG view of Rosetta's tail excursion at comet 67P/Churyumov-Gerasimenko

NASA Astrophysics Data System (ADS)

Volwerk, Martin; Goetz, Charlotte; Richter, Ingo; Delva, Magda; Ostaszewski, Katharina; Schwingenschuh, Konrad; Glassmeier, Karl-Heinz

2018-06-01

Context. The Rosetta Plasma Consortium (RPC) magnetometer (MAG) data during the tail excursion in March-April 2016 are used to investigate the magnetic structure of and activity in the tail region of the weakly outgassing comet 67P/Churyumov-Gerasimenko (67P). Aims: The goal of this study is to compare the large scale (near) tail structure with that of earlier missions to strong outgassing comets, and the small scale turbulent energy cascade (un)related to the singing comet phenomenon. Methods: The usual methods of space plasma physics are used to analyse the magnetometer data, such as minimum variance analysis, spectral analysis, and power law fitting. Also the cone angle and clock angle of the magnetic field are calculated to interpret the data. Results: It is found that comet 67P does not have a classical draped magnetic field and no bi-lobal tail structure at this late stage of the mission when the comet is already at 2.7 AU distance from the Sun. The main magnetic field direction seems to be more across the tail direction, which may implicate an asymmetric pick-up cloud. During periods of singing comet activity the propagation direction of the waves is at large angles with respect to the magnetic field and to the radial direction towards the comet. Turbulent cascade of magnetic energy from large to small scales is different in the presence of singing as without it.

VLA observations of the OH emission from Comet Wilson (1986) - The value of high resolution in both spatial and velocity coordinates

NASA Technical Reports Server (NTRS)

Palmer, Patrick; De Pater, Imke; Snyder, Lewis E.

1989-01-01

In comparison with Comet Halley, the radio OH emission from Comet Wilson behaved very erratically, changing rapidly in position as well as in velocity, while the emission and brightness distribution from Comet Halley displayed apparent stability. A few months later, nearer perihelion, just the opposite behavior was observed at UV wavelengths. Another difference between the two comets is that the OH emission from Comet Halley seemed confined to a region a few times 100.000 km in size, while the emission from Comet Wilson showed up in sporadic blobs, with variable intensities and velocities, at distances as far as 10 to the 6th km from the nucleus. This behavior in Comet Wilson may be associated with the disintegration of the outer frosting associated with new comets and possibly with the fragmentation and ejection of cometesimals from the nucleus. As part of the data analysis, it is demonstrated that lengthening the integration time and lowering the velocity resolution affects the symmetry of the OH images and spectral-line profiles. As a consequence, asymmetric cometary OH line profiles may be more common than previously thought.

Apoptosis after gamma irradiation. Is it an important cell death modality?

PubMed Central

Siles, E.; Villalobos, M.; Jones, L.; Guerrero, R.; Eady, J. J.; Valenzuela, M. T.; Núñez, M. I.; McMillan, T. J.; Ruiz de Almodóvar, J. M.

1998-01-01

Apoptosis and necrosis are two different forms of cell death that can be induced by cytotoxic stress, such as ionizing radiation. We have studied the importance of apoptotic death induced after treatment with 6 Gy of gamma-irradiation in a panel of eight human tumour cell lines of different radiosensitivities. Three different techniques based on the detection of DNA fragmentation have been used, a qualitative one--DNA ladder formation --and two quantitative approaches--in situ tailing and comet assay. No statistically significant relationship between the two quantitative assays was found (r= 0.327, P = 0.159) so these methods seem to show different aspects of the process of cell death. The presence of the DNA ladder related well to the end-labelling method in that the least amount of end labelling was seen in samples in which necrotic degradation rather than apoptotic ladders were seen. However, as the results obtained by the comet assay are not in agreement with the DNA ladder experiments, we suggest that the distinction between the degraded DNA produced by apoptosis and necrosis may be difficult by this technique. Finally, although apoptosis has been proposed to be dependent on p53 functionality, and this may explain differences in cellular radiosensitivity, no statistically significant relationship was found between these parameters and apoptosis in the eight cell lines studied. PMID:9862569

Both base excision repair and nucleotide excision repair in humans are influenced by nutritional factors.

PubMed

Brevik, Asgeir; Karlsen, Anette; Azqueta, Amaya; Tirado, Anna Estaban; Blomhoff, Rune; Collins, Andrew

2011-01-01

Lack of reliable assays for DNA repair has largely prevented measurements of DNA repair from being included in human biomonitoring studies. Using newly developed modifications of the comet assay we tested whether a fruit- and antioxidant-rich plant-based intervention could affect base excision repair (BER) and nucleotide excision repair (NER) in a group of 102 male volunteers. BER and NER repair capacities were measured in lymphocytes before and after a dietary intervention lasting 8 weeks. The study had one control group, one group consuming three kiwifruits per day and one group consuming a variety of antioxidant-rich fruits and plant products in addition to their normal diet. DNA strand breaks were reduced following consumption of both kiwifruits (13%, p = 0.05) and antioxidant-rich plant products (20%, p = 0.02). Increased BER (55%, p = 0.01) and reduced NER (-39%, p < 0.01) were observed in the group consuming a wide variety of plant products. Reduced NER was also observed in the kiwifruit group (-38%, p = 0.05), but BER was not affected in this group. Here we have demonstrated that DNA repair is affected by diet and that modified versions of the comet assay can be used to assess activity of different DNA repair pathways in human biomonitoring studies. Copyright © 2010 John Wiley & Sons, Ltd.

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.

Thermal structure analyses for CSM testbed (COMET)

NASA Technical Reports Server (NTRS)

Xue, David Y.; Mei, Chuh

1994-01-01

This document is the final report for the project entitled 'Thermal Structure Analyses for CSM Testbed (COMET),' for the period of May 16, 1992 - August 15, 1994. The project was focused on the investigation and development of finite element analysis capability of the computational structural mechanics (CSM) testbed (COMET) software system in the field of thermal structural responses. The stages of this project consisted of investigating present capabilities, developing new functions, analysis demonstrations, and research topics. The appendices of this report list the detailed documents of major accomplishments and demonstration runstreams for future references.

Composition/Structure/Dynamics of comet and planetary satellite atmospheres

NASA Technical Reports Server (NTRS)

Combi, Michael R. (Principal Investigator)

1995-01-01

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

Model-based engineering for laser weapons systems

NASA Astrophysics Data System (ADS)

Panthaki, Malcolm; Coy, Steve

2011-10-01

The Comet Performance Engineering Workspace is an environment that enables integrated, multidisciplinary modeling and design/simulation process automation. One of the many multi-disciplinary applications of the Comet Workspace is for the integrated Structural, Thermal, Optical Performance (STOP) analysis of complex, multi-disciplinary space systems containing Electro-Optical (EO) sensors such as those which are designed and developed by and for NASA and the Department of Defense. The CometTM software is currently able to integrate performance simulation data and processes from a wide range of 3-D CAD and analysis software programs including CODE VTM from Optical Research Associates and SigFitTM from Sigmadyne Inc. which are used to simulate the optics performance of EO sensor systems in space-borne applications. Over the past year, Comet Solutions has been working with MZA Associates of Albuquerque, NM, under a contract with the Air Force Research Laboratories. This funded effort is a "risk reduction effort", to help determine whether the combination of Comet and WaveTrainTM, a wave optics systems engineering analysis environment developed and maintained by MZA Associates and used by the Air Force Research Laboratory, will result in an effective Model-Based Engineering (MBE) environment for the analysis and design of laser weapons systems. This paper will review the results of this effort and future steps.

Planetary research at Lowell Observatory

NASA Technical Reports Server (NTRS)

Baum, William A.

1988-01-01

Scientific goals include a better determination of the basic physical characteristics of cometary nuclei, a more complete understanding of the complex processes in the comae, a survey of abundances and gas/dust ratios in a large number of comets, and measurement of primordial (12)C/(13)C and (14)N/(15)N ratios. The program also includes the observation of Pluto-Charon mutual eclipses to derive dimensions. Reduction and analysis of extensive narrowband photometry of Comet Halley from Cerro Tololo Inter-American Observatory, Perth Observatory, Lowell Observatory, and Mauna Kea Observatory were completed. It was shown that the 7.4-day periodicity in the activity of Comet Halley was present from late February through at least early June 1986, but there is no conclusive evidence of periodic variability in the preperihelion data. Greatly improved NH scalelengths and lifetimes were derived from the Halley data which lead to the conclusion that the abundance of NH in comets is much higher than previously believed. Simultaneous optical and thermal infrared observations were obtained of Comet P/Temple 2 using the MKO 2.2 m telescope and the NASA IRTF. Preliminary analysis of these observations shows that the comet's nucleus is highly elongated, very dark, and quite red.

Evidence of Some Natural Products with Antigenotoxic Effects. Part 1: Fruits and Polysaccharides

PubMed Central

Izquierdo-Vega, Jeannett Alejandra; Morales-González, José Antonio; Sánchez-Gutiérrez, Manuel; Betanzos-Cabrera, Gabriel; Sosa-Delgado, Sara M.; Sumaya-Martínez, María Teresa; Morales-González, Ángel; Paniagua-Pérez, Rogelio; Madrigal-Bujaidar, Eduardo; Madrigal-Santillán, Eduardo

2017-01-01

Cancer is one of the leading causes of deaths worldwide. The agents capable of causing damage to genetic material are known as genotoxins and, according to their mode of action, are classified into mutagens, carcinogens or teratogens. Genotoxins are involved in the pathogenesis of several chronic degenerative diseases including hepatic, neurodegenerative and cardiovascular disorders, diabetes, arthritis, cancer, chronic inflammation and ageing. In recent decades, researchers have found novel bioactive phytocompounds able to counteract the effects of physical and chemical mutagens. Several studies have shown potential antigenotoxicity in a variety of fruits. In this review (Part 1), we present an overview of research conducted on some fruits (grapefruit, cranberries, pomegranate, guava, pineapple, and mango) which are frequently consumed by humans, as well as the analysis of some phytochemicals extracted from fruits and yeasts which have demonstrated antigenotoxic capacity in various tests, including the Ames assay, sister chromatid exchange, chromosomal aberrations, micronucleus and comet assay. PMID:28157162

Cytotoxicity and genotoxicity property of hydroxyapatite-mullite eluates.

PubMed

Kalmodia, Sushma; Sharma, Vyom; Pandey, Alok K; Dhawan, Alok; Basu, Bikramjit

2011-02-01

Long-term biomedical applications of implant materials may cause osteolysis, aseptic losing and toxicity. Therefore, we investigated the cytotoxic and genotoxic potential of hydroxyapatite (HA) mullite eluates in L929 mouse fibroblast cells. The spark plasma sintered HA-20% mullite biocomposite (HA20M) were ground using mortar and pestle as well as ball milling. The cells were exposed for 6 h to varying concentrations (10, 25, 50, 75 and 100%) of the eluates of HA-20% mullite (87 nm), HA (171 nm) and mullite (154 nm). The scanning electron microscopy and MTT assay revealed the concentration dependent toxicity of H20M eluate at and above 50%. The analysis of the DNA damaging potential of HA, mullite and HA20M eluates using Comet assay demonstrated a significant DNA damage by HA20M which was largely related to the presence of mullite. The results collectively demonstrate the cytotoxic and genotoxic potential of HA20M eluate in L929 cells is dependent on particle size, concentration and composition.

Apoptogenic effects of β-sitosterol glucoside from Castanopsis indica leaves.

PubMed

Dolai, Narayan; Kumar, Ashish; Islam, Aminul; Haldar, Pallab K

2016-01-01

β-Sitosterol glucoside (BSSG) is a natural biologically active substance isolated from the Castanopsis indica leaves. This study explored the apoptogenic mechanistic studies of BSSG against Ehrlich's ascites carcinoma (EAC) treated mice through morphological study, comet assay, flow cytometry (FACS) and Western blotting assay method. AO/EB staining and FACS analysis showed that BSSG possessed apoptosis induction activities on EAC cells. Dose dependent induction of DNA damage was observed after BSSG treatment. Increase the expression of apoptotic protein p53 and p21 in EAC, multiple downstream factors contributing to apoptosis pathway. The increase of caspase-9 and caspase-3 activities revealed that caspase was a key mediator of the apoptotic pathway induced by BSSG, and up-regulation of Bax and down-regulation of anti-apoptotic protein Bcl-2 resulted in the decrease of Bcl-2/Bax ratio. Owing to the combination of significant antitumour activity by inducing apoptosis, BSSG holds the promise of being an interesting chemo-preventive agent active in cancer therapy.

In Vivo Genotoxic Evaluation of D-003, a Mixture of Very Long Chain Aliphatic Acids.

PubMed

Gámez, Rafael; González, Jorge E.; Rodeiro, Idania; Fernández, Ivonne; Alemán, Celia; Rodríguez, María D.; Acosta, Pilar C.; García, Haydee

2001-01-01

D-003 is a mixture of very long chain aliphatic acids purified from sugar cane wax with cholesterol-lowering effects. The present study was undertaken to investigate the in vivo cytotoxic and genotoxic potential of D-003 using three established assays: bone marrow micronucleus, sperm morphology, and single cell gel electrophoresis (Comet) assay. In a first experimental series, CEN/NMRI mice (6-8 animals per sex per group) were administered D-003 by gastric gavage at 5, 50, or 500 mg/kg for 90 days, then sacrificed 24 hours after the last administration. The effects on bone marrow micronucleus were evaluated only in female mice. D-003 (5-500 mg/kg) did not increase the frequency of micronucleated polychromatic erythrocytes, nor the ratio of polychromatic to normochromatic erythrocytes, compared with the controls. The assessment of the effects on sperm morphology showed that D-003 did not change the sperm count or the frequency of all types of abnormal head shapes, compared with the controls. In a second series, the micronucleus assay was performed in mice of both sexes given 2,000 mg/kg for 6 days. Likewise, in this series, neither cytotoxic nor genotoxic effects were found. Finally, five male Sprague-Dawley rats were treated with D-003 (1,250 mg/kg) by oral gavage for 90 days, and Comet assay on liver cells was performed. No single-strand breaks or alkali-labile site induction on DNA was observed. These results indicate that D-003 does not show evidence of cytotoxic or genotoxic activity on either somatic or germ cells in rodents.

A novel comprehensive evaluation platform to assess nanoparticle toxicity in vitro

NASA Astrophysics Data System (ADS)

Hirsch, C.; Kaiser, J.-P.; Wessling, F.; Fischer, K.; Roesslein, M.; Wick, P.; Krug, H. F.

2011-07-01

The amount of engineered nanomaterials (ENM) is constantly increasing. Their unique properties, compared to their bulk counterparts, render them suitable for various applications in many areas of life. Hence, nanomaterials appear in a variety of different consumer products leading to the exposure of human beings and the environment during their lifecycle. Even though results on biological effects of ENM are available, harmonized and validated test systems are still missing. One major problem concerning the reliable and robust toxicity testing arises from interactions of ENM with different assay systems. Modifications or damage to DNA can have fatal consequences, such as the formation of tumor cells and hence carcinogenesis. Therefore we focused on the re-evaluation of two genotoxicity assays concerning their nanomaterial compatibility; namely the cytokinesis-block micronucleus cytome assay (MN-assay) and the alkaline single cell gel electorphoresis assay (comet assay). We demonstrate the interference of ENM agglomerates with the read-out of both assays and discuss possibilities how to acquire relevant genotoxicity data.

Pulsewidth-dependent nature of laser-induced DNA damage in RPE cells

NASA Astrophysics Data System (ADS)

Hall, Rebecca M.; Glickman, Randolph D.; Rockwell, Benjamin A.; Kumar, Neeru; Noojin, Gary D.

2001-07-01

Ultrashort pulse laser radiation may produce cellular damage through unique mechanisms. Primary cultures of bovine retinal pigment epithelial (RPE) cells were exposed to the out put of a Ti:Sapphire laser producing 30 fs (mode-locked) pulses, 44 amplified fs pulses, or continuous wave exposures at 800 nm. Laser exposures at and below the damage threshold were studied. DNA damage was detected using single cell gel electrophoresis (comet assay). Unexposed (control) cells produced short tails with low tail moments. In contrast, all laser-exposed cells showed some degree of DNA fragmentation, but the size and shape of the resulting comets differed among the various modalities. CW-exposed cells produced generally light and relatively compact tails, suggesting fewer and larger DNA fragments, while mode-locked laser exposures (30 fs pulses) resulted in large and diffuse comets, indicating the DNA was fragmented into many very small pieces. Work is continuing to define the relationship of laser pulsewidth and intensity with the degree of DNA fragmentation. These results suggest that DNA damage may result from multiple mechanisms of laser-cell interaction, including multiphoton absorption.

Groundbased investigation of comet 67p/churyumov- gerasimenko, target of the spacecraft Mission Rosetta

NASA Astrophysics Data System (ADS)

de Almeida, A. A.; Trevisan Sanzovo, D.; Sanzovo, G. C.; Boczko, R.; Miguel Torres, R.

In this work, we make a comparative study of Comet 67P/Churyumov-Gerasimenko, target of Mission Rosetta, with Comets 1P/Halley and Hyakutake(C/1996 B2). Water and gas) release rates are derived from visual magnitudes (mv), determined mostly by amateur astronomers, and listed in several issues of International Comet Quarterly(ICQ). We make a systematic and uniform analysis of continuum fluxes obtained at visual wavelengths and, using the framework of photometric theory of Newburn & Spinrad (1985, 1989), we estimate dust release rates, qd (in g/s), effective particle sizes, a (in micron), and dust-to-gas mass ratios, for this important sample of comets. We also determine the color excess of the dust particles, CE, relative to the Sun at wavelength ranges 477.0-524.0 nm in the 1996 return of Comet 67P/Churyumov-Gerasimenko, and 365.0-484.5 nm for Comets 1P/Halley and C/1996 B2.

Reservoirs for Comets: Compositional Differences Based on Infrared Observations

NASA Astrophysics Data System (ADS)

Disanti, Michael A.; Mumma, Michael J.

Tracing measured compositions of comets to their origins continues to be of keen interest to cometary scientists and to dynamical modelers of Solar System formation and evolution. This requires building a taxonomy of comets from both present-day dynamical reservoirs: the Kuiper Belt (hereafter KB), sampled through observation of ecliptic comets (primarily Jupiter Family comets, or JFCs), and the Oort cloud (OC), represented observationally by the long-period comets and by Halley Family comets (HFCs). Because of their short orbital periods, JFCs are subjected to more frequent exposure to solar radiation compared with OC comets. The recent apparitions of the JFCs 9P/Tempel 1 and 73P/Schwassmann-Wachmann 3 permitted detailed observations of material issuing from below their surfaces—these comets added significantly to the compositional database on this dynamical class, which is under-represented in studies of cometary parent volatiles. This chapter reviews the latest techniques developed for analysis of high-resolution spectral observations from ˜2-5 μm, and compares measured abundances of native ices among comets. While no clear compositional delineation can be drawn along dynamical lines, interesting comparisons can be made. The sub-surface composition of comet 9P, as revealed by the Deep Impact ejecta, was similar to the majority of OC comets studied. Meanwhile, 73P was depleted in all native ices except HCN, similar to the disintegrated OC comet C/1999 S4 (LINEAR). These results suggest that 73P may have formed in the inner giant planets' region while 9P formed farther out or, alternatively, that both JFCs formed farther from the Sun but with 73P forming later in time.

Reservoirs for Comets: Compositional Differences Based on Infrared Observations

NASA Astrophysics Data System (ADS)

Disanti, Michael A.; Mumma, Michael J.

2008-07-01

Tracing measured compositions of comets to their origins continues to be of keen interest to cometary scientists and to dynamical modelers of Solar System formation and evolution. This requires building a taxonomy of comets from both present-day dynamical reservoirs: the Kuiper Belt (hereafter KB), sampled through observation of ecliptic comets (primarily Jupiter Family comets, or JFCs), and the Oort cloud (OC), represented observationally by the long-period comets and by Halley Family comets (HFCs). Because of their short orbital periods, JFCs are subjected to more frequent exposure to solar radiation compared with OC comets. The recent apparitions of the JFCs 9P/Tempel 1 and 73P/Schwassmann-Wachmann 3 permitted detailed observations of material issuing from below their surfaces—these comets added significantly to the compositional database on this dynamical class, which is under-represented in studies of cometary parent volatiles. This chapter reviews the latest techniques developed for analysis of high-resolution spectral observations from ˜2 5 μm, and compares measured abundances of native ices among comets. While no clear compositional delineation can be drawn along dynamical lines, interesting comparisons can be made. The sub-surface composition of comet 9P, as revealed by the Deep Impact ejecta, was similar to the majority of OC comets studied. Meanwhile, 73P was depleted in all native ices except HCN, similar to the disintegrated OC comet C/1999 S4 (LINEAR). These results suggest that 73P may have formed in the inner giant planets’ region while 9P formed farther out or, alternatively, that both JFCs formed farther from the Sun but with 73P forming later in time.

End-of-mission ROSINA/COPS measurements as a probe of the innermost coma of comet 67P/Churyumov-Gerasimenko

NASA Astrophysics Data System (ADS)

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

2017-12-01

A cometary coma is a unique phenomenon in the Solar system that represents an example of a planetary atmosphere influenced by little or no gravity. Due to the negligible gravity of a comet's nucleus, a coma has a characteristic size that exceeds that of the nucleus itself by many orders of magnitude. An extended dusty gas cloud that forms a coma is affected mainly by molecular collisions, radiative cooling, and photolytic, charge-exchange, and impact-ionization reactions. Such an environment has been extensively observed during the recent Rosetta mission, which was the first mission that escorts a comet along its way through the Solar system for an extended amount of time with the main scientific objectives of characterizing comet's nucleus, determining the surface composition, and studying the comet's activity development. The ROSINA (Rosetta Orbiter Spectrometer for Ion and Neutral Analysis) Comet Pressure Sensor (COPS) onboard the Rosetta spacecraft has performed one of the most exciting observations of the innermost coma during the spacecraft descend maneuver during the last ten hours of the mission when the random and outflow directed pressures in the coma have been measured all the way down to the comet's surface. Performed at such close proximity to the nucleus, these observations can help to characterize effects due to topological features and/or the gas local conditions at the surface of the nucleus. The major focus of the presented study is analyzing of the end-of-mission pressure measurements by the ROSINA/COPS instrument. Because the coma at a heliocentric distance of 3.8 AU was in a collisionless regime, it can be described by solving the Liouville equation, as we have done in our analysis. We have used the SHAP5 nucleus model to account for the topology of the volatile source. Spacecraft trajectory and the instrument pointing with respect to the comet's nucleus have been obtained with the SPICE library. Here, we present results of our analysis and discuss the effects of the surface topology and that of the local surface volatile injection on the distribution of gas in the innermost coma of comet 67P/Churyumov-Gerasimenko.

The Possible Interrelation of TNO and Long-Period Comets by MOID Distribution

NASA Astrophysics Data System (ADS)

Guliyev, A. S.; Nabiyev, Sh. A.; Guliyev, R. A.; Dadashov, A. S.

2015-03-01

The study objects of our work were 91 TNO with diameters greater than 200 km. On the other hand, the paper used the data for 1048 and comets with aphelion and perihelion distances Q > 30 AU and q > 0.1AU, respectively, were observed until 2012. Short-perihelion comets (sporadic and concentrated in separate groups) were excluded from the analysis. If some comet split into several parties, we have taken data for only one fragment, which is marked with the letter A. Data for comets are taken from the catalog [4] and the individual Circulars International Astronomical Union, issued in period after 2008. The data for TNO, mostly borrowed from the website [5], as well as the issues of the same circulars.

Antigenotoxic effect of acute, subacute and chronic treatments with Amazonian camu-camu (Myrciaria dubia) juice on mice blood cells.

PubMed

da Silva, Francisco Carlos; Arruda, Andrelisse; Ledel, Alexandre; Dauth, Cíntia; Romão, Nathalia Faria; Viana, Rafaele Nazário; de Barros Falcão Ferraz, Alexandre; Picada, Jaqueline Nascimento; Pereira, Patrícia

2012-07-01

Myrciaria dubia, a plant native to the Amazon region, stands out as a fruit rich in vitamin C and other metabolites with nutritional potential. We evaluated the antioxidant, genotoxic and antigenotoxic potential of M. dubia juice on blood cells of mice after acute, subacute and chronic treatments. Flavonoids and vitamin C present in the fruit of M. dubia were quantified. In vitro antioxidant activity was evaluated by DPPH assay. Blood samples were collected for analysis after treatment, and the alkaline comet assay was used to analyze the genotoxic and antigenotoxic activity (ex vivo analysis using H(2)O(2)). The amount of vitamin C per 100mL of M. dubia was 52.5mg. DPPH assay showed an antioxidant potential of the fruit. No M. dubia concentration tested exerted any genotoxic effect on mice blood cells. In the ex vivo test, the juice demonstrated antigenotoxic effect, and acute treatment produced the most significant results. After the treatments, there was no evidence of toxicity or death. In conclusion, our data show that M. dubia juice has antigenotoxic and antioxidant activities, though with no genotoxicity for blood cells. Nevertheless, more in-depth studies should be conducted to assess the safety of this fruit for human consumption. Copyright © 2012 Elsevier Ltd. All rights reserved.

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.

DNA damage protection by bulk and nano forms of quercetin in lymphocytes of patients with chronic obstructive pulmonary disease exposed to the food mutagen 2-amino-3-methylimidazo [4,5-f]quinolone (IQ).

PubMed

Habas, Khaled; Abdulmwli, Mhamoued; Demir, Eşref; Jacob, Badie K; Najafzadeh, Mojgan; Anderson, Diana

2018-05-25

Chronic obstructive pulmonary disease (COPD) in humans, describes a group of lung conditions characterised by airflow limitation that is poorly reversible. The airflow limitation usually progresses slowly and is related to an abnormal inflammatory response of the lung to toxic particles. COPD is characterised by oxidative stress and an increased risk of lung carcinoma. The 2-amino-3-methylimidazo [4,5-f]quinoline (IQ) is one of a number of mutagenic/carcinogenic heterocyclic amines found mainly in well-cooked meats which are thus part of the regular diet. Antioxidants are very important in order to protect the cells against oxidative damage. The aim of the present study was to assess the effects of IQ on the level of DNA damage and susceptibility to a potent mutagen in peripheral blood cells of COPD patients. DNA damage and the frequency of micronuclei (MNi) were evaluated using the Comet and micronucleus assays, respectively. Differential expressions of both mRNA and protein of the endogenous antioxidant enzyme catalase were evaluated with quantitative polymerase chain reaction (qPCR) and Western blot analysis, respectively. Furthermore, the effect of bulk and nano forms of quercetin and their combination with IQ were examined. Results of the present study clearly demonstrated that MNi frequency in the peripheral blood lymphocytes exhibited a positive correlation with the DNA damage as evident from the different Comet assay parameters. Increase of the endogenous antioxidant catalase also showed there was a stimulation of this enzyme system by IQ. Whereas, the endogenous antioxidant quercetin significantly reduced oxidative stress in COPD patients and healthy individuals. Copyright © 2018 Elsevier Inc. All rights reserved.

Investigating the anti-proliferative activity of styrylazanaphthalenes and azanaphthalenediones.

PubMed

Mrozek-Wilczkiewicz, Anna; Kalinowski, Danuta S; Musiol, Robert; Finster, Jacek; Szurko, Agnieszka; Serafin, Katarzyna; Knas, Magdalena; Kamalapuram, Sishir K; Kovacevic, Zaklina; Jampilek, Josef; Ratuszna, Alicja; Rzeszowska-Wolny, Joanna; Richardson, Des R; Polanski, Jaroslaw

2010-04-01

A group of styrylazanaphthalenes and azanaphthalenediones were synthesized and tested for their anti-proliferative activity. Most of the compounds were obtained with the use of microwave-assisted synthesis. The lipophilicity of the compounds was measured by RP-HPLC and their anti-proliferative activity was assayed against the human SK-N-MC neuroepithelioma and HCT116 human colon carcinoma cell lines. Active compounds were also tested in clonogenity and comet assays. Several quinazolinone and styrylquinazoline analogues were found to have markedly greater anti-proliferative activity than desferoxamine and cis-platin. Copyright 2010 Elsevier Ltd. All rights reserved.

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.

New catalogue of single-apparition comets discovered in the years 1901-1950. Part I

NASA Astrophysics Data System (ADS)

Królikowska, M.; Sitarski, G.; Pittich, E.; Szutowicz, S.; Ziołkowski, K.; Rickman, H.; Gabryszewski, R.; Rickman, B.

2014-07-01

A new catalogue of cometary orbits derived using a completely homogeneous method of data treatment, accurate methods of numerical integration, and modern model of the Solar System is presented. We constructed a sample of near-parabolic comets from the first half of the twentieth century with original reciprocals of semimajor axes less than 0.000130 au^{-1} in the Marsden and Williams Catalogue of Cometary Orbits (2008, hereafter MW08), i.e., comets of original semimajor axes larger than 7700 au. We found 38 such comets in MW08, where 32 have first-quality orbits (class 1A or 1B) and the remaining 6 have second-quality orbits (2A or 2B). We presented satisfactory non-gravitational (hereafter NG) models for thirteen of the investigated comets. The four main features, distinguishing this catalogue of orbits of single- apparition comets discovered in the early twentieth century from other catalogues of orbits of similarly old objects, are the following. 1. Old cometary positional observations require a very careful analysis. For the purpose of this new catalogue, great emphasis has been placed in collecting sets of observations as complete as possible for the investigated comets. Moreover, for many observations, comet-minus-star-type measurements were also available. This type of data was particularly valuable as the most original measurements of comet positions and has allowed us to recalculate new positions of comets using the PPM star catalogue. 2. Old cometary observations were prepared by observers usually as apparent positions in Right Ascension and Declination or as reduced positions for the epoch of the beginning of the year of a given observation. This was a huge advantage of these data, because this allows us to uniformly take into account all necessary corrections associated with the data reduction to the standard epoch. 3. The osculating orbits of single-apparition comets discovered more than sixty years ago have been formerly determined with very different numerical methods and assumptions on the model of the Solar System, including the number of planets taken into account. This new catalogue changes this situation. We offer a new catalogue of cometary orbits derived using completely homogeneous methods of data treatment, accurate methods of numerical integration, and a modern model of the Solar System. 4. The osculating, original, and future sets of orbits are presented for each catalogue comet. In the case of a comet with detectable NG effects, we give both types of orbit: purely gravitational and non- gravitational. We concluded, however, that all thirteen NG orbital solutions given in the catalogue better represent the actual motions of the investigated comets. Surprisingly, the NG effects were detectable in data for five comets of second-quality-class orbits. Among these five are three comets with hyperbolic original, barycentric GR orbits. This publication will be accompanied by an online catalogue available at ssdp.cbk.waw.pl/LPCs, providing entries to orbital elements of considered comets as well as to full swarms of original and future virtual comets that formed the basis for the further analysis of dynamical evolution.

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

PubMed

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

2014-10-10

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

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

PubMed Central

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

2014-01-01

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

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.

Effects of the anti-malarial compound cryptolepine and its analogues in human lymphocytes and sperm in the Comet assay.

PubMed

Gopalan, Rajendran C; Emerce, Esra; Wright, Colin W; Karahalil, Bensu; Karakaya, Ali E; Anderson, Diana

2011-12-15

Malaria is a mosquito-borne infectious disease caused by the genus Plasmodium. It causes one million deaths per year in African children under the age of 5 years. There is an increasing development of resistance of malarial parasites to chloroquine and other currently used anti-malarial drugs. Some plant products such as the indoloquinoline alkaloid cryptolepine have been shown to have potent activity against P. falciparum in vitro. On account of its toxicity, cryptolepine is not suitable for use as an antimalarial drug but a number of analogues of cryptolepine have been synthesised in an attempt to find compounds that have reduced cytotoxicity and these have been investigated in the present study in human sperm and lymphocytes using the Comet assay. The results suggest that cryptolepine and the analogues cause DNA damage in lymphocytes, but appear to have no effect on human sperm at the assessed doses. In the context of antimalarial drug development, the data suggest that all cryptolepine compounds and in particular 2,7-dibromocryptolepine cause DNA damage and therefore may not be suitable for pre clinical development as antimalarial agents. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Terahertz electromagnetic fields (0.106 THz) do not induce manifest genomic damage in vitro.

PubMed

Hintzsche, Henning; Jastrow, Christian; Kleine-Ostmann, Thomas; Kärst, Uwe; Schrader, Thorsten; Stopper, Helga

2012-01-01

Terahertz electromagnetic fields are non-ionizing electromagnetic fields in the frequency range from 0.1 to 10 THz. Potential applications of these electromagnetic fields include the whole body scanners, which currently apply millimeter waves just below the terahertz range, but future scanners will use higher frequencies in the terahertz range. These and other applications will bring along human exposure to these fields. Up to now, only a limited number of investigations on biological effects of terahertz electromagnetic fields have been performed. Therefore, research is strongly needed to enable reliable risk assessment.Cells were exposed for 2 h, 8 h, and 24 h with different power intensities ranging from 0.04 mW/cm(2) to 2 mW/cm(2), representing levels below, at, and above current safety limits. Genomic damage on the chromosomal level was measured as micronucleus formation. DNA strand breaks and alkali-labile sites were quantified with the comet assay. No DNA strand breaks or alkali-labile sites were observed as a consequence of exposure to terahertz electromagnetic fields in the comet assay. The fields did not cause chromosomal damage in the form of micronucleus induction.

Terahertz Electromagnetic Fields (0.106 THz) Do Not Induce Manifest Genomic Damage In Vitro

PubMed Central

Hintzsche, Henning; Jastrow, Christian; Kleine-Ostmann, Thomas; Kärst, Uwe; Schrader, Thorsten; Stopper, Helga

2012-01-01

Terahertz electromagnetic fields are non-ionizing electromagnetic fields in the frequency range from 0.1 to 10 THz. Potential applications of these electromagnetic fields include the whole body scanners, which currently apply millimeter waves just below the terahertz range, but future scanners will use higher frequencies in the terahertz range. These and other applications will bring along human exposure to these fields. Up to now, only a limited number of investigations on biological effects of terahertz electromagnetic fields have been performed. Therefore, research is strongly needed to enable reliable risk assessment. Cells were exposed for 2 h, 8 h, and 24 h with different power intensities ranging from 0.04 mW/cm2 to 2 mW/cm2, representing levels below, at, and above current safety limits. Genomic damage on the chromosomal level was measured as micronucleus formation. DNA strand breaks and alkali-labile sites were quantified with the comet assay. No DNA strand breaks or alkali-labile sites were observed as a consequence of exposure to terahertz electromagnetic fields in the comet assay. The fields did not cause chromosomal damage in the form of micronucleus induction. PMID:23029508

Genotoxic potential and heart rate disorders in the Mediterranean mussel Mytilus galloprovincialis exposed to Superdispersant-25 and dispersed diesel oil.

PubMed

Martinović, Rajko; Kolarević, Stoimir; Kračun-Kolarević, Margareta; Kostić, Jovana; Marković, Sandra; Gačić, Zoran; Kljajić, Zoran; Vuković-Gačić, Branka

2015-07-01

The effects of ex situ exposure of Mytilus galloprovincialis to Superdispersant-25 (S-25), diesel oil and dispersed diesel oil mixtures were studied by the impact on level of DNA damage in haemocytes (comet assay) and the cardiac activity patterns of mussels. Specimens were exposed for 72 h in a static system to diesel oil (100 μL/L and 1 mL/L), S-25 (5 and 50 μL/L), and dispersed diesel oil mixtures M1 (diesel oil 100 μL/L + S-25 5 μL/L) and M2 (diesel oil 1 mL/L + S-25 50 μL/L). For positive control 40 μM CdCl2 was used. The comet assay results indicated genotoxic potential of S-25 while the effects of diesel oil alone were not observed. The highest response was detected for M1 while the effects of M2 were not detected. The heart rate disorders were recorded for the diesel oil (1 mL/L), S-25 (50 μL/L) and both dispersed diesel oil mixtures. Copyright © 2015 Elsevier Ltd. All rights reserved.

Assessment of DNA damage in a group of professional dancers during a 10-month dancing season.

PubMed

Esteves, Filipa; Teixeira, Eduardo; Amorim, Tânia; Costa, Carla; Pereira, Cristiana; Fraga, Sónia; De Andrade, Vanessa Moraes; Teixeira, João Paulo; Costa, Solange

2017-01-01

Despite the numerous health benefits of physical activity, some studies reported that increased intensity and duration may induce oxidative stress in several cellular components including DNA. The aim of this study was to assess the level of basal DNA damage as well as oxidative DNA damage in a group of professional dancers before and after a 10-month dancing season. A group of individuals from general population was also assessed as a control. The alkaline version of the comet assay was the method selected to measure both basal DNA damage and oxidative stress, since this method quantifies both endpoints. In order to measure oxidative stress, the comet assay was coupled with a lesion-specific endonuclease (formamidopyrimidine glycosylase) to detect oxidized purines. The levels of oxidative DNA damage in dancers were significantly increased after the dancing season. Pre-season levels of oxidative DNA damage were lower in dancers than those obtained from the general population, suggesting an adaptation of antioxidant system in dancers. Results of the present biomonitoring study indicate the need for more effective measures to protect ballet dancers from potentially occupational health risks related to regular intensive physical exercise.

Comparative study of icy patches on comet nuclei

NASA Astrophysics Data System (ADS)

Oklay, Nilda; Pommerol, Antoine; Barucci, Maria Antonietta; Sunshine, Jessica; Sierks, Holger; Pajola, Maurizio

2016-07-01

Cometary missions Deep Impact, EPOXI and Rosetta investigated the nuclei of comets 9P/Tempel 1, 103P/Hartley 2 and 67P/Churyumov-Gerasimenko respectively. Bright patches were observed on the surfaces of each of these three comets [1-5]. Of these, the surface of 67P is mapped at the highest spatial resolution via narrow angle camera (NAC) of the Optical, Spectroscopic, and Infrared Remote Imaging System (OSIRIS, [6]) on board the Rosetta spacecraft. OSIRIS NAC is equipped with twelve filters covering the wavelength range of 250 nm to 1000 nm. Various filters combinations are used during surface mapping. With high spatial resolution data of comet 67P, three types of bright features were detected on the comet surface: Clustered, isolated and bright boulders [2]. In the visible spectral range, clustered bright features on comet 67P display bluer spectral slopes than the average surface [2, 4] while isolated bright features on comet 67P have flat spectra [4]. Icy patches observed on the surface of comets 9P and 103P display bluer spectral slopes than the average surface [1, 5]. Clustered and isolated bright features are blue in the RGB composites generated by using the images taken in NIR, visible and NUV wavelengths [2, 4]. This is valid for the icy patches observed on comets 9P and 103P [1, 5]. Spectroscopic observations of bright patches on comets 9P and 103P confirmed the existence of water [1, 5]. There were more than a hundred of bright features detected on the northern hemisphere of comet 67P [2]. Analysis of those features from both multispectral data and spectroscopic data is an ongoing work. Water ice is detected in eight of the bright features so far [7]. Additionally, spectroscopic observations of two clustered bright features on the surface of comet 67P revealed the existence of water ice [3]. The spectral properties of one of the icy patches were studied by [4] using OSIRIS NAC images and compared with the spectral properties of the active regions observed on comet 67P. Additionally jets rising from the same clustered bright feature were detected visually [4]. We analyzed bright patches on the surface of comets 9P, 103P and 67P using multispectral data obtained by the high-resolution instrument (HRI), medium- resolution instrument (MRI) and OSIRIS NAC using various spectral analysis techniques. Clustered bright features on comet 67P have similar visible spectra to the bright patches on comets 9P and 103P. The comparison of the bright patches includes the published results of the IR spectra. References: [1] Sunshine et al., 2006, Science, 311, 1453 [2] Pommerol et al., 2015, A&A, 583, A25 [3] Filacchione et al., 2016, Nature, 529, 368-372 [4] Oklay et al., 2016, A&A, 586, A80 [5] Sunshine et al. 2012, ACM [6] Keller et al., 2007, Space Sci. Rev., 128, 433 [7] Barucci et al., 2016, COSPAR, B04

Oort spike comets with large perihelion distances

NASA Astrophysics Data System (ADS)

Królikowska, Małgorzata; Dybczyński, Piotr A.

2017-12-01

The complete sample of large-perihelion nearly-parabolic comets discovered during the period 1901-2010 is studied, starting with their orbit determination. Next, an orbital evolution that includes three perihelion passages (previous-observed-next) is investigated in which a full model of Galactic perturbations and perturbations from passing stars is incorporated. We show that the distribution of planetary perturbations suffered by actual large-perihelion comets during their passage through the Solar system has a deep, unexpected minimum around zero, which indicates a lack of 'almost unperturbed' comets. Using a series of simulations we show that this deep well is moderately resistant to some diffusion of the orbital elements of the analysed comets. It seems reasonable to assert that the observed stream of these large-perihelion comets experienced a series of specific planetary configurations when passing through the planetary zone. An analysis of the past dynamics of these comets clearly shows that dynamically new comets can appear only when their original semimajor axes are greater than 20 000 au. On the other hand, dynamically old comets are completely absent for semimajor axes longer than 40 000 au. We demonstrate that the observed 1/aori-distribution exhibits a local minimum separating dynamically new from dynamically old comets. Long-term dynamical studies reveal a wide variety of orbital behaviour. Several interesting examples of the action of passing stars are also described, in particular the impact of Gliese 710, which will pass close to the Sun in the future. However, none of the obtained stellar perturbations is sufficient to change the dynamical status of the analysed comets.

Interaction of Comets and the Solar Wind

NASA Technical Reports Server (NTRS)

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

2004-01-01

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

Enhanced reactive oxygen species overexpression by CuO nanoparticles in poorly differentiated hepatocellular carcinoma cells

NASA Astrophysics Data System (ADS)

Kung, Mei-Lang; Hsieh, Shu-Ling; Wu, Chih-Chung; Chu, Tian-Huei; Lin, Yu-Chun; Yeh, Bi-Wen; Hsieh, Shuchen

2015-01-01

Copper oxide nanoparticles (CuO NPs) are known to exhibit toxic effects on a variety of cell types and organs. To determine the oxidative impact of CuO NPs on hepatocellular carcinoma (HCC) cells, well-differentiated (HepG2) and poorly differentiated (SK-Hep-1) cells were exposed to CuO NPs. Cell viability assay showed that the median inhibition concentration (IC50) for SK-Hep-1 and HepG2 cells was 25 μg ml-1 and 85 μg ml-1, respectively. Cellular fluorescence intensity using DCFH-DA staining analysis revealed significant intracellular reactive oxygen species (ROS) generation of up to 242% in SK-Hep-1 cells, compared with 86% in HepG2 cells. HPLC analysis demonstrated that a CuO NP treatment caused cellular GSH depletion of 58% and a GSH/GSSG ratio decrease to ~0.1 in SK-Hep-1 cells. The oxidative stress caused by enhanced superoxide anion production was observed in both HepG2 (146%) and SK-Hep-1 (192%) cells. The Griess assay verified that CuO NPs induced NO production (170%) in SK-Hep-1 cells. Comet assay and western blot further demonstrated that CuO NPs induced severe DNA strand breakage (70%) in SK-Hep-1 cells and caused DNA damage via increased γ-H2AX levels. These results suggest that well-differentiated HepG2 cells possess a robust antioxidant defense system against CuO NP-induced ROS stress and exhibit more tolerance to oxidative stress. Conversely, poorly differentiated SK-Hep-1 cells exhibited a deregulated antioxidant defense system that allowed accumulation of CuO NP-induced ROS and resulted in severe cytotoxicity.Copper oxide nanoparticles (CuO NPs) are known to exhibit toxic effects on a variety of cell types and organs. To determine the oxidative impact of CuO NPs on hepatocellular carcinoma (HCC) cells, well-differentiated (HepG2) and poorly differentiated (SK-Hep-1) cells were exposed to CuO NPs. Cell viability assay showed that the median inhibition concentration (IC50) for SK-Hep-1 and HepG2 cells was 25 μg ml-1 and 85 μg ml-1, respectively. Cellular fluorescence intensity using DCFH-DA staining analysis revealed significant intracellular reactive oxygen species (ROS) generation of up to 242% in SK-Hep-1 cells, compared with 86% in HepG2 cells. HPLC analysis demonstrated that a CuO NP treatment caused cellular GSH depletion of 58% and a GSH/GSSG ratio decrease to ~0.1 in SK-Hep-1 cells. The oxidative stress caused by enhanced superoxide anion production was observed in both HepG2 (146%) and SK-Hep-1 (192%) cells. The Griess assay verified that CuO NPs induced NO production (170%) in SK-Hep-1 cells. Comet assay and western blot further demonstrated that CuO NPs induced severe DNA strand breakage (70%) in SK-Hep-1 cells and caused DNA damage via increased γ-H2AX levels. These results suggest that well-differentiated HepG2 cells possess a robust antioxidant defense system against CuO NP-induced ROS stress and exhibit more tolerance to oxidative stress. Conversely, poorly differentiated SK-Hep-1 cells exhibited a deregulated antioxidant defense system that allowed accumulation of CuO NP-induced ROS and resulted in severe cytotoxicity. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr05843g

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

NASA Astrophysics Data System (ADS)

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

2015-01-01

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

COMETS2: An advanced MATLAB toolbox for the numerical analysis of electric fields generated by transcranial direct current stimulation.

PubMed

Lee, Chany; Jung, Young-Jin; Lee, Sang Jun; Im, Chang-Hwan

2017-02-01

Since there is no way to measure electric current generated by transcranial direct current stimulation (tDCS) inside the human head through in vivo experiments, numerical analysis based on the finite element method has been widely used to estimate the electric field inside the head. In 2013, we released a MATLAB toolbox named COMETS, which has been used by a number of groups and has helped researchers to gain insight into the electric field distribution during stimulation. The aim of this study was to develop an advanced MATLAB toolbox, named COMETS2, for the numerical analysis of the electric field generated by tDCS. COMETS2 can generate any sizes of rectangular pad electrodes on any positions on the scalp surface. To reduce the large computational burden when repeatedly testing multiple electrode locations and sizes, a new technique to decompose the global stiffness matrix was proposed. As examples of potential applications, we observed the effects of sizes and displacements of electrodes on the results of electric field analysis. The proposed mesh decomposition method significantly enhanced the overall computational efficiency. We implemented an automatic electrode modeler for the first time, and proposed a new technique to enhance the computational efficiency. In this paper, an efficient toolbox for tDCS analysis is introduced (freely available at http://www.cometstool.com). It is expected that COMETS2 will be a useful toolbox for researchers who want to benefit from the numerical analysis of electric fields generated by tDCS. Copyright © 2016. Published by Elsevier B.V.

Genotoxicity of air borne particulates assessed by comet and the Salmonella mutagenicity test in Jeddah, Saudi Arabia.

PubMed

Elassouli, Sufian M; Alqahtani, Mohamed H; Milaat, Waleed

2007-09-01

Fine airborne respirable particulates less than 10 micrometer (PM10) are considered one of the top environmental public health concerns, since they contain polycyclic aromatic hydrocarbons (PAHs) which are among the major carcinogenic compounds found in urban air. The objective of this study is to assess the genotoxicity of the ambient PM10 collected at 11 urban sites in Jeddah, Saudi Arabia. The PM10 extractable organic matter (EOM) was examined for its genotoxicity by the single cell gel electrophoresis (SCGE) comet assay and the Salmonella mutagenicity (Ames) test .Gas chromatography-mass spectrometry was used to quantify 16 PAH compounds in four sites. Samples from oil refinery and heavy diesel vehicles traffic sites showed significant DNA damage causing comet in 20-44% of the cells with tail moments ranging from 0.5-2.0 compared to samples from petrol driven cars and residential area, with comet in less than 2% of the cells and tail moments of < 0.02. In the Ames test, polluted sites showed indirect mutagenic response and caused 20-56 rev/ m3, mean while residential and reference sites caused 2-15 rev /m3. The genotoxicity of the EOM in both tests directly correlated with the amount of organic particulate and the PAHs concentrations in the air samples. The PAHs concentrations ranged between 0.83 ng/m3 in industrial and heavy diesel vehicles traffic sites to 0.18 ng /m3 in the residential area. Benzo(ghi)pyrene was the major PAH components and at one site it represented 65.4 % of the total PAHs. Samples of the oil refinery site were more genotoxic in the SCGE assay than samples from the heavy diesel vehicles traffic site, despite the fact that both sites contain almost similar amount of PAHs. The opposite was true for the mutagenicity in the Ames test. This could be due to the nature of the EOM in both sites. These findings confirm the genotoxic potency of the PM10 organic extracts to which urban populations are exposed.

DNA damage in haemocytes and midgut gland cells of Steatoda grossa (Theridiidae) spiders exposed to food contaminated with cadmium.

PubMed

Stalmach, Monika; Wilczek, Grażyna; Wilczek, Piotr; Skowronek, Magdalena; Mędrzak, Monika

2015-03-01

The aim of this study was to assess the genotoxic effects of Cd on haemocytes and midgut gland cells of web-building spiders, Steatoda grossa (Theridiidae), exposed to the metal under laboratory conditions. Analyzes were conducted on adult females and males, fed for four weeks with cadmium-contaminated Drosophila hydei flies, grown on a medium suplemented with 0.25 mM CdCl2. The comet assay, providing a quantitative measure of DNA strand breaks, was used to evaluate the DNA damage caused by the metal. Cadmium content was measured in whole spider bodies by the AAS method. Metal body burden was significantly lower in females (0.25 µgg(-1) dry weight) than in males (3.03 µgg(-1) dry weight), suggesting that females may have more effective mechanisms controlling the uptake of metal, via the digestive tract, or its elimination from the body. Irrespectively of sex, spiders fed prey contaminated with cadmium showed significantly higher values of comet parameters: tail DNA (TDNA), tail length (TL) and olive tail moment (OTM), in comparison with the control. In midgut gland cells, the level of DNA damage was higher for males than females, while in haemocytes the genotoxic effect of cadmium was greater in females. The obtained results indicate that in spiders cadmium displays strong genotoxic effects and may cause DNA damage even at low concentrations, however the severity of damage seems to be sex- and internal organ-dependent. The comet assay can be considered a sensitive tool for measuring the deleterious effect of cadmium on DNA integrity in spiders. Copyright © 2014 Elsevier Inc. All rights reserved.

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.

Dietary protection against free radicals: a case for multiple testing to establish structure-activity relationships for antioxidant potential of anthocyanic plant species.

PubMed

Philpott, Martin; Lim, Chiara Cheng; Ferguson, Lynnette R

2009-03-01

DNA damage by reactive species is associated with susceptibility to chronic human degenerative disorders. Anthocyanins are naturally occurring antioxidants, that may prevent or reverse such damage. There is considerable interest in anthocyanic food plants as good dietary sources, with the potential for reducing susceptibility to chronic disease. While structure-activity relationships have provided guidelines on molecular structure in relation to free hydroxyl-radical scavenging, this may not cover the situation in food plants where the anthocyanins are part of a complex mixture, and may be part of complex structures, including anthocyanic vacuolar inclusions (AVIs). Additionally, new analytical methods have revealed new structures in previously-studied materials. We have compared the antioxidant activities of extracts from six anthocyanin-rich edible plants (red cabbage, red lettuce, blueberries, pansies, purple sweetpotato skin, purple sweetpotato flesh and Maori potato flesh) using three chemical assays (DPPH, TRAP and ORAC), and the in vitro Comet assay. Extracts from the flowering plant, lisianthus, were used for comparison. The extracts showed differential effects in the chemical assays, suggesting that closely related structures have different affinities to scavenge different reactive species. Integration of anthocyanins to an AVI led to more sustained radical scavenging activity as compared with the free anthocyanin. All but the red lettuce extract could reduce endogenous DNA damage in HT-29 colon cancer cells. However, while extracts from purple sweetpotato skin and flesh, Maori potato and pansies, protected cells against subsequent challenge by hydrogen peroxide at 0 degrees C, red cabbage extracts were pro-oxidant, while other extracts had no effect. When the peroxide challenge was at 37 degrees C, all of the extracts appeared pro-oxidant. Maori potato extract, consistently the weakest antioxidant in all the chemical assays, was more effective in the Comet assays. These results highlight the dangers of generalising to potential health benefits, based solely on identification of high anthocyanic content in plants, results of a single antioxidant assay and traditional approaches to structure activity relationships. Subsequent studies might usefully consider complex mixtures and a battery of assays.

The renoprotective effects of mannitol and udenafil in renal ischemia-reperfusion injury model.

PubMed

Özlülerden, Yusuf; Toktaş, Cihan; Aybek, Hülya; Küçükatay, Vural; Şen Türk, Nilay; Zumrutbas, Ali Ersin

2017-07-01

The aim of this study was to investigate and compare the effects of udenafil and mannitol in an experimental renal ischemia-reperfusion (I/R) injury model. A total of 64 female Wister Albino rats were used. Right nephrectomy was performed in all groups. In the control group; I/R injury was not performed. In the I/R group; left renal pedicle was clamped for 45 minutes and then underwent 60 minutes and 24 hours of reperfusion. In the mannitol group; 1 mL 20% mannitol was given intravenously 15 minutes before clamping. In the udenafil group; 10-mg/kg udenafil was given orally 1 hour before clamping. Creatinine (Cr), blood urea nitrogen (BUN), Cr clearance, malondialdehyde, neutrophil gelatinase associated lipocalin (NGAL), histological examination and DNA damage (Comet Assay method) levels were compared in tissue, serum and urine samples. Udenafil had a better protective effect than mannitol according to biochemical parameters (Cr, BUN, Cr clearance, and NGAL levels) and histopathological findings when compared with the I/R group. In the Comet sampling analysis no significant difference was detected. Udenafil has a better renoprotective effect than mannitol against I/R injury and this effect supports more functional improvements. Further clinical trials are needed to demonstrate those effects and clinical utility of udenafil for that purpose in humans.

Genotoxic effects of old landfill leachate on HepG2 cells after nitration/ultrafiltration/reverse osmosis membrane treatment process.

PubMed

Cheng, Rong; Zhao, Ling; Yin, Pinghe

2017-12-01

Toxicity assessment of nitration/ultrafiltration/reverse osmosis (nitration/UF/RO) project, which has recently been widely used as an efficient process with applications in practical leachate treatment, was very limited. In the present study, DNA damage of leachates was investigated before and after the nitration/UF/RO process by a battery of assays with human hepatoma cells. Methyletrazolium assay showed a high cytotoxicity of 97.1% after being exposed to the highest concentration of raw leachate for 24 h, and a cytotoxicity of 26% in effluent at a concentration of 30% (v/v). Both comet assay (24 h) and γH2AX flow cytometer assay (3 h) showed increased levels of DNA damage in cells exposed to raw leachate and after nitration/UF-treated leachate followed by a significant increase of 7-ethoxyresorufin-O-deethylase activity. However, the effluent after nitration/UF/RO treatment showed no significant difference compared to negative control for γH2AX flow cytometer assay but slight DNA damage at concentrations of 20% and 30% (v/v) as well as increase of 7-ethoxyresorufin-O-deethylase. Analysis showed that nitration/UF/RO process exhibited high removal of physicochemical indexes and significant reduction of toxic and genotoxic effects of leachate, but still demands an improvement to reduce all possible negative risks to the environment and humans. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Silver nanoparticles: correlating nanoparticle size and cellular uptake with genotoxicity

PubMed Central

Butler, Kimberly S.; Peeler, David J.; Casey, Brendan J.; Dair, Benita J.; Elespuru, Rosalie K.

2015-01-01

The focus of this research was to develop a better understanding of the pertinent physico-chemical properties of silver nanoparticles (AgNPs) that affect genotoxicity, specifically how cellular uptake influences a genotoxic cell response. The genotoxicity of AgNPs was assessed for three potential mechanisms: mutagenicity, clastogenicity and DNA strand-break-based DNA damage. Mutagenicity (reverse mutation assay) was assessed in five bacterial strains of Salmonella typhimurium and Echerichia coli, including TA102 that is sensitive to oxidative DNA damage. AgNPs of all sizes tested (10, 20, 50 and 100nm), along with silver nitrate (AgNO3), were negative for mutagenicity in bacteria. No AgNPs could be identified within the bacteria cells using transmission electron microscopy (TEM), indicating these bacteria lack the ability to actively uptake AgNPs 10nm or larger. Clastogenicity (flow cytometry-based micronucleus assay) and intermediate DNA damage (DNA strand breaks as measured in the Comet assay) were assessed in two mammalian white blood cell lines: Jurkat Clone E6-1 and THP-1. It was observed that micronucleus and Comet assay end points were inversely correlated with AgNP size, with smaller NPs inducing a more genotoxic response. TEM results indicated that AgNPs were confined within intracellular vesicles of mammalian cells and did not penetrate the nucleus. The genotoxicity test results and the effect of AgNO3 controls suggest that silver ions may be the primary, and perhaps only, cause of genotoxicity. Furthermore, since AgNO3 was not mutagenic in the gram-negative bacterial Ames strains tested, the lack of bacterial uptake of the AgNPs may not be the major reason for the lack of genotoxicity observed. PMID:25964273

An investigation into the potential of phenothiazinium-based photo-sensitisers to act as PDT agents.

PubMed

Harris, F; Sayed, Z; Hussain, S; Phoenix, D A

2004-11-01

There is an urgent need for effective cancer treatments and increasingly, photo-dynamic therapy (PDT) is being used to fulfil this need as it offers a number of advantages over traditional cancer treatments. Here, the potential of a series of phenothiazinium-based photo-sensitisers (PhBPs) as PDT agents is tested. PhBPs were incubated with EMT-6 tumour cells and erythrocytes respectively under dark and light conditions (3.15Jcm(-2) over 30min). "Comet assay" and haemolytic assay were then used to assess cellular photo-damage induced by these PhBPs. Additionally, in vitro assays were used to determine light adsorption characteristics, singlet oxygen yields (ΦΔPhBP) and lipophilicity (logP) of these PhBPs. "Comet assay" showed EMT-6 incubation with PhBPs under light conditions to produce DNA "tails", which were circa 35μm long, indicating the presence of DNA photo-damage. Corresponding incubations under dark conditions led to no such damage. The majority of the PhBPs tested possessed significant singlet oxygen yields (ΦΔPhBP>0.7), suggesting the general use of type II mechanisms for photo-sensitization, and were generally lipophilic (logP>0). Incubation of erythrocytes with these PhBPs in the dark produced between 6% and 19% haemolysis. These levels were generally unaffected by illumination except in the case of DMMB, which showed haemolytic levels increasing from 11% to 61%. It is suggested that DNA may be the primary target for the photo-dynamic anti-tumour activity of the PhBPs tested with the exception of DMMB, which may potentially also target tumour cell membranes.

Toxicological characterization of the landfill leachate prior/after chemical and electrochemical treatment: a study on human and plant cells.

PubMed

Garaj-Vrhovac, Vera; Oreščanin, Višnja; Gajski, Goran; Gerić, Marko; Ruk, Damir; Kollar, Robert; Radić Brkanac, Sandra; Cvjetko, Petra

2013-10-01

In this research, toxicological safety of two newly developed methods for the treatment of landfill leachate from the Piškornica (Croatia) sanitary landfill was investigated. Chemical treatment procedure combined chemical precipitation with CaO followed by coagulation with ferric chloride and final adsorption by clinoptilolite. Electrochemical treatment approach included pretreatment with ozone followed by electrooxidation/electrocoagulation and final polishing by microwave irradiation. Cell viability of untreated/treated landfill leachate was examined using fluorescence microscopy. Cytotoxic effect of the original leachate was obtained for both exposure periods (4 and 24 h) while treated samples showed no cytotoxic effect even after prolonged exposure time. The potential DNA damage of the untreated/treated landfill leachate was evaluated by the comet assay and cytokinesis-block micronucleus (CBMN) assay using either human or plant cells. The original leachate exhibited significantly higher comet assay parameters compared to negative control after 24 h exposure. On the contrary, there was no significant difference between negative control and chemically/electrochemically treated leachate for any of the parameters tested. There was also no significant increase in either CBMN assay parameter compared to the negative control following the exposure of the lymphocytes to the chemically or electrochemically treated landfill leachate for both exposure periods while the original sample showed significantly higher number of micronuclei, nucleoplasmic bridges and nuclear buds for both exposure times. Results suggest that both methods are suitable for the treatment of such complex waste effluent due to high removal efficiency of all measured parameters and toxicological safety of the treated effluent. Copyright © 2013 Elsevier Ltd. All rights reserved.

Evaluation of Cytotoxicity and Genotoxicity of Acacia aroma Leaf Extracts

PubMed Central

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

2014-01-01

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

[Cytotoxicity and genotoxicity of drinking water of two networks supplied by surface water].

PubMed

Pellacani, Claudia; Branchi, Elisa; Buschini, Annamaria; Furlini, Mariangela; Poli, Paola; Rossi, Carlo

2005-01-01

Evaluation of cytotoxic and genotoxic load of drinking water in relationship to the source of supplies, the disinfection process, and the piping system. Two treatment/distribution networks of drinking water, the first (#1) located near the source, the second (#2) located near the mouth of a river supplying the plants. Water samples were collected before (F) and after (A) the disinfection process and in two points (R1 and R2) of the piping system. The samples, concentrated on C18, were tested for DNA damage in human leukocytes by the Comet assay and for gene conversion, reversion and mitochondrial mutability in Saccharomyces cerevisiae D7 strain. The approach used in this study is able to identify genotoxic compounds at low concentration and evaluate their antagonism/synergism in complex mixtures. Comet assay results show that the raw water quality depends on the sampling point, suggesting that a high input of environmental pollutants occurred during river flowing; they also show that the disinfection process can both detoxify or enhance biological activity of raw water according to its quality and that the piping systems do not affect tap water cytotoxic/genotoxic load. The yeast tests indicate the presence of some disinfection by-products effective on mitochondrial DNA. The biological assays used in this study are proven to be able to detect the presence of low concentrations of toxic/genotoxic compounds and assess the sources of their origin/production.

Association of HSP70 and genotoxic damage in lymphocytes of workers exposed to coke-oven emission

PubMed Central

Xiao, Chengfeng; Chen, Sheng; Li, Jizhao; Hai, Tao; Lu, Qiaofa; Sun, Enling; Wang, Ruibo; Tanguay, Robert M.; Wu, Tangchun

2002-01-01

Heat shock proteins (Hsps) have been reported to protect cells, tissues, and organisms against damage from a wide variety of stressful stimuli. Whether they protect against deoxyribonucleic acid (DNA) damage in individuals exposed to environmental stresses and chemical carcinogens is unknown. In the study, we investigated the association between Hsp70 levels (the most abundant mammalian Hsp) and genotoxic damage in lymphocytes of workers exposed to coke-oven emission using Western dot blot and 2 DNA damage assays, the comet assay and the micronucleus test. The data show that there is a significant increase in Hsp70 levels, DNA damage score, and micronucleus rates in lymphocytes of workers exposed to coke-oven emission as compared with the control subjects. Furthermore, there was a significant negative correlation of Hsp70 levels with DNA damage scores in the comet assay (r = −0.663, P < 0.01) and with micronucleus rates (r = −0.461, P < 0.01) in the exposed group. In the control group, there was also a light negative correlation between Hsp70 with DNA damage and micronuclei rate (r = −0.236 and r = 0.242, respectively), but it did not reach a statistically significant level (P > 0.05). Our results show that individuals who had high Hsp70 levels generally showed lower genotoxic damage than others. These results suggest a role of Hsp70 in the protection of DNA from genotoxic damage induced by coke-oven emission. PMID:12653484

Biomarkers of early genotoxicity and oxidative stress for occupational risk assessment of exposure to styrene in the fibreglass reinforced plastic industry.

PubMed

Cavallo, Delia; Tranfo, Giovanna; Ursini, Cinzia Lucia; Fresegna, Anna Maria; Ciervo, Aureliano; Maiello, Raffaele; Paci, Enrico; Pigini, Daniela; Gherardi, Monica; Gatto, Maria Pia; Buresti, Giuliana; Iavicoli, Sergio

2018-06-10

This study aimed to identify sensitive and not-invasive biomarkers of early genotoxic/oxidative effect for exposure to styrene in the fibreglass reinforced plastic manufacture. We studied 11 workers of a plastic manufacture using open molding process (A), 16 workers of a manufacture using closed process (B) and 12 controls. We evaluated geno/cytotoxic effects on buccal cells by Buccal Micronucleus Cytome (BMCyt) assay and genotoxic/oxidative effects on lymphocytes by Fpg-comet test. On A workers we also evaluated urinary 8oxoGua, 8oxodGuo and 8oxoGuo to investigate oxidative stress. Personal inhalation exposure to styrene was monitored by passive air sampling and GC/MS. Biological monitoring included urinary metabolites mandelic acid (MA) and phenylglyoxylic acid (PGA). The findings show higher styrene exposure, urinary MA + PGA levels and micronucleus frequency in manufacture A. Higher buccal karyolytic cell frequency vs controls were found in both exposed populations. We found in exposed workers, no induction of direct DNA damage but oxidative DNA damage. Fpg-comet assay and urinary oxidized guanine seem to be sensitive biomarkers of oxidative stress and BMCyt assay a good-not invasive biomarker of cyto-genotoxicity at target organ. The study, although limited by the small number of studied subjects, shows the usefulness of used biomarkers in risk assessment of styrene-exposed workers. Copyright © 2018 Elsevier B.V. All rights reserved.

Silica nanoparticles and biological dispersants: genotoxic effects on A549 lung epithelial cells

NASA Astrophysics Data System (ADS)

Brown, David M.; Varet, Julia; Johnston, Helinor; Chrystie, Alison; Stone, Vicki

2015-10-01

Silica nanoparticle exposure could be intentional (e.g. medical application or food) or accidental (e.g. occupational inhalation). On entering the body, particles become coated with specific proteins depending on the route of entry. The ability of silica particles of different size and charge (non-functionalized 50 and 200 nm and aminated 50 and 200 nm) to cause genotoxic effects in A549 lung epithelial cells was investigated. Using the modified comet assay and the micronucleus assay, we examined the effect of suspending the particles in different dispersion media [RPMI or Hanks' balanced salt solution (HBSS), supplemented with bovine serum albumin (BSA), lung lining fluid (LLF) or serum] to determine if this influenced the particle's activity. Particle characterisation suggested that the particles were reasonably well dispersed in the different media, with the exception of aminated 50 nm particles which showed evidence of agglomeration. Plain 50, 200 nm and aminated 50 nm particles caused significant genotoxic effects in the presence of formamidopyrimidine-DNA glycosylase when dispersed in HBSS or LLF. These effects were reduced when the particles were dispersed in BSA and serum. There was no significant micronucleus formation produced by any of the particles when suspended in any of the dispersants. The data suggest that silica particles can produce a significant genotoxic effect according to the comet assay in A549 cells, possibly driven by an oxidative stress-dependent mechanism which may be modified depending on the choice of dispersant employed.

Mutagenicity and genotoxicity of coal fly ash water leachate

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

Chakraborty, R.; Mukherjee, A.

2009-03-15

Fly ash is a by-product of coal-fired electricity generation plants. The prevalent practice of disposal is as slurry of ash and water to storage or ash ponds located near power stations. This has lain to waste thousands of hectares of land all over the world. Since leaching is often the cause of off-site contamination and pathway of introduction into the human environment, a study on the genotoxic effects of fly ash leachate is essential. Leachate prepared from the fly ash sample was analyzed for metal content, and tested for mutagenicity and genotoxicity. Analyses of metals show predominance of the metalsmore » - sodium, silicon, potassium, calcium, magnesium, iron, manganese, zinc, and sulphate. The Ames Salmonella mutagenicity assay, a short-term bacterial reverse mutation assay, was conducted on two-tester strains of Salmonella typhimurium strains TA97a and TA102. For genotoxicity, the alkaline version of comet assay on fly ash leachate was carried in vitro on human blood cells and in vivo on Nicotiana plants. The leachate was directly mutagenic and induced significantconcentration-dependent increases in DNA damage in whole blood cells, lymphocytes, and in Nicotiana plants. The comet parameters show increases in tail DNA percentage (%), tail length (mu m), and olive tail moment (arbitrary units). Our results indicate that leachate from fly ash dumpsites has the genotoxic potential and may lead to adverse effects on vegetation and on the health of exposed human populations.« less

In vivo genotoxicity evaluation of an artichoke (Cynara scolymus L.) aqueous extract.

PubMed

Zan, Meriele A; Ferraz, Alexandre B F; Richter, Marc F; Picada, Jaqueline N; de Andrade, Heloisa H R; Lehmann, Mauricio; Dihl, Rafael R; Nunes, Emilene; Semedo, Juliane; Da Silva, Juliana

2013-02-01

The Cynara scolymus (artichoke) is widely consumed as tea or food and shows important therapeutic properties. However, few studies have assessed the possible toxic effects of artichoke extracts. This study evaluates genotoxic and mutagenic activities of artichoke leaf aqueous extract in mice using the comet assay and the micronucleus test. Leaf extracts were given by gavage (500 mg/kg, 1000 mg/kg, and 2000 mg/kg) for 3 consecutive days. Extract composition was investigated using phytochemical screening and high-performance liquid chromatography (HPLC). In addition, antioxidant capacity was analyzed through the diphenyl-picrylhydrazyl (DPPH) and xanthine oxidase assay. Phytochemical screening detected the presence of phenolic compounds, flavonoids, and saponins. HPLC analyses indicated the presence of chlorogenic acid, caffeic acid, isoquercetrin, and rutin. Extracts showed a dose-dependent free radical scavenging effect of DPPH and an inhibitory effect of xanthine oxidase. The genotoxic results showed that leaf extracts did not increase micronuclei in peripheral blood cells. Compared to the control group, a significant increase in comet assay values was observed only in bone marrow of group treated with 2000 mg/kg, the highest dose tested, indicating that artichoke tea should be consumed with moderation. This is the first report of in vivo mutagenic and genotoxic evaluation with C. scolymus. The present study revealed leaf aqueous extract from artichoke shows lack of mutagenicity in vivo, and low genotoxicity and antioxidant activity; indicating that artichoke tea should be consumed with moderation. © 2013 Institute of Food Technologists®

Pb low doses induced genotoxicity in Lactuca sativa plants.

PubMed

Silva, S; Silva, P; Oliveira, H; Gaivão, I; Matos, M; Pinto-Carnide, O; Santos, C

2017-03-01

Soil and water contamination by lead (Pb) remains a topic of great concern, particularly regarding crop production. The admissible Pb values in irrigation water in several countries range from ≈0.1 to ≈5 mg L -1 . In order to evaluate putative effects of Pb within legal doses on crops growth, we exposed Lactuca sativa seeds and seedlings to increasing doses of Pb(NO 3 ) 2 up to 20 mg L -1 . The OECD parameter seed germination and seedling/plant growth were not affected by any of the Pb-concentrations used. However, for doses higher than 5 mg L -1 significant DNA damage was detected: Comet assay detected DNA fragmentation at ≥ 5 mg L -1 and presence of micronuclei (MN) were detected for 20 mg L -1 . Also, cell cycle impairment was observed for doses as low as 0.05 mg L -1 and 0.5 mg L -1 (mostly G 2 arrest). Our data show that for the low doses of Pb used, the OECD endpoints were not able to detect toxicity, while more sensitive endpoints (related with DNA damage and mitotic/interphase disorders) identified genotoxic and cytostatic effects. Furthermore, the nature of the genotoxic effect was dependent on the concentration. Finally, we recommend that MN test and the comet assay should be included as sensitive endpoints in (eco)toxicological assays. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Toxic effects of environmental pollutants: Comparative investigation using Allium cepa L. and Lactuca sativa L.

PubMed

Silveira, Graciele Lurdes; Lima, Maria Gabriela Franco; Reis, Gabriela Barreto Dos; Palmieri, Marcel José; Andrade-Vieria, Larissa Fonseca

2017-07-01

Studies that help understand the mechanisms of action of environmental pollutants are extremely important in environmental toxicology. In this context, assays using plants as models stand out for their simplicity and low performance cost. Among the plants used for this purpose, Allium cepa L. is the model most commonly applied for cytogenotoxic tests, while Lactuca sativa L., already widely used in phytotoxic investigations, has been gaining prominence in cytotoxic analyses. The present study aimed to compare the responses of A. cepa and L. sativa via macroscopic (root growth) and microscopic analyses (cell cycle and DNA fragmentation via TdT-mediated deoxy-uracil nick and labeling (TUNEL) and comet assays) after exposure of their roots to environmental pollutants with known cytogenotoxic mechanisms. Both species presented sensitive and efficient response to the applied tests after exposure to the DNA-alkylating agent Methyl Methanesulfonate (MMS), the heavy metal Cadmium, the aluminum industry waste Spent Potliner (SPL) and the herbicide Atrazine. However, they differed regarding the responses to the evaluated endpoints. Overall, A. cepa was more efficient in detecting clastogenic changes, arising from DNA breakage, while L. sativa rather detected aneugenic alterations, related to chromosome segregation in mitosis. In the tests applied to verify DNA fragmentation (comet and TUNEL assays), A. cepa presented higher sensitivity. In conclusion, both models are efficient to evaluate toxicological risks of environmental pollutants. Copyright © 2017 Elsevier Ltd. All rights reserved.

In vitro toxicological assessment of iron oxide, aluminium oxide and copper nanoparticles in prokaryotic and eukaryotic cell types.

PubMed

Sadiq, Rakhshinda; Khan, Qaiser Mahmood; Mobeen, Ameena; Hashmat, Amer Jamal

2015-04-01

Metallic nanoparticles (NPs) have a variety of applications in different industries including pharmaceutical industry where these NPs are used mainly for image analysis and drug delivery. The increasing interest in nanotechnology is largely associated with undefined risks to the human health and to the environment. Therefore, in the present study cytotoxic and genotoxic effects of iron oxide, aluminium oxide and copper nanoparticles were evaluated using most commonly used assays i.e. Ames assay, in vitro cytotoxicity assay, micronucleus assay and comet assay. Cytotoxicity to bacterial cells was assessed in terms of colony forming units by using Escherichia coli (gram negative) and Bacillus subtilis (gram positive). Ames assay was carried out using two bacterial strains of Salmonella typhimurium TA98 and TA100. Genotoxicity of these NPs was evaluated following exposure to monkey kidney cell line, CHS-20. No cytotoxic and genotoxic effects were observed for iron oxide, and aluminium oxide NPs. Copper NPs were found mutagenic in TA98 and in TA100 and also found cytotoxic in dose dependent manner. Copper NPs induced significant (p < 0.01) increase in number of binucleated cells with micronuclei (96.6 ± 5.40) at the highest concentration (25 µg/mL). Copper NPs also induced DNA strand breaks at 10 µg/mL and oxidative DNA damage at 5 and 10 µg/mL. We consider these findings very useful in evaluating the genotoxic potential of NPs especially because of their increasing applications in human health and environment with limited knowledge of their toxicity and genotoxicity.

Evaluation of the cytotoxic and genotoxic effects of benchmark multi-walled carbon nanotubes in relation to their physicochemical properties.

PubMed

Louro, Henriqueta; Pinhão, Mariana; Santos, Joana; Tavares, Ana; Vital, Nádia; Silva, Maria João

2016-11-16

To contribute with scientific evidence to the grouping strategy for the safety assessment of multi-walled carbon nanotubes (MWCNTs), this work describes the investigation of the cytotoxic and genotoxic effects of four benchmark MWCNTs in relation to their physicochemical characteristics, using two types of human respiratory cells. The cytotoxic effects were analysed using the clonogenic assay and replication index determination. A 48h-exposure of cells revealed that NM-401 was the only cytotoxic MWCNT in both cell lines, but after 8-days exposure, the clonogenic assay in A549 cells showed cytotoxic effects for all the tested MWCNTs. Correlation analysis suggested an association between the MWCNTs size in cell culture medium and cytotoxicity. No induction of DNA damage was observed after any MWCNTs in any cell line by the comet assay, while the micronucleus assay revealed that both NM-401 and NM-402 were genotoxic in A549 cells. NM-401 and NM-402 are the two longest MWCNTs analyzed in this work, suggesting that length may be determinant for genotoxicity. No induction of micronuclei was observed in BBEAS-2Beas-2B cell line and the different effect in both cell lines is explained in view of the size-distribution of MWCNTs in the cell culture medium, rather than cell's specificities. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

De la boule de neige sale au canard en caoutchouc noir, 67P/ Churyumov Gerasimenko, From the dirty snowball to the rubber black duck, 67P/ Churyumov Gerasimenko

NASA Astrophysics Data System (ADS)

Borg, Janet

2016-12-01

Rosetta has ended its mission on 30 September 2016 with a controlled impact in the Ma'at region on the small lobe of comet 67P/ Churyumov Gerasimenko. Since its arrival in the vicinity of the comet in august 2014, Rosetta has spent more than two years living with the comet, investigating its nucleus and its environment. It has witnessed how a comet changes as it approached perihelia and after, returned to the outer solar system. This paper is a summary of the main results obtained from the returned data that give a completely new image of what is a comet. In the future, information obtained from the analysis of more data will of course lead to more progress in a better understanding of comets, and more generally of the formation of the solar system and even of the emergence of live on Earth.