Comet assay optimization for assessment of DNA damage due to radiation exposure

International Nuclear Information System (INIS)

Dwi Ramadhani; Devita Tetriana; Viria Agesti Suvifan

2016-01-01

Comet assay can be used to measure the deoxyribonucleic acid (DNA) damage level caused by ionizing radiation exposure in peripheral blood lymphocytes. The principle of the comet assay is based on the amount of denatured DNA fragments that migrated out of the cell nucleus during electrophoresis. There are several aspects that must be concerned when doing the comet assay. For example the agarose concentration, duration of alkaline incubation, electrophoresis conditions (time, temperature, and voltage gradient), and the measurement parameters that used in analyze the comet. Percentage of DNA in the comet tail (% tail DNA) is strongly recommended as a parameter when analyze the comet because it can be converted to lesions per 106 base pairs (bp) using calibration curve that show relationship between the dose of ionizing radiation and % tail DNA. To obtain an accurate result, the calibration curve must be made and comet should be analyzing using image processing analysis software since it can be increase the precision and reduce the subjectivity of the measurement process. (author)

Variation in assessment of oxidatively damaged DNA in mononuclear blood cells by the comet assay with visual scoring

DEFF Research Database (Denmark)

Forchhammer, Lykke; Bräuner, Elvira Vaclavik; Folkmann, Janne Kjaersgaard

2008-01-01

The comet assay is popular for assessments of genotoxicity, but the comparison of results between studies is challenging because of differences in experimental procedures and reports of DNA damage in different units. We investigated the variation of DNA damage in mononuclear blood cells (MNBCs......) measured by the comet assay with focus on the variation related to alkaline unwinding and electrophoresis time, number of cells scored, as well as the putative benefits of transforming the primary end points to common units by the use of reference standards and calibration curves. Eight experienced......, our results indicate that inter-investigator difference in scoring is a strong determinant of DNA damage levels measured by the comet assay....

Factors influencing heterogeneity of radiation-induced DNA-damage measured by the alkaline comet assay

International Nuclear Information System (INIS)

Seidel, Clemens; Lautenschläger, Christine; Dunst, Jürgen; Müller, Arndt-Christian

2012-01-01

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

Factors influencing heterogeneity of radiation-induced DNA-damage measured by the alkaline comet assay

Directory of Open Access Journals (Sweden)

Seidel Clemens

2012-04-01

Full Text Available Abstract 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.

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

Science.gov (United States)

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

2016-09-01

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

In vitro and in vivo assay of radio-induced damage in Escherichia Coli, DNA labelled on thymidilic fragment

International Nuclear Information System (INIS)

Bonicel, A.

1977-01-01

A technique of rapid assay for a particular and very important damage, N-formamido (DNA), is described. Using this technique, the importance of radio-induced DNA damage can be evaluated before the repair enzymatic system takes place [fr

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

Science.gov (United States)

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

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

Science.gov (United States)

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.

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

Science.gov (United States)

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

Detection of DNA damage in mussels and sea urchins exposed to crude oil using comet assay

International Nuclear Information System (INIS)

Taban, I.C.; Bechmann, R.K.; Torgrimsen, S.; Baussant, T.; Sanni, S.

2004-01-01

The single-cell microgel electrophoresis assay or the comet assay was used to evaluate DNA damage of dispersed crude oil on sea urchins (Strongylocentrotus droebachiensis) and mussels (Mytilus edulis L.). Sea urchins were exposed to 0.06 and 0.25 mg/L dispersed crude oil in a continuous flow system, while the mussels were exposed to 0.015, 0.06 and 0.25 mg/L dispersed crude oil. Sea urchin coelomocytes and mussel haemocytes were sampled after 4 and 5 weeks exposure, respectively. In the sea urchin coelomocytes, there was a significant concentration-related increase in the percentage of DNA in comet tail. In mussel haemocytes, there was a significantly higher percentage of DNA in comet tail for all treatments compared to the control. The responses were concentration-related up to 0.06 mg/L oil. The two highest exposure concentrations of mussels were not significantly different from each other. These results indicate that the comet assay can be used for biomonitoring of DNA damage in marine invertebrates following oil contamination. (author)

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

International Nuclear Information System (INIS)

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.

2014-01-01

Highlights: • Possible genotoxic effect of accidental exposure of aquatic fauna to γ radiation. • Relative sensitivity of bivalves to γ radiation is also analyzed using comet assay. • γ radiation induced significant genetic damage in both the species of bivalves. • P. malabarica and M. casta exhibited a similar level of sensitivity to γ radiation. • Comet assay may be used as a biomarker for the environmental biomonitoring. - Abstract: 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

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

Energy Technology Data Exchange (ETDEWEB)

Praveen Kumar, M.K., E-mail: here.praveen@gmail.com [Department of Zoology, Goa University, Goa 403206 (India); Shyama, S.K., E-mail: skshyama@gmail.com [Department of Zoology, Goa University, Goa 403206 (India); Sonaye, B.S. [Department of Radiation Oncology, Goa Medical College, Goa (India); Naik, U Roshini; Kadam, S.B.; Bipin, P.D.; D’costa, A. [Department of Zoology, Goa University, Goa 403206 (India); Chaubey, R.C. [Radiation Biology and Health Science Division, Bhabha Atomic Research Centre, Mumbai (India)

2014-05-01

Highlights: • Possible genotoxic effect of accidental exposure of aquatic fauna to γ radiation. • Relative sensitivity of bivalves to γ radiation is also analyzed using comet assay. • γ radiation induced significant genetic damage in both the species of bivalves. • P. malabarica and M. casta exhibited a similar level of sensitivity to γ radiation. • Comet assay may be used as a biomarker for the environmental biomonitoring. - Abstract: 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

DNA damage and repair assessed by comet assay in workers exposed to lead in a battery recycling.

Directory of Open Access Journals (Sweden)

Mahara Valverde

2015-05-01

In addition to these findings, DNA damage determined by comet assay was sensible to reflect lead exposure levels related to specific activities inside this factory. Human biomonitoring studies through comet assay could be robust when additional biomarkers are determined at time.

Evaluation on diminishing effects of DNA damaging potential by humic substances using the bacillus subtilis rec-assay; Karekusakin rec-assay wo mochiita fuminsan ni yoru DNA sonshosei kaizen koka no kento

Energy Technology Data Exchange (ETDEWEB)

Yamamoto, H.; Takigami, H.; Shimizu, Y.; Matsui, S. [Kyoto University, Kyoto (Japan). Faculty of Engineering

1998-05-22

Antimutagenic effect of humic substances has been reported by various investigators. In this research, the diminishing effect of DNA damaging toxicity by humic acid was evaluated for the influent and effluent of activated sludge tank receiving municipal wastewater and several DNA damaging chemicals (e.g., pyrene, 1-aminopyrene and benzo(a)pyrene) using Bacillus subtilis rec-assay. The diminishing effect was not apparent for influent but observed in the effluent. Among the DNA damaging chemicals, the DNA toxicity was effectively suppressed by humic acid for pyrene and benzo(a)pyrene. However, the effect was relatively smaller for 1-aminopyrene. 19 refs., 6 figs., 6 tabs.

Development of laser-induced fluorescence detection to assay DNA damage

International Nuclear Information System (INIS)

Sharma, M.; Freund, H.G.

1991-01-01

A precolumn derivation method has been developed for high performance liquid chromatographic (HPLC) analysis of DNA damage using fluorescence detection. The modified nucleotide, having excised enzymatically from the exposed DNA, is enriched from the normal nucleotides and labeled with a fluorescent reagent. The labeling procedure involves phosphoramidation of the nucleotide with ethylenediamine (EDA) followed by conjugation of the free amino end of the phosphoramidate with 5-dimethylaminonaphthalene 1-sulfonyl chloride, commonly known as Dansyl chloride. The dansylated nucleotide can be analyzed with a sub-picomole limit of detection (LOD) by conventional HPLC using a conventional fluorescence detector. By combining microbore HPLC with laser-induced fluorescence (LIF) detection, the authors present the development of an analytical system that has sub-femtomole LOD for real-time analysis of the dansylated nucleotide. In this paper the application of the developed system in fluorescence postlabeling assay of a small alkyl-modified nucleotide (5-methyl CMP) in calf-thymus DNA is discussed

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

Science.gov (United States)

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; Pcomet assay (r=-0.73; Pcomet 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.

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

Science.gov (United States)

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 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.

Analysis of DNA damage in lizards D.raddei, from areas with different levels of soil contamination using comet assay

International Nuclear Information System (INIS)

Simonyan, A.E.; Gevorgyan, A.L.; Sargsyan, A.A.; Arakelyan, M.S.; Minasyan, S.H.

2015-01-01

The levels of DNA damage in erythrocytes of rock lizards Darevskia raddei from reserve Shikahogh and Kajaran (Republic of Armenia) and Zuar (Nagorno-Karabakh Republic), were assessed using the comet assay. Female lizards were more sensitive to environmental pollutants than males. Significant positive correlation was found between DNA damage in female lizards and content of Cu, Mo, Pb, Cd, V and As in soil

Chromatin structure influence of DNA damage measurements by four assays: pulsed- and constant-field gel electrophoresis, DNA precipitation and non-denaturing filter elution

International Nuclear Information System (INIS)

Wlodek, D.; Olive, P.L.

1996-01-01

The of elution of DNA during non-denaturing filter elution (NFE) often correlates with cell sensitivity to radiation. The elution rate is influenced by two cellular factors: chromatin structure and the number of DNA-strand breaks (DSBs) produced in an intact cell by ionizing radiation. To determine which of the above factors is relevant to cell radiosensitivity, four assays were used to measure induction of DNA damage in three cell lines varying in radiosensitivity (V79, CHO, and L5178Y-R). Each of the assays, neutral filter elution (NFE), DNA precipitation, constant (CFGE) and pulsed field gel electrophoresis (PFGE) have different physical basis for DNA damage measurement and might be differently affected by chromatin structure. Three of the methods used to measure DNA double-strand breaks gave different results: NFE was dependent on cell type and location of DNA relative to the replication fork, gel electrophoresis was independent of cell type but was affected by proximity to the replication fork, and the precipitation assay was independent of both cell type and replication status. Pulsed field gel electrophoresis produced the same results and constant field gel electrophoresis for 3 cell lines examined. Only NFE showed differences in sensitivity which correlated with cell survival following irradiation. The results suggest that three is the same initial amount of DSBs in cells from all three lines and that the sensitivity to radiation is determined by some additional factors, probably chromatin structure. (author). 18 refs, 5 figs

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

Directory of Open Access Journals (Sweden)

Adriana Díaz

2009-01-01

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

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

Science.gov (United States)

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

Immunoassay of DNA damage

International Nuclear Information System (INIS)

Gasparro, F.P.; Santella, R.M.

1988-01-01

The direct photomodification of DNA by ultraviolet light or the photo-induced addition of exogenous compounds to DNA components results in alterations of DNA structure ranging from subtle to profound. There are two consequences of these conformational changes. First, cells in which the DNA has been damaged are capable of executing repair steps. Second, the DNA which is usually of very low immunogenicity now becomes highly antigenic. This latter property has allowed the production of a series of monoclonal antibodies that recognize photo-induced DNA damage. Monoclonal antibodies have been generated that recognize the 4',5'-monoadduct and the crosslink of 8-methoxypsoralen in DNA. In addition, another antibody has been prepared which recognizes the furan-side monoadduct of 6,4,4'-trimethylangelicin in DNA. These monoclonal antibodies have been characterized as to sensitivity and specificity using non-competitive and competitive enzyme-linked-immunosorbent assays (ELISA). (author)

Immunoassay of DNA damage

Energy Technology Data Exchange (ETDEWEB)

Gasparro, F P; Santella, R M

1988-09-01

The direct photomodification of DNA by ultraviolet light or the photo-induced addition of exogenous compounds to DNA components results in alterations of DNA structure ranging from subtle to profound. There are two consequences of these conformational changes. First, cells in which the DNA has been damaged are capable of executing repair steps. Second, the DNA which is usually of very low immunogenicity now becomes highly antigenic. This latter property has allowed the production of a series of monoclonal antibodies that recognize photo-induced DNA damage. Monoclonal antibodies have been generated that recognize the 4',5'-monoadduct and the crosslink of 8-methoxypsoralen in DNA. In addition, another antibody has been prepared which recognizes the furan-side monoadduct of 6,4,4'-trimethylangelicin in DNA. These monoclonal antibodies have been characterized as to sensitivity and specificity using non-competitive and competitive enzyme-linked-immunosorbent assays (ELISA).

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

Science.gov (United States)

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.

Assessment of DNA damage by panmasala, gutkha chewing and ...

African Journals Online (AJOL)

In the present study the comet assay was performed in buccal epithelial cells to evaluate DNA damage among pan masala or gutkha chewers and smokers. The assay is a rapid, suitable and sensitive method for detecting various forms of DNA damage at individual cell level. The study comprises 300 individuals of which 50 ...

Gene polymorphisms against DNA damage induced by hydrogen peroxide in leukocytes of healthy humans through comet assay: a quasi-experimental study

Directory of Open Access Journals (Sweden)

Klautau-Guimarães Maria N

2010-05-01

Full Text Available Abstract Background Normal cellular metabolism is well established as the source of endogenous reactive oxygen species which account for the background levels of oxidative DNA damage detected in normal tissue. Hydrogen peroxide imposes an oxidative stress condition on cells that can result in DNA damage, leading to mutagenesis and cell death. Several potentially significant genetic variants related to oxidative stress have already been identified, and angiotensin I-converting enzyme (ACE inhibitors have been reported as possible antioxidant agents that can reduce vascular oxidative stress in cardiovascular events. Methods We investigate the influences of haptoglobin, manganese superoxide dismutase (MnSOD Val9Ala, catalase (CAT -21A/T, glutathione peroxidase 1 (GPx-1 Pro198Leu, ACE (I/D and gluthatione S-transferases GSTM1 and GSTT1 gene polymorphisms against DNA damage and oxidative stress. These were induced by exposing leukocytes from peripheral blood of healthy humans (N = 135 to hydrogen peroxide (H2O2, and the effects were tested by comet assay. Blood samples were submitted to genotyping and comet assay (before and after treatment with H2O2 at 250 μM and 1 mM. Results After treatment with H2O2 at 250 μM, the GPx-1 polymorphism significantly influenced results of comet assay and a possible association of the Pro/Leu genotype with higher DNA damage was found. The highest or lowest DNA damage also depended on interaction between GPX-1/ACE and Hp/GSTM1T1 polymorphisms when hydrogen peroxide treatment increased oxidative stress. Conclusions The GPx-1 polymorphism and the interactions between GPX-1/ACE and Hp/GSTM1T1 can be determining factors for DNA oxidation provoked by hydrogen peroxide, and thus for higher susceptibility to or protection against oxidative stress suffered by healthy individuals.

The alkaline comet assay as a biomarker of primary DNA damage in peripheral blood leukocytes of nuclear medicine personnel

International Nuclear Information System (INIS)

Kopjar, N.; Garaj-Vrhovac, V.

2003-01-01

The aim of this study was to assess whether occupational exposure to chronic low doses of ionizing radiation in nuclear medicine departments may lead to genotoxicity. The alkaline comet assay was selected as a bio-marker of exposure to evaluate the levels of primary DNA damage in peripheral blood leukocytes of exposed and corresponding control subjects. Statistically significant differences were found between comet tail length and tail moment values measured in leukocytes from the exposed and control groups. Within exposed group significant inter-individual differences in DNA damage were assessed, indicating different genome sensitivity. In majority of exposed subjects the levels of DNA damage were in positive correlation with the duration of occupational exposure, while the influences of age and dosimeter readings could be excluded. However, the levels of primary DNA damage detected both in control and exposed subjects were significantly influenced by smoking. The present study indicates the possibility of genotoxic risks related to occupational exposure in nuclear medicine departments. Therefore, the exposed personnel should carefully apply the radiation protection procedures to minimize, as low as possible, radiation exposure to avoid possible genotoxic effects. According to results obtained, the alkaline comet assay could be usefully applied as a sensitive additional bio-marker in the regular health screening of workers occupationally exposed to low doses of ionizing radiation. (authors)

Detection of xenobiotic-induced DNA damage by the comet assay applied to human and rat precision-cut liver slices

NARCIS (Netherlands)

Plazar, Janja; Hrejac, Irena; Pirih, Primoz; Filipic, Metka; Groothuis, Geny M. M.

The comet assay is a simple and sensitive method for measuring DNA damage at the level of individual cells and is extensively used in genotoxicity studies. It is commonly applied to cultured cells. The aim of this study was to apply the comet assay for use in fresh liver tissue, where metabolic

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

Science.gov (United States)

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

2016-01-01

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

Cellular radiosensitivity and DNA damage in primary human fibroblasts

International Nuclear Information System (INIS)

Wurm, R.; Burnet, N.G.; Duggal, N.

1994-01-01

To evaluate the relationship between radiation-induced cell survival and DNA damage in primary human fibroblasts to decide whether the initial or residual DNA damage levels are more predictive of normal tissue cellular radiosensitivity. Five primary human nonsyndromic and two primary ataxia telangiectasia fibroblast strains grown in monolayer were studied. Cell survival was assessed by clonogenic assay. Irradiation was given at high dose rate (HDR) 1-2 Gy/min. DNA damage was measured in stationary phase cells and expressed as fraction released from the well by pulsed-field gel electrophoresis (PFGE). For initial damage, cells were embedded in agarose and irradiated at HDR on ice. Residual DNA damage was measured in monolayer by allowing a 4-h repair period after HDR irradiation. Following HDR irradiation, cell survival varied between SF 2 0.025 to 0.23. Measurement of initial DNA damage demonstrated linear induction up to 30 Gy, with small differences in the slope of the dose-response curve between strains. No correlation between cell survival and initial damage was found. Residual damage increased linearly up to 80 Gy with a variation in slope by a factor of 3.2. Cell survival correlated with the slope of the dose-response curves for residual damage of the different strains (p = 0.003). The relationship between radiation-induced cell survival and DNA damage in primary human fibroblasts of differing radiosensitivity is closest with the amount of DNA damage remaining after repair. If assays of DNA damage are to be used as predictors of normal tissue response to radiation, residual DNA damage provides the most likely correlation with cell survival. 52 refs., 5 figs., 2 tabs

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

Science.gov (United States)

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

2011-09-01

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

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

Science.gov (United States)

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

2016-01-01

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

Alpha-phellandrene-induced DNA damage and affect DNA repair protein expression in WEHI-3 murine leukemia cells in vitro.

Science.gov (United States)

Lin, Jen-Jyh; Wu, Chih-Chung; Hsu, Shu-Chun; Weng, Shu-Wen; Ma, Yi-Shih; Huang, Yi-Ping; Lin, Jaung-Geng; Chung, Jing-Gung

2015-11-01

Although there are few reports regarding α-phellandrene (α-PA), a natural compound from Schinus molle L. essential oil, there is no report to show that α-PA induced DNA damage and affected DNA repair associated protein expression. Herein, we investigated the effects of α-PA on DNA damage and repair associated protein expression in murine leukemia cells. Flow cytometric assay was used to measure the effects of α-PA on total cell viability and the results indicated that α-PA induced cell death. Comet assay and 4,6-diamidino-2-phenylindole dihydrochloride staining were used for measuring DNA damage and condensation, respectively, and the results indicated that α-PA induced DNA damage and condensation in a concentration-dependent manner. DNA gel electrophoresis was used to examine the DNA damage and the results showed that α-PA induced DNA damage in WEHI-3 cells. Western blotting assay was used to measure the changes of DNA damage and repair associated protein expression and the results indicated that α-PA increased p-p53, p-H2A.X, 14-3-3-σ, and MDC1 protein expression but inhibited the protein of p53, MGMT, DNA-PK, and BRCA-1. © 2014 Wiley Periodicals, Inc.

Vitamin C for DNA damage prevention

International Nuclear Information System (INIS)

Sram, Radim J.; Binkova, Blanka; Rossner, Pavel

2012-01-01

The ability of vitamin C to affect genetic damage was reviewed in human studies that used molecular epidemiology methods, including analysis of DNA adducts, DNA strand breakage (using the Comet assay), oxidative damage measured as levels of 8-oxo-7,8-dihydroxy-2′-deoxyguanosine (8-oxodG), cytogenetic analysis of chromosomal aberrations and micronuclei, and the induction of DNA repair proteins. The protective effect of vitamin C was observed at plasma levels > 50 μmol/l. Vitamin C supplementation decreased the frequency of chromosomal aberrations in groups with insufficient dietary intake who were occupationally exposed to mutagens, and also decreased the sensitivity to mutagens as assessed using the bleomycin assay. High vitamin C levels in plasma decreased the frequency of genomic translocations in groups exposed to ionizing radiation or c-PAHs in polluted air. The frequency of micronuclei was decreased by vitamin C supplementation in smokers challenged with γ-irradiation, and higher vitamin C levels in plasma counteracted the damage induced by air pollution. The prevalence of DNA adducts inversely correlated with vitamin C levels in groups environmentally exposed to high concentrations of c-PAHs. Increased vitamin C levels decreased DNA strand breakage induced by air pollution. Oxidative damage (8-oxodG levels) was decreased by vitamin C supplementation in groups with plasma levels > 50 μmol/l exposed to PM2.5 and c-PAHs. Modulation of DNA repair by vitamin C supplementation was observed both in poorly nourished subjects and in groups with vitamin C plasma levels > 50 μmol/l exposed to higher concentrations of c-PAHs. It is possible that the impact of vitamin C on DNA damage depends both on background values of vitamin C in the individual as well as on the level of exposure to xenobiotics or oxidative stress.

Vitamin C for DNA damage prevention

Energy Technology Data Exchange (ETDEWEB)

Sram, Radim J., E-mail: sram@biomed.cas.cz [Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, 14220 Prague 4 (Czech Republic); Binkova, Blanka; Rossner, Pavel [Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, 14220 Prague 4 (Czech Republic)

2012-05-01

The ability of vitamin C to affect genetic damage was reviewed in human studies that used molecular epidemiology methods, including analysis of DNA adducts, DNA strand breakage (using the Comet assay), oxidative damage measured as levels of 8-oxo-7,8-dihydroxy-2 Prime -deoxyguanosine (8-oxodG), cytogenetic analysis of chromosomal aberrations and micronuclei, and the induction of DNA repair proteins. The protective effect of vitamin C was observed at plasma levels > 50 {mu}mol/l. Vitamin C supplementation decreased the frequency of chromosomal aberrations in groups with insufficient dietary intake who were occupationally exposed to mutagens, and also decreased the sensitivity to mutagens as assessed using the bleomycin assay. High vitamin C levels in plasma decreased the frequency of genomic translocations in groups exposed to ionizing radiation or c-PAHs in polluted air. The frequency of micronuclei was decreased by vitamin C supplementation in smokers challenged with {gamma}-irradiation, and higher vitamin C levels in plasma counteracted the damage induced by air pollution. The prevalence of DNA adducts inversely correlated with vitamin C levels in groups environmentally exposed to high concentrations of c-PAHs. Increased vitamin C levels decreased DNA strand breakage induced by air pollution. Oxidative damage (8-oxodG levels) was decreased by vitamin C supplementation in groups with plasma levels > 50 {mu}mol/l exposed to PM2.5 and c-PAHs. Modulation of DNA repair by vitamin C supplementation was observed both in poorly nourished subjects and in groups with vitamin C plasma levels > 50 {mu}mol/l exposed to higher concentrations of c-PAHs. It is possible that the impact of vitamin C on DNA damage depends both on background values of vitamin C in the individual as well as on the level of exposure to xenobiotics or oxidative stress.

Ginsenoside Rg3 induces DNA damage in human osteosarcoma cells and reduces MNNG-induced DNA damage and apoptosis in normal human cells.

Science.gov (United States)

Zhang, Yue-Hui; Li, Hai-Dong; Li, Bo; Jiang, Sheng-Dan; Jiang, Lei-Sheng

2014-02-01

Panax ginseng is a Chinese medicinal herb. Ginsenosides are the main bioactive components of P. ginseng, and ginsenoside Rg3 is the primary ginsenoside. Ginsenosides can potently kill various types of cancer cells. The present study was designed to evaluate the potential genotoxicity of ginsenoside Rg3 in human osteosarcoma cells and the protective effect of ginsenoside Rg3 with respect to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced DNA damage and apoptosis in a normal human cell line (human fibroblasts). Four human osteosarcoma cell lines (MG-63, OS732, U-2OS and HOS cells) and a normal human cell line (human fibroblasts) were employed to investigate the cytotoxicity of ginsenosides Rg3 by MTT assay. Alkaline comet assay and γH2AX focus staining were used to detect the DNA damage in MG-63 and U-2OS cells. The extent of cell apoptosis was determined by flow cytometry and a DNA ladder assay. Our results demonstrated that the cytotoxicity of ginsenoside Rg3 was dose-dependent in the human osteosarcoma cell lines, and MG-63 and U-2OS cells were the most sensitive to ginsenoside Rg3. As expected, compared to the negative control, ginsenoside Rg3 significantly increased DNA damage in a concentration-dependent manner. In agreement with the comet assay data, the percentage of γH2AX-positive MG-63 and U-2OS cells indicated that ginsenoside Rg3 induced DNA double-strand breaks in a concentration-dependent manner. The results also suggest that ginsenoside Rg3 reduces the extent of MNNG-induced DNA damage and apoptosis in human fibroblasts.

Sperm DNA damage in relation to lipid peroxidation following ...

African Journals Online (AJOL)

This study investigated the relationships between lipid peroxidation (LPO) and sperm DNA damage following freezing-thawing of boar semen in different extenders. The comet assay was used to measure the extent of sperm DNA damage in a cryoprotectant-free extender or in cryoprotectant-based extenders after single ...

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

Science.gov (United States)

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

Evaluation of irradiation in foods using DNA comet assay

International Nuclear Information System (INIS)

Khawar, Affaf; Bhatti, Ijaz Ahmad; Khan, Q.M.; Ali, T.; Khan, A.I.; Asi, M.R.

2011-01-01

Comet assay is a rapid, inexpensive and sensitive biological technique to detect DNA damage in food stuffs by irradiation. In this study the Comet assay is applied on foods of plant and animal origins. Samples were irradiated by using 60 Co gamma-radiation source. The applied doses were 2, 6 and 10 kGy for food of plant origin and 0.5, 1 and 2 kGy for meat items. The un-irradiated and irradiated samples were clearly differentiated on the basis of DNA fragmentation. During the electrophoresis study, it was found that in un-irradiated cells DNA remained intact and appeared as Comets without tail whereas in irradiated cells Comets with tails were visible due to stretching of fragmented DNA. Moreover, it was also revealed that the DNA tail length was dose dependent. Dry food stuffs (seeds) showed good results as compared to moist foods (meat, fruits and vegetables) due to the absence of background damage. (author)

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

Science.gov (United States)

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 (P0.05). 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.

A biological evaluation of DNA damage detected by comet assay in healthy populations residing in areas that differ in lung cancer incidence.

Science.gov (United States)

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.

DNA Strand Breaks in Mitotic Germ Cells of Caenorhabditis elegans Evaluated by Comet Assay

Science.gov (United States)

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

DNA damage by the cobalt (II) and zinc (II) complexes of ...

African Journals Online (AJOL)

STORAGESEVER

2008-09-03

Sep 3, 2008 ... distributed in grade 3. The results indicated that Co(II)-L induced a relatively high level of DNA damage in comparison with the level of damage induced by Zn(II)-L. Key words: Tetraazamacrocycle Zn(II) complex, tetraazamacrocycle Co(II) complex, Tetrahymena thermophila, DNA damage, the comet assay.

FIBER OPTIC BIOSENSOR FOR DNA DAMAGE

Science.gov (United States)

This paper describes a fiber optic biosensor for the rapid and sensitive detection of radiation-induced or chemically-induced oxidative DNA damage. The assay is based on the hybridization and temperature-induced dissociation (melting curves) of synthetic oligonucleotides. The...

DETECTION OF DNA DAMAGE USING A FIBEROPTIC BIOSENSOR

Science.gov (United States)

A rapid and sensitive fiber optic biosensor assay for radiation-induced DNA damage is reported. For this assay, a biotin-labeled capture oligonucleotide (38 mer) was immobilized to an avidin-coated quartz fiber. Hybridization of a dye-labeled complementary sequence was observed...

Quantitative measurement of ultraviolet-induced damage in cellular DNA by an enzyme immunodot assay

International Nuclear Information System (INIS)

Wakizaka, A.; Nishizawa, Y.; Aiba, N.; Okuhara, E.; Takahashi, S.

1989-01-01

A simple enzyme immunoassay procedure was developed for the quantitative determination of 254-nm uv-induced DNA damage in cells. With the use of specific antibodies to uv-irradiated DNA and horseradish peroxidase-conjugated antibody to rabbit IgG, the extent of damaged DNA in uv-irradiated rat spleen mononuclear cells was quantitatively measurable. Through the use of this method, the amount of damaged DNA present in 2 X 10(5) cells irradiated at a dose of 75 J/m2 was estimated to be 7 ng equivalents of the standard uv-irradiated DNA. In addition, when the cells, irradiated at 750 J/m2, were incubated for 1 h, the antigenic activity of DNA decreased by 40%, suggesting that a repair of the damaged sites in DNA had proceeded to some extent in the cells

Nucleobase-Based Barbiturates: Their Protective Effect against DNA Damage Induced by Bleomycin-Iron, Antioxidant, and Lymphocyte Transformation Assay

Directory of Open Access Journals (Sweden)

Bhaveshkumar D. Dhorajiya

2014-01-01

Full Text Available A number of nucleobase-based barbiturates have been synthesized by combination of nucleic acid bases and heterocyclic amines and barbituric acid derivatives through green and efficient multicomponent route and one pot reaction. This approach was accomplished efficiently using aqueous medium to give the corresponding products in high yield. The newly synthesized compounds were characterized by spectral analysis (FT-IR, 1H NMR, 13C NMR, HMBC, and UV spectroscopy and elemental analysis. Representative of all synthesized compounds was tested and evaluated for antioxidant, bleomycin-dependent DNA damage, and Lymphocyte Transformation studies. Compounds TBC > TBA > TBG showed highest lymphocyte transformation assay, TBC > TBA > BG showed inhibitory antioxidant activity using ABTS methods, and TBC > BPA > BAMT > TBA > 1, 3-TBA manifested the best protective effect against DNA damage induced by bleomycin.

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

Science.gov (United States)

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.

DNA damage assessment by visualization and quantification of DNA damage response

International Nuclear Information System (INIS)

Matsuda, Shun; Matsuda, Tomonari; Ikura, Tsuyoshi

2017-01-01

DNA damage response (DDR) carries out signal transduction for DNA repair, activation of cell cycle checkpoint, and apoptosis to maintain genome integrity, in response to DNA damage. Many proteins and their post-translational modifications participate in the process. Especially, S139-phosphorylated histone H2AX (γH2AX), which is formed by DNA double-strand breaks (DSBs), is an important factor to bring and retain other DDR proteins to DSB sites, Thus, γH2AX is used as a good indicator of DSBs in clinical study and pharmacology for efficacy evaluation of chemotherapy and radiotherapy, detection of precancerous regions, and others. In regulatory science, γH2AX is also a useful biomarker of genotoxicity of chemicals, since a wide range of genotoxic chemicals induce γH2AX. However, conventional detection methods of γH2AX absolutely require anti-γH2AX antibody whose staining is burdensome and time-consuming, and some of these methods are not so superior in quantitativity. In this review, we introduce two new methods to overcome these limitations, involving an easy-to-use genotoxicity assay using DDR-visualizing cells and an absolute quantification method of γH2AX using liquid chromatography-tandem mass spectrometry (LC/MS/MS). (author)

Endogenous melatonin and oxidatively damaged guanine in DNA

DEFF Research Database (Denmark)

Davanipour, Zoreh; Poulsen, Henrik E; Weimann, Allan

2009-01-01

overnight guanine DNA damage. 8-oxodG and 8-oxoGua were measured using a high-performance liquid chromatography-electrospray ionization tandem mass spectrometry assay. The mother, father, and oldest sampled daughter were used for these analyses. Comparisons between the mothers, fathers, and daughters were...... attack and increase the rate of repair of that damage. This paper reports the results of a study relating the level of overnight melatonin production to the overnight excretion of the two primary urinary metabolites of the repair of oxidatively damaged guanine in DNA. METHODS: Mother...

Evaluation through comet assay of DNA damage induced in human lymphocytes by alpha particles. Comparison with protons and Co-60 gamma rays

International Nuclear Information System (INIS)

Di Giorgio, M.; Kreiner, A. J.; Schuff, J. A.; Vallerga, M. B.; Taja, M. R.; Lopez, F. O.; Alvarez, D. E.; Saint Martin, G.; Burton, A.; Debray, M. E.; Kesque, J. M.; Somacal, H.; Stoliar, P.; Valda, A.; Davidson, J.; Davidson, M.; Ozafran, M. J.; Vazquez, M. E.

2004-01-01

Several techniques with different sensitivity to single-strand breaks and/or double strand breaks were applied to detect DNA breaks generated by high LET particles. Tests that assess DNA damage in single cells might be the appropriate tool to estimate damage induced by particles, facilitating the assessment of heterogeneity of damage in a cell population. The microgel electrophoresis (comet) assay is a sensitive method for measuring DNA damage in single cells. The objective of this work was to evaluate the proficiency of comet assay to assess the effect of high LET radiation on peripheral blood lymphocytes, compared to protons and Co-60 gamma rays. Materials and methods: Irradiations of blood samples were performed at TANDAR laboratory (Argentina). Thin samples of human peripheral blood were irradiated with different doses (0-2.5 Gy) of 20.2 MeV helium-4 particles in the track segment mode, at nearly constant LET. Data obtained were compared with the effect induced by a MeV protons and Co-60 gamma rays. Alkaline comet assay was applied. Comets were quantified by the Olive tail moment. Distribution of the helium-4 particle and protons were evaluated considering Poisson distribution in lymphocyte nuclei. The mean dose per nucleus per particle result 0.053 Gy for protons and 0.178 Gy for helium-4 particles. When cells are exposed to a dose of 0.1 Gy, the hit probability model predicts that 43% of the nuclei should have experienced and alpha traversal while with protons, 85% of the nuclei should be hit. The experimental results show a biphasic response for helium-4 particles (0.1 Gy), indicating the existence of two subpopulations: unhit and hit. Distributions of tail moment as a function of fluence and experimental dose for comets induced by helium-4 particles, protons and Co-60 gamma rays were analyzed. With helium-4 irradiations, lymphocyte nuclei show an Olive tail moment distribution flattened to higher tail moments a dose increase. However, for irradiations with

Ultraviolet radiation-mediated damage to cellular DNA

International Nuclear Information System (INIS)

Cadet, Jean; Sage, Evelyne; Douki, Thierry

2005-01-01

Emphasis is placed in this review article on recent aspects of the photochemistry of cellular DNA in which both the UVB and UVA components of solar radiation are implicated individually or synergistically. Interestingly, further mechanistic insights into the UV-induced formation of DNA photoproducts were gained from the application of new accurate and sensitive chromatographic and enzymic assays aimed at measuring base damage. Thus, each of the twelve possible dimeric photoproducts that are produced at the four main bipyrimidine sites can now be singled out as dinucleoside monophosphates that are enzymatically released from UV-irradiated DNA. This was achieved using a recently developed high-performance liquid chromatography-tandem mass spectrometry assay (HPLC-MS/MS) assay after DNA extraction and appropriate enzymic digestion. Interestingly, a similar photoproduct distribution pattern is observed in both isolated and cellular DNA upon exposure to low doses of either UVC or UVB radiation. This applies more specifically to the DNA of rodent and human cells, the cis-syn cyclobutadithymine being predominant over the two other main photolesions, namely thymine-cytosine pyrimidine (6-4) pyrimidone adduct and the related cyclobutyl dimer. UVA-irradiation was found to generate cyclobutane dimers at TT and to a lower extent at TC sites as a likely result of energy transfer mechanism involving still unknown photoexcited chromophore(s). Oxidative damage to DNA is also induced although less efficiently by UVA-mediated photosensitization processes that mostly involved 1 O 2 together with a smaller contribution of hydroxyl radical-mediated reactions through initially generated superoxide radicals

Metformin (dimethyl-biguanide induced DNA damage in mammalian cells

Directory of Open Access Journals (Sweden)

Rubem R. Amador

2012-01-01

Full Text Available Metformin (dimethyl-biguanide is an insulin-sensitizing agent that lowers fasting plasma-insulin concentration, wherefore it's wide use for patients with a variety of insulin-resistant and prediabetic states, including impaired glucose tolerance. During pregnancy it is a further resource for reducing first-trimester pregnancy loss in women with the polycystic ovary syndrome. We tested metformin genotoxicity in cells of Chinese hamster ovary, CHO-K1 (chromosome aberrations; comet assays and in mice (micronucleus assays. Concentrations of 114.4 µg/mL and 572 µg/mL were used in in vitro tests, and 95.4 mg/kg, 190.8 mg/kg and 333.9 mg/kg in assaying. Although the in vitro tests revealed no chromosome aberrations in metaphase cells, DNA damage was detected by comet assaying after 24 h of incubation at both concentrations. The frequency of DNA damage was higher at concentrations of 114.4 µg/mL. Furthermore, although mortality was not observed in in vitro tests, the highest dose of metformin suppressed bone marrow cells. However, no statistically significant differences were noted in micronuclei frequencies between treatments. In vitro results indicate that chronic metformin exposure may be potentially genotoxic. Thus, pregnant woman undergoing treatment with metformin should be properly evaluated beforehand, as regards vulnerability to DNA damage.

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

Science.gov (United States)

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...

Measurement of oxidative damage to DNA in nanomaterial exposed cells and animals

DEFF Research Database (Denmark)

Møller, Peter; Jensen, Ditte Marie; Christophersen, Daniel Vest

2015-01-01

-reactivity with other molecules in cells. This review provides an overview of efforts to reliably detect oxidatively damaged DNA and a critical assessment of the published studies on DNA damage levels. Animal studies with high baseline levels of oxidatively damaged DNA are more likely to show positive associations...... of oxidatively damaged DNA in lung tissue. Oral exposure to nanosized carbon black, TiO2 , carbon nanotubes and ZnO is associated with elevated levels of oxidatively damaged DNA in tissues. These observations are supported by cell culture studies showing concentration-dependent associations between ENM exposure...... and oxidatively damaged DNA measured by the comet assay. Cell culture studies show relatively high variation in the ability of ENMs to oxidatively damage DNA; hence, it is currently impossible to group ENMs according to their DNA damaging potential. Environ. Mol. Mutagen., 2014. © 2014 Wiley Periodicals, Inc....

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

Science.gov (United States)

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.

Assessment of evidence for nanosized titanium dioxide-generated DNA strand breaks and oxidatively damaged DNA in cells and animal models

DEFF Research Database (Denmark)

Møller, Peter; Jensen, Ditte Marie; Wils, Regitze Sølling

2017-01-01

Nanosized titanium dioxide (TiO2) has been investigated in numerous studies on genotoxicity, including comet assay endpoints and oxidatively damaged DNA in cell cultures and animal models. The results have been surprisingly mixed, which might be attributed to physico-chemical differences...... culture studies also demonstrate increased levels of oxidatively damaged DNA after exposure to TiO2. There are relatively few studies on animal models where DNA strand breaks and oxidatively damaged DNA have been tested with reliable methods. Collectively, this review shows that exposure to nanosized TiO2...... of the tested TiO2. In the present review, we assess the role of certain methodological issues and publication bias. The analysis shows that studies on DNA strand breaks without proper assay controls or very low intra-group variation tend to show statistically significant effects. Levels of oxidatively damaged...

Single Molecule Scanning of DNA Radiation Oxidative Damage, Phase I

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National Aeronautics and Space Administration — This proposal will develop an assay to map genomic DNA, at the single molecule level and in a nanodevice, for oxidative DNA damage arising from radiation exposure;...

Kaempferol induces DNA damage and inhibits DNA repair associated protein expressions in human promyelocytic leukemia HL-60 cells.

Science.gov (United States)

Wu, Lung-Yuan; Lu, Hsu-Feng; Chou, Yu-Cheng; Shih, Yung-Luen; Bau, Da-Tian; Chen, Jaw-Chyun; Hsu, Shu-Chun; Chung, Jing-Gung

2015-01-01

Numerous evidences have shown that plant flavonoids (naturally occurring substances) have been reported to have chemopreventive activities and protect against experimental carcinogenesis. Kaempferol, one of the flavonoids, is widely distributed in fruits and vegetables, and may have cancer chemopreventive properties. However, the precise underlying mechanism regarding induced DNA damage and suppressed DNA repair system are poorly understood. In this study, we investigated whether kaempferol induced DNA damage and affected DNA repair associated protein expression in human leukemia HL-60 cells in vitro. Percentages of viable cells were measured via a flow cytometry assay. DNA damage was examined by Comet assay and DAPI staining. DNA fragmentation (ladder) was examined by DNA gel electrophoresis. The changes of protein levels associated with DNA repair were examined by Western blotting. Results showed that kaempferol dose-dependently decreased the viable cells. Comet assay indicated that kaempferol induced DNA damage (Comet tail) in a dose-dependent manner and DAPI staining also showed increased doses of kaempferol which led to increased DNA condensation, these effects are all of dose-dependent manners. Western blotting indicated that kaempferol-decreased protein expression associated with DNA repair system, such as phosphate-ataxia-telangiectasia mutated (p-ATM), phosphate-ataxia-telangiectasia and Rad3-related (p-ATR), 14-3-3 proteins sigma (14-3-3σ), DNA-dependent serine/threonine protein kinase (DNA-PK), O(6)-methylguanine-DNA methyltransferase (MGMT), p53 and MDC1 protein expressions, but increased the protein expression of p-p53 and p-H2AX. Protein translocation was examined by confocal laser microscopy, and we found that kaempferol increased the levels of p-H2AX and p-p53 in HL-60 cells. Taken together, in the present study, we found that kaempferol induced DNA damage and suppressed DNA repair and inhibited DNA repair associated protein expression in HL-60

Biologically important radiation damage in DNA

International Nuclear Information System (INIS)

Ward, J.F.

1994-01-01

Most DNA damage by the hydroxyl radical is confined to the bases, and this base damage represents an important component of locally multiply demanded sites (LMOS). The yields of the major damaged bases have been determined by gas chromatography mass spectrometry. For our propose, it was necessary to convert a known fraction of these damaged bases to strand breaks and then assay these labile sites as the increase in strand break yield over the normally observed level. Three potential agents by which this strategy of conversion of base damage to strand break could be implemented were identified in the original application: 1, Sl nuclease; 2, piperidine; and 3, base damage specific enzymes

A non-isotopic assay uses bromouridine and RNA synthesis to detect DNA damage responses.

Science.gov (United States)

Hasegawa, Mayu; Iwai, Shigenori; Kuraoka, Isao

2010-06-17

Individuals with inherited xeroderma pigmentosum (XP) disorder and Cockayne syndrome (CS) are deficient in nucleotide excision repair and experience hypersensitivity to sunlight. Although there are several diagnostic assays for these disorders, the recovery of RNA synthesis (RRS) assay that can discriminate between XP cells and CS cells is very laborious. Here, we report on a novel non-radioisotope RRS assay that uses bromouridine (a uridine analog) as an alternative to (3)H-uridine. This assay can easily detect RNA polymerase I transcription in nucleoli and RNA polymerase II transcription in nuclei. The non-RI RSS assay also can rapidly detect normal RRS activity in HeLa cells. Thus, this assay is useful as a novel and easy technique for CS diagnosis. Because RRS is thought to be related to transcription-coupled DNA repair, which is triggered by the blockage of transcriptional machinery by DNA lesions, this assay may be of use for analysis of DNA repair, transcription, and/or genetic toxicity. Copyright 2010 Elsevier B.V. All rights reserved.

DNA damage and repair in age-related macular degeneration

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Szaflik, Jacek P. [Department of Ophthalmology, Medical University of Warsaw and Samodzielny Publiczny Szpital Okulistyczny, Sierakowskiego 13, 03-710 Warsaw (Poland); Janik-Papis, Katarzyna; Synowiec, Ewelina; Ksiazek, Dominika [Department of Molecular Genetics, University of Lodz, Banacha 12/16, 90-237 Lodz (Poland); Zaras, Magdalena [Department of Ophthalmology, Medical University of Warsaw and Samodzielny Publiczny Szpital Okulistyczny, Sierakowskiego 13, 03-710 Warsaw (Poland); Wozniak, Katarzyna [Department of Molecular Genetics, University of Lodz, Banacha 12/16, 90-237 Lodz (Poland); Szaflik, Jerzy [Department of Ophthalmology, Medical University of Warsaw and Samodzielny Publiczny Szpital Okulistyczny, Sierakowskiego 13, 03-710 Warsaw (Poland); Blasiak, Janusz, E-mail: januszb@biol.uni.lodz.pl [Department of Molecular Genetics, University of Lodz, Banacha 12/16, 90-237 Lodz (Poland)

2009-10-02

Age-related macular degeneration (AMD) is a retinal degenerative disease that is the main cause of vision loss in individuals over the age of 55 in the Western world. Clinically relevant AMD results from damage to the retinal pigment epithelial (RPE) cells thought to be mainly caused by oxidative stress. The stress also affects the DNA of RPE cells, which promotes genome instability in these cells. These effects may coincide with the decrease in the efficacy of DNA repair with age. Therefore individuals with DNA repair impaired more than average for a given age may be more susceptible to AMD if oxidative stress affects their RPE cells. This may be helpful in AMD risk assessment. In the present work we determined the level of basal (measured in the alkaline comet assay) endogenous and endogenous oxidative DNA damage, the susceptibility to exogenous mutagens and the efficacy of DNA repair in lymphocytes of 100 AMD patients and 110 age-matched individuals without visual disturbances. The cells taken from AMD patients displayed a higher extent of basal endogenous DNA damage without differences between patients of dry and wet forms of the disease. DNA double-strand breaks did not contribute to the observed DNA damage as checked by the neutral comet assay and pulsed field gel electrophoresis. The extent of oxidative modification to DNA bases was grater in AMD patients than in the controls, as probed by DNA repair enzymes NTH1 and Fpg. Lymphocytes from AMD patients displayed a higher sensitivity to hydrogen peroxide and UV radiation and repaired lesions induced by these factors less effectively than the cells from the control individuals. We postulate that the impaired efficacy of DNA repair may combine with enhanced sensitivity of RPE cells to blue and UV lights, contributing to the pathogenesis of AMD.

DNA damage and repair in age-related macular degeneration

International Nuclear Information System (INIS)

Szaflik, Jacek P.; Janik-Papis, Katarzyna; Synowiec, Ewelina; Ksiazek, Dominika; Zaras, Magdalena; Wozniak, Katarzyna; Szaflik, Jerzy; Blasiak, Janusz

2009-01-01

Age-related macular degeneration (AMD) is a retinal degenerative disease that is the main cause of vision loss in individuals over the age of 55 in the Western world. Clinically relevant AMD results from damage to the retinal pigment epithelial (RPE) cells thought to be mainly caused by oxidative stress. The stress also affects the DNA of RPE cells, which promotes genome instability in these cells. These effects may coincide with the decrease in the efficacy of DNA repair with age. Therefore individuals with DNA repair impaired more than average for a given age may be more susceptible to AMD if oxidative stress affects their RPE cells. This may be helpful in AMD risk assessment. In the present work we determined the level of basal (measured in the alkaline comet assay) endogenous and endogenous oxidative DNA damage, the susceptibility to exogenous mutagens and the efficacy of DNA repair in lymphocytes of 100 AMD patients and 110 age-matched individuals without visual disturbances. The cells taken from AMD patients displayed a higher extent of basal endogenous DNA damage without differences between patients of dry and wet forms of the disease. DNA double-strand breaks did not contribute to the observed DNA damage as checked by the neutral comet assay and pulsed field gel electrophoresis. The extent of oxidative modification to DNA bases was grater in AMD patients than in the controls, as probed by DNA repair enzymes NTH1 and Fpg. Lymphocytes from AMD patients displayed a higher sensitivity to hydrogen peroxide and UV radiation and repaired lesions induced by these factors less effectively than the cells from the control individuals. We postulate that the impaired efficacy of DNA repair may combine with enhanced sensitivity of RPE cells to blue and UV lights, contributing to the pathogenesis of AMD.

A damage-responsive DNA binding protein regulates transcription of the yeast DNA repair gene PHR1

International Nuclear Information System (INIS)

Sebastian, J.; Sancar, G.B.

1991-01-01

The PHR1 gene of Saccharomyces cerevisiae encodes the DNA repair enzyme photolyase. Transcription of PHR1 increases in response to treatment of cells with 254-nm radiation and chemical agents that damage DNA. The authors here the identification of a damage-responsive DNA binding protein, termed photolyase regulatory protein (PRP), and its cognate binding site, termed the PHR1 transcription after DNA damage. PRP activity, monitored by electrophoretic-mobility-shift assay, was detected in cells during normal growth but disappeared within 30 min after irradiation. Copper-phenanthroline footprinting of PRP-DNA complexes revealed that PRP protects a 39-base-pair region of PHR1 5' flanking sequence beginning 40 base pairs upstream from the coding sequence. Thus these observations establish that PRP is a damage-responsive repressor of PHR1 transcription

The Assessment of Primary DNA Damage in Medical Personnel Occupationally Exposed to Ionizing Radiation

International Nuclear Information System (INIS)

Kopjar, N.; Garaj-Vrhovac, V.

2003-01-01

In physico-chemical interaction with cellular DNA ionizing radiation produces a variety of primary lesions, such as single-strand breaks (SSB), alkali-labile sites, double-strand breaks (DSB), DNA-DNA and DNA-protein crosslinks, and damage to purine and pyrimidine bases. The effects of low-level exposure to ionising radiation are of concern to large number of people, including workers receiving radiation exposure on the job. It is very important to estimate absorbed doses from individuals occupationally exposed to ionising radiation for carrying out radioprotection procedures and restrict the hazards to human health. A wide range of methods is presently used for the detection of early biological effects of DNA-damaging agents in environmental and occupational settings. Currently, unstable chromosomal aberrations in peripheral blood lymphocytes, in particularly dicentrics, are the most fully developed biological indicators of ionizing radiation exposure. This methodology usually complements data obtained by physical dosimetry. As a routine, it is used whenever the individual dosimeter shows an exposure to penetrating radiation above its limit of detection. One of the advantages of cytogenetic dosimetry is that this biological dosimeter can be assessed at any moment whereas physical dosimeters are not always present in the subject. During the last years, the single cell gel electrophoresis (SCGE) or comet assay has gained widespread acceptance for genotoxicity testing. In molecular epidemiology studies DNA damage evaluated by the comet assay is utilized as a biomarker of exposure. The comet assay permits the detection of primary DNA damage and the study of repair kinetics at the level of single cells. The aim of the present study was to assess and quantificate the levels of DNA damage in peripheral blood leukocytes of medical workers occupationally exposed to ionizing radiation and corresponding unexposed control subjects. As a sensitive biomarker of exposure the

Protective effects of the exopolysaccharide Lasiodiplodan against DNA damage and inflammation induced by doxorubicin in rats: Cytogenetic and gene expression assays

International Nuclear Information System (INIS)

Mello, M.B.; Machado, C.S.; Ribeiro, D.L.; Aissa, A.F.; Burim, R.V.; Alves da Cunha, M.A.; Barcelos, G.R.M.

2017-01-01

The lasiodiplodan (LS) is a β-(1 → 6)-D-glucan produced by the fungus Lasiodiplodia theobromae and some of the biological activities of LS were reported as hypoglycemic, anticoagulant, anti-proliferative and anticancer action; however, its effects on DNA instability and modulation of gene expression are still unclear. Aims of study were investigate the genotoxic effects of lasiodiplodan, and its protective activity against DNA damage induced by doxorubicin (DXR) and its impact on the expression of genes associated with DNA damage and inflammatory response pathways. Therefore, Wistar rats were treated (15 days) orally with LS (5.0; 10 and 20 mg/kg bw) alone and in combination with DXR (15 mg/kg bw; administrated intraperitoneally on 14th day) as well as their respective controls: distilled water and DXR. Monitoring of DNA damage was assessed by comet and micronucleus (MN) assays and gene expression was evaluated by PCR-Arrays. Treatments with LS alone did not induce disturbances on DNA; when LS was given in combination with DXR, comet and MN formations were reduced to those found in the respective controls. Moreover, LS was able to reduce the disturbances on gene expressions induced by DXR treatment, since the animals that receive LS associated with DXR showed no alteration in the expression of genes related to DNA damage response. Also, DXR induced several up- and down-regulation of several genes associated to inflammatory process, while the animals that received LS + DXR had their gene expression patterns similar to those found in the control group. In conclusion, our results showed that LS did not induce disturbances on DNA stability and significantly reduce the DNA damage and inflammation caused by DXR exposure. In addition, we give further information concerning the molecular mechanisms associated to LS protective effects which seems to be a promising nutraceutical with chemopreventive potential.

Ultraviolet and chemical induced DNA repair in human cells assayed by bromodeoxyuridine photolysis or cytosine arabinoside arrest

International Nuclear Information System (INIS)

Regan, J.D.; Dunn, W.C.

1979-01-01

The bromodeoxyuridine photolysis assay of DNA damage in human cells permits an estimate of both the number of repaired regions in the DNA and the size of the average repaired region - the patch size. The antineoplastic agent arabinofuranosyl cytosine (ara-C) can also be employed to assay the magnitude of repair since this agent appears to block rejoining of single-strand incisions made in the DNA during the initial step of repair. Thus, the number of incisions can be accumulated. The ara-C effect is dependent on the presence of hydroxyurea. Both assays can be employed for the study of physical or chemical DNA damages. Results comparing these assays are presented

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

International Nuclear Information System (INIS)

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

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 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. 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

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

Energy Technology Data Exchange (ETDEWEB)

Franken, Carmen, E-mail: carmen.franken@vito.be [Flemish Institute for Technological Research (VITO), Mol (Belgium); Department of Biomedical Sciences, University of Antwerp, Antwerp (Belgium); Koppen, Gudrun; Lambrechts, Nathalie; Govarts, Eva [Flemish Institute for Technological Research (VITO), Mol (Belgium); Bruckers, Liesbeth [Interuniversity Institute for Biostatistics and Statistical Bioinformatics, Hasselt University, Hasselt (Belgium); Den Hond, Elly [Flemish Institute for Technological Research (VITO), Mol (Belgium); Loots, Ilse [Political and Social Sciences, University of Antwerp, Antwerp (Belgium); Nelen, Vera [Provincial Institute for Hygiene, Antwerp (Belgium); Sioen, Isabelle [Department of Public Health, Ghent University, Ghent (Belgium); Department of Food Safety and Food Quality, Ghent University, Ghent (Belgium); Nawrot, Tim S. [Centre for Environmental Sciences, Hasselt University, Diepenbeek (Belgium); Department of Public Health & Primary Care, Leuven University, Leuven (Belgium); Baeyens, Willy [Department of Analytical and Environmental Chemistry, Vrije Universiteit Brussel, Brussels (Belgium); Van Larebeke, Nicolas [Department of Analytical and Environmental Chemistry, Vrije Universiteit Brussel, Brussels (Belgium); Department of Radiotherapy and Experimental Cancerology, Ghent University, Ghent (Belgium); Boonen, Francis; Ooms, Daniëlla; Wevers, Mai; Jacobs, Griet [Flemish Institute for Technological Research (VITO), Mol (Belgium); Covaci, Adrian [Toxicological Centre, Department of Pharmaceutical Sciences, University of Antwerp, Antwerp (Belgium); Schettgen, Thomas [Department of Occupational and Social Medicine, RWTH Aachen University, Aachen (Germany); Schoeters, Greet [Flemish Institute for Technological Research (VITO), Mol (Belgium); Department of Biomedical Sciences, University of Antwerp, Antwerp (Belgium); University of Southern Denmark, Institute of Public Health, Department of Environmental Medicine, Odense (Denmark)

2017-01-15

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 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. 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

Assessment of folic acid and DNA damage in cleft lip and cleft palate

Directory of Open Access Journals (Sweden)

Sivakumar Brooklyin

2014-03-01

Full Text Available Studies have identified the risk factors like folic acid deficiency during gestational period, family history for orofacial clefts, drugs like antiepileptic, vitamin A. But, the data regarding the folic acid status in children with cleft lip/palate is hardly evaluated in depth. Here, an assessment of folic acid and DNA damage were carried out in children with orofacial anomalies. Folic acid level and DNA damage were evaluated by folic acid assay (direct chemiluminescent technology and single cell gel electrophoresis or comet assay method respectively. The mean value of plasma folic acid by direct chemiluminescent technology was 6.5±3.6 nmol/L and the normal value in children ranges from 11.3 to 47.6 nmol/L. The amount of damaged DNA, measured as the tail length of the comet in cases, was 19.4±8.9 ?m and the mean percentage of DNA in tail was 16.5±3.7. Folic acid deficiency could be the reason for DNA damage.

DNA Damage in Melania Snail (Semisulcospira libertine) Irradiated with Gamma Radiation

Energy Technology Data Exchange (ETDEWEB)

Ryu, Tae Ho; Kim, Jin Kyu [Korea Atomic Energy Research Institute, Advanced Radiation Technology Institute, Jeongeup (Korea, Republic of); An, Kwang Guk [Chungnam National University, Daejeon (Korea, Republic of); Nili, Mohammad [Dawnesh Radiation Research Institute, Barcelona (Spain)

2010-10-15

Generally radiological protection has focused on human. But International Commission on Radiological Protection (ICRP) requires the effect data of ionizing radiation on nonhuman biota for the radiological protection of the environment. The choice of a melania snail as a model for environmental biomonitoring of radiation genotoxicity took into account that invertebrates represent one of aquatic species. The comet assay or single cell gel electrophoresis (SCGE) assay, first introduced by Ostling and Johanson, was used to detect DNA single strand breaks and to investigate the application of this technique as a tool for aquatic biomonitoring. Comet assay offers considerable advantages over some other assays used in DNA damage detection, such as chromosomal aberrations, sister chromatid Exchange and the micronucleus test, since there is no need for cells to be in a dividing state. Other advantages are its rapidity, relatively low coast, and wide applicability to virtually any nucleated cell type. In this study, we evaluated DNA damage in cells of Semisulcospira libertina after irradiation with {sup 60}Co gamma radiation by using the comet assay

Aging of hematopoietic stem cells: DNA damage and mutations?

Science.gov (United States)

Moehrle, Bettina M; Geiger, Hartmut

2016-10-01

Aging in the hematopoietic system and the stem cell niche contributes to aging-associated phenotypes of hematopoietic stem cells (HSCs), including leukemia and aging-associated immune remodeling. Among others, the DNA damage theory of aging of HSCs is well established, based on the detection of a significantly larger amount of γH2AX foci and a higher tail moment in the comet assay, both initially thought to be associated with DNA damage in aged HSCs compared with young cells, and bone marrow failure in animals devoid of DNA repair factors. Novel data on the increase in and nature of DNA mutations in the hematopoietic system with age, the quality of the DNA damage response in aged HSCs, and the nature of γH2AX foci question a direct link between DNA damage and the DNA damage response and aging of HSCs, and rather favor changes in epigenetics, splicing-factors or three-dimensional architecture of the cell as major cell intrinsic factors of HSCs aging. Aging of HSCs is also driven by a strong contribution of aging of the niche. This review discusses the DNA damage theory of HSC aging in the light of these novel mechanisms of aging of HSCs. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

The Protective Effect of Whole Honey and Phenolic Extract on Oxidative DNA Damage in Mice Lymphocytes Using Comet Assay.

Science.gov (United States)

Cheng, Ni; Wang, Yuan; Cao, Wei

2017-12-01

In this study, the antioxidant activity and the protective effect against hydrogen peroxide-induced DNA damage were assessed for five honeys of different botanical origin. Seven phenolic acids were detected in the honey samples. Ferulic acid was the most abundant phenolic acid detected in longan honey, jujube honey and buckwheat honey. Ellagic acid, p-hydroxybenzoic acid and protocatechuic acid were the main phenolic acids detected in vitex honey. Of all honey samples tested, the highest total phenolic content and antioxidant activity were found in buckwheat honey, whereas the lowest total phenolic content and antioxidant activity were found in locust honey. Treatment with hydrogen peroxide induced a 62% increase in tail DNA in mice lymphocytes, and all studied honeys significantly inhibited this effect (P Phenolic extracts of honey displayed greater protective effects than whole honey in comet assay. The hydrogen peroxide-generated increase in 8-hydroxy-2-deoxyguanosine (8-OHdG) was effectively inhibited by the honeys studied (P phenolic acids of honey can penetrate into lymphocytes and protect DNA from oxidative damage by scavenging hydrogen peroxide and/or chelating ferrous ions.

DNA damage and autophagy

International Nuclear Information System (INIS)

Rodriguez-Rocha, Humberto; Garcia-Garcia, Aracely; Panayiotidis, Mihalis I.; Franco, Rodrigo

2011-01-01

Both exogenous and endogenous agents are a threat to DNA integrity. Exogenous environmental agents such as ultraviolet (UV) and ionizing radiation, genotoxic chemicals and endogenous byproducts of metabolism including reactive oxygen species can cause alterations in DNA structure (DNA damage). Unrepaired DNA damage has been linked to a variety of human disorders including cancer and neurodegenerative disease. Thus, efficient mechanisms to detect DNA lesions, signal their presence and promote their repair have been evolved in cells. If DNA is effectively repaired, DNA damage response is inactivated and normal cell functioning resumes. In contrast, when DNA lesions cannot be removed, chronic DNA damage triggers specific cell responses such as cell death and senescence. Recently, DNA damage has been shown to induce autophagy, a cellular catabolic process that maintains a balance between synthesis, degradation, and recycling of cellular components. But the exact mechanisms by which DNA damage triggers autophagy are unclear. More importantly, the role of autophagy in the DNA damage response and cellular fate is unknown. In this review we analyze evidence that supports a role for autophagy as an integral part of the DNA damage response.

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

Science.gov (United States)

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

DNA Damage by Radiation in Tradescantia Leaf Cells

International Nuclear Information System (INIS)

Han, Min; Hyun, Kyung Man; Ryu, Tae Ho; Kim, Jin Kyu; Nili, Mohammad

2010-01-01

The comet assay is currently used in different areas of biological sciences to detect DNA damage. The comet assay, due to its simplicity, sensitivity and need of a few cells, is ideal as a short-term genotoxicity test. The comet assay can theoretically be applied to every type of eukaryotic cell, including plant cells. Plants are very useful as monitors of genetic effects caused by pollution in the atmosphere, water and soil. Tradescantia tests are very useful tools for screening the mutagenic potential in the environment. Experiments were conducted to study the genotoxic effects of ionizing radiations on the genome integrity, particularly of Tradescantia. The increasingly frequent use of Tradescantia as a sensitive environmental bioindicator of genotoxic effects. This study was designed to assess the genotoxicity of ionizing radiation using Tradescnatia-comet assay. The development of comet assay has enabled investigators to detect DNA damage at the levels of cells. To adapt this assay to plant cells, nuclei were directly obtained from Tradescantia leaf samples. A significant dose-dependent increase in the average tail moment values over the negative control was observed. Recently the adaptation of this technique to plant cells opens new possibilities for studies in variety area. The future applications of the comet assay could impact some other important areas, certainly, one of the limiting factors to its utility is the imagination of the investigator.

DNA Damage by Radiation in Tradescantia Leaf Cells

Energy Technology Data Exchange (ETDEWEB)

Han, Min; Hyun, Kyung Man; Ryu, Tae Ho; Kim, Jin Kyu [Korea Atomic Energy Research Institute, Advanced Radiation Technology Institute, Jeongeup (Korea, Republic of); Nili, Mohammad [Dawnesh Radiation Research Institute, Barcelona (Spain)

2010-04-15

The comet assay is currently used in different areas of biological sciences to detect DNA damage. The comet assay, due to its simplicity, sensitivity and need of a few cells, is ideal as a short-term genotoxicity test. The comet assay can theoretically be applied to every type of eukaryotic cell, including plant cells. Plants are very useful as monitors of genetic effects caused by pollution in the atmosphere, water and soil. Tradescantia tests are very useful tools for screening the mutagenic potential in the environment. Experiments were conducted to study the genotoxic effects of ionizing radiations on the genome integrity, particularly of Tradescantia. The increasingly frequent use of Tradescantia as a sensitive environmental bioindicator of genotoxic effects. This study was designed to assess the genotoxicity of ionizing radiation using Tradescnatia-comet assay. The development of comet assay has enabled investigators to detect DNA damage at the levels of cells. To adapt this assay to plant cells, nuclei were directly obtained from Tradescantia leaf samples. A significant dose-dependent increase in the average tail moment values over the negative control was observed. Recently the adaptation of this technique to plant cells opens new possibilities for studies in variety area. The future applications of the comet assay could impact some other important areas, certainly, one of the limiting factors to its utility is the imagination of the investigator.

Primary DNA damage in chrome-plating workers.

Science.gov (United States)

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).

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.

Science.gov (United States)

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.

Sevoflurane Induces DNA Damage Whereas Isoflurane Leads to Higher Antioxidative Status in Anesthetized Rats

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Thalita L. A. Rocha

2015-01-01

Full Text Available Taking into account that there are controversial antioxidative effects of inhalational anesthetics isoflurane and sevoflurane and absence of comparison of genotoxicity of both anesthetics in animal model, the aim of this study was to compare DNA damage and antioxidant status in Wistar rats exposed to a single time to isoflurane or sevoflurane. The alkaline single-cell gel electrophoresis assay (comet assay was performed in order to evaluate DNA damage in whole blood cells of control animals (unexposed; n = 6 and those exposed to 2% isoflurane (n = 6 or 4% sevoflurane (n = 6 for 120 min. Plasma antioxidant status was determined by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay. There was no statistically significant difference between isoflurane and sevoflurane groups regarding hemodynamic and temperature variables (P > 0.05. Sevoflurane significantly increased DNA damage compared to unexposed animals (P = 0.02. In addition, Wistar rats anesthetized with isoflurane showed higher antioxidative status (MTT than control group (P = 0.019. There were no significant differences in DNA damage or antioxidant status between isoflurane and sevoflurane groups (P > 0.05. In conclusion, our findings suggest that, in contrast to sevoflurane exposure, isoflurane increases systemic antioxidative status, protecting cells from DNA damage in rats.

Studying the synergistic damage effects induced by 1.8 GHz radiofrequency field radiation (RFR) with four chemical mutagens on human lymphocyte DNA using comet assay in vitro

International Nuclear Information System (INIS)

Wang Baohong; He Jiliang; Jin Lifen; Lu Deqiang; Zheng Wei; Lou Jianlin; Deng Hongping

2005-01-01

The aim of this investigation was to study the synergistic DNA damage effects in human lymphocytes induced by 1.8 GHz radiofrequency field radiation (RFR, SAR of 3 W/kg) with four chemical mutagens, i.e. mitomycin C (MMC, DNA crosslinker), bleomycin (BLM, radiomimetic agent), methyl methanesulfonate (MMS, alkylating agent), and 4-nitroquinoline-1-oxide (4NQO, UV-mimetic agent). The DNA damage of lymphocytes exposed to RFR and/or with chemical mutagens was detected at two incubation time (0 or 21 h) after treatment with comet assay in vitro. Three combinative exposure ways were used. Cells were exposed to RFR and chemical mutagens for 2 and 3 h, respectively. Tail length (TL) and tail moment (TM) were utilized as DNA damage indexes. The results showed no difference of DNA damage indexes between RFR group and control group at 0 and 21 h incubation after exposure (P > 0.05). There were significant difference of DNA damage indexes between MMC group and RFR + MMC co-exposure group at 0 and 21 h incubation after treatment (P 0.05). The experimental results indicated 1.8 GHz RFR (SAR, 3 W/kg) for 2 h did not induce the human lymphocyte DNA damage effects in vitro, but could enhance the human lymphocyte DNA damage effects induced by MMC and 4NQO. The synergistic DNA damage effects of 1.8 GHz RFR with BLM or MMS were not obvious

Phototherapy causes DNA damage in peripheral mononuclear leukocytes in term infants.

Science.gov (United States)

Aycicek, Ali; Kocyigit, Abdurrahim; Erel, Ozcan; Senturk, Hakan

2008-01-01

Our aim was to determine whether endogenous mononuclear leukocyte DNA strand is a target of phototherapy. The study included 65 term infants aged between 3-10 days that had been exposed to intensive (n = 23) or conventional (n = 23) phototherapy for at least 48 hours due to neonatal jaundice, and a control group (n = 19). DNA damage was assayed by single-cell alkaline gel electrophoresis (comet assay). Plasma total antioxidant capacity and total oxidant status levels were also measured, and correlation between DNA damage and oxidative stress was investigated. Mean values of DNA damage scores in both the intensive and conventional phototherapy groups were significantly higher than those in the control group (p Total oxidant status levels in both the intensive and conventional phototherapy groups were significantly higher than those in the control group (p = 0.005). Mean (standard deviation) values were 18.1 (4.2), 16.9 (4.4), 13.5 (4.2) micromol H2O2 equivalent/L, respectively. Similarly, oxidative stress index levels in both the intensive and conventional phototherapy groups were significantly higher than those in the control group (p = 0.041). Plasma total antioxidant capacity and total bilirubin levels did not differ between the groups (p > 0.05). There were no significant correlations between DNA damage scores and bilirubin, total oxidant status and oxidative stress levels in either phototherapy group (p > 0.05). Both conventional phototherapy and intensive phototherapy cause endogenous mononuclear leukocyte DNA damage in jaundiced term infants.

Protective effects of Brussels sprouts towards B[a]P-induced DNA damage: a model study with the single-cell gel electrophpresis (SCGE)/Hep G2 assay

NARCIS (Netherlands)

Laky, B.; Knasmuller, S.; Gminski, R.; Mersch-Sundermann, V.; Scharf, G.; Verkerk, R.; Freywald, C.; Uhl, M.; Kassie, F.

2002-01-01

The aim of this study was to investigate the chemoprotective effects of Brussels sprouts juice towards benzo[a]pyrene (B(a)P)-induced DNA damage in the single-cell gel electrophoresis (SCGE)/Hep G2 test system. This assay combines the advantages of the SCGE assay with that of the use of

p53 protein expression versus micronucleus induction as an indicator of DNA damage

International Nuclear Information System (INIS)

Hickman, A.W.; Carpenter, T.R.; Johnson, N.F.

1994-01-01

In vitro assays for detecting DNA damage play an important role in evaluating the possible adverse health effects of chemical compounds. Exposure to many DNA-damaging agents in vitro has been shown to cause elevated levels of the tumor-suppressor protein p53. Work in our laboratory has shown that induction of the p53 protein is useful as a biodosimeter for determining the radiation dose to cells. The purpose of this investigation was to compare the sensitivity of this assay to that of micronucleus induction, which is commonly used as a marker of radiation-induced damage

A comparative study of radiation induced DNA damage and repair in buccal cells and lymphocytes assessed by single cell gel electrophoresis (comet) assay

International Nuclear Information System (INIS)

Dhillon, V.S.; Fenech, M.

2003-01-01

Full text: During the past few years, there has been increasing interest in epithelial cells from buccal mucosa for genotoxicity evaluation of different chemical and/or physical agents. In the present study we used the buccal and sublingual epithelial cells to detect both inter- and intra-individual variation in radiation induced DNA damage and repair. For this purpose we used the single cell gel electrophoresis assay which over the years has gained wide spread acceptance as a simple, sensitive and reliable assay to measure genotoxicity related effects as well as kinetics of DNA repair. Buccal and sublingual epithelial cells from six individuals (3 male and 3 females; 35-45 years old) were collected. Cells were then irradiated for 0, 2 and 4 Gy doses using 137 Cs-source (5.58 Gy min-1). After irradiation the cells were either placed immediately on ice or incubated at 37 deg C for 2 1/2 hour to allow cellular repair. We also studied G0 and G1 lymphocytes from the same individuals to compare the radiation-induced DNA damage and repair potential with the two types of buccal cells. Baseline DNA damage rate was significantly greater (p < 0.001) in buccal (28.18%) and sublingual epithelial cells (30.66) as compared to G0 (22.02%) and G1 (21.46%) lymphocytes. Radiation-induced DNA damage in buccal (19.34%, 2Gy; 21.41%, 4 Gy) and sublingual epithelial cells (18.11% and 20.60%) was very similar and significantly lower than that observed in lymphocytes (29.76%, 56.77% for G0 and 32.66%, 59.32% for G1). The extent of DNA repair in buccal and sublingual epithelial cells was significantly lower than that observed in lymphocytes. The results for buccal and sublingual epithelial cells were highly correlated with each other (r 0.9541) as were those of G0 and G1 lymphocytes (r 0.9868). The results suggest a much reduced capacity for cellular repair in buccal and sublingual epithelial cells

Flavonoids can protect maize DNA from the induction of ultraviolet radiation damage

International Nuclear Information System (INIS)

Stapleton, A.E.; Walbot, V.

1994-01-01

Diverse flavonoid compounds are widely distributed in angiosperm families. Flavonoids absorb radiation in the ultraviolet (UV) region of the spectrum, and it has been proposed that these compounds function as UV filters. We demonstrate that the DNA in Zea mays plants that contain flavonoids (primarily anthocyanins) is protected from the induction of damage caused by UV radiation relative to the DNA in plants that are genetically deficient in these compounds. DNA damage was measured with a sensitive and simple assay using individual monoclonal antibodies, one specific for cyclobutane pyrimidine dimer damage and the other specific for pyrimidine(6,4)pyrimidone damage. (author)

DNA damage by ethylbenzenehydroperoxide formed from carcinogenic ethylbenzene by sunlight irradiation

International Nuclear Information System (INIS)

Toda, Chitose; Uchida, Takafumi; Midorikawa, Kaoru; Murata, Mariko; Hiraku, Yusuke; Okamoto, Yoshinori; Ueda, Koji; Kojima, Nakao; Kawanishi, Shosuke

2003-01-01

Ethylbenzene, widely used in human life, is a non-mutagenic carcinogen. Sunlight-irradiated ethylbenzene caused DNA damage in the presence of Cu 2+ , but unirradiated ethylbenzene did not. A Cu + -specific chelator bathocuproine inhibited DNA damage and catalase showed a little inhibitory effect. The scopoletin assay revealed that peroxides and H 2 O 2 were formed in ethylbenzene exposed to sunlight. These results suggest that Cu + and alkoxyl radical mainly participate in DNA damage, and H 2 O 2 partially does. When catalase was added, DNA damage at thymine and cytosine was inhibited. Ethylbenzenehydroperoxide, identified by GC/MS analysis, induced the formation of 8-oxo-7,8-dihydro-2 ' -deoxyguanosine and caused DNA damage at consecutive guanines, as observed with cumenehydroperoxide. Equimolar concentrations of H 2 O 2 and acetophenone were produced by the sunlight-irradiation of 1-phenylethanol, a further degraded product of ethylbenzene. These results indicate a novel pathway that oxidative DNA damage induced by the peroxide and H 2 O 2 derived from sunlight-irradiated ethylbenzene may lead to expression of the carcinogenicity

DNA damage and cytotoxicity in pathology laboratory technicians exposed to organic solvents

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TATIANE DE AQUINO

2016-03-01

Full Text Available The aim of this study was to evaluate potential DNA damage and cytotoxicity in pathology laboratory technicians exposed to organic solvents, mainly xylene. Peripheral blood and buccal cells samples were collected from 18 technicians occupationally exposed to organic solvents and 11 non-exposed individuals. The technicians were sampled at two moments: Monday and Friday. DNA damage and cytotoxicity were evaluated using the Comet Assay and the Buccal Micronucleus Cytome assay. Fifteen subjects (83.5% of the exposed group to solvents complained about some symptom probably related to contact with vapours of organic solvents. DNA damage in the exposed group to solvents was nearly 2-fold higher on Friday than on Monday, and in both moments the individuals of this group showed higher levels of DNA damage in relation to controls. No statistical difference was detected in buccal cell micronucleus frequency between the laboratory technicians and the control group. However, in the analysis performed on Friday, technicians presented higher frequency (about 3-fold of karyolytic and apoptotic-like cells (karyorrhectic and pyknotic in relation to control group. Considering the damage frequency and the working time, a positive correlation was found in the exposed group to solvents (r=0.468; p=0.05. The results suggest that pathology laboratory workers inappropriately exposed to organic solvents have increased levels of DNA damage.

Comet assay for rapid detection of base damage in foods

International Nuclear Information System (INIS)

Al-Zubaidi, I. A.; Abdullah, T. S.; Qasim, S. R.

2012-12-01

Single cell gel electrophoresis (SCGE) or comet assay technique a sensitive, reliable and rapid method for DNA double and single strand break, alkali- labile site and delayed repair site detection in individual cells. In recent years, this method has been widely used for studies of DNA repair, genetic toxicology, and environmental biomontoring, however, this technique serves as an important tool for detection of DNA damage in living organism and is increasing being used in genetic testing of industrial chemicals, environmental agent's contaminations. This research paper helps to evaluate the oxidant agent's effects of exposure to organic pollutants by using comet assay techniques. This study used five samples of each food sample (Meat, Chicken, Rice, Fruits, Vegetables and Tea) to evaluate the genotoxic effects of exposure, to environmental agent's pollutants. The experimental data suggest that the DNA damage parameters ( Tail length, Tail width 1 ) were found higher value in exposed population when compared with the ratio of the length to width that cells exhibiting no migration having a ratio of 1. The percentage and distribution of cells in exposed population of cells also increases with the increase in values. This study demonstrates that, using sensitive techniques, it is possible to detect environmental agent's risks at an early stage. (Author)

Lymphocyte DNA damage and oxidative stress in patients with iron deficiency anemia.

Science.gov (United States)

Aslan, Mehmet; Horoz, Mehmet; Kocyigit, Abdurrahim; Ozgonül, Saadet; Celik, Hakim; Celik, Metin; Erel, Ozcan

2006-10-10

Oxidant stress has been shown to play an important role in the pathogenesis of iron deficiency anemia. The aim of this study was to investigate the association between lymphocyte DNA damage, total antioxidant capacity and the degree of anemia in patients with iron deficiency anemia. Twenty-two female with iron deficiency anemia and 22 healthy females were enrolled in the study. Peripheral DNA damage was assessed using alkaline comet assay and plasma total antioxidant capacity was determined using an automated measurement method. Lymphocyte DNA damage of patients with iron deficiency anemia was significantly higher than controls (ptotal antioxidant capacity was significantly lower (ptotal antioxidant capacity and hemoglobin levels (r=0.706, ptotal antioxidant capacity and hemoglobin levels were negatively correlated with DNA damage (r=-0.330, p<0.05 and r=-0.323, p<0.05, respectively). In conclusion, both oxidative stress and DNA damage are increased in IDA patients. Increased oxidative stress seems as an important factor that inducing DNA damage in those IDA patients. The relationships of oxidative stress and DNA damage with the severity of anemia suggest that both oxidative stress and DNA damage may, in part, have a role in the pathogenesis of IDA.

Does the recommended lymphocyte cytokinesis-block micronucleus assay for human biomonitoring actually detect DNA damage induced by occupational and environmental exposure to genotoxic chemicals?

Science.gov (United States)

Speit, Günter

2013-07-01

This commentary challenges the paradigm that the cytokinesis-block micronucleus assay (CBMN assay) with cultured human lymphocytes, as it is performed currently, is a sensitive and useful tool for detecting genotoxic effects in populations exposed occupationally or environmentally to genotoxic chemicals. Based on the principle of the assay and the available data, increased micronucleus (MN) frequencies in binucleated cells (BNC) are mainly due to MN produced in vitro during the cultivation period (i.e. MN produced in vivo do not substantially contribute to the MN frequency measured in BNC). The sensitivity of the assay for the detection of induced MN in BNC after an in vivo exposure to a genotoxic chemical is limited because cytochalasin B (Cyt-B) is added relatively late during the culture period and, therefore, the BNC that are scored do not always represent cells that have completed one cell cycle only. Furthermore, this delay means that damaged cells can be eliminated by apoptosis and/or that DNA damage induced in vivo can be repaired prior to the production of a MN in the presence of Cyt-B. A comparison with the in vitro CBMN assay used for genotoxicity testing leads to the conclusion that it is highly unlikely that DNA damage induced in vivo is the cause for increased MN frequencies in BNC after occupational or environmental exposure to genotoxic chemicals. This commentary casts doubt on the usefulness of the CBMN assay as an indicator of genotoxicity in human biomonitoring and questions the relevance of many published data for hazard identification and risk assessment. Thus, it seems worthwhile to reconsider the use of the CBMN assay as presently conducted for the detection of genotoxic exposure in human biomonitoring.

Biomarkers of oxidative stress and DNA damage in agricultural workers: A pilot study

International Nuclear Information System (INIS)

Muniz, Juan F.; McCauley, Linda; Scherer, J.; Lasarev, M.; Koshy, M.; Kow, Y.W.; Nazar-Stewart, Valle; Kisby, G.E.

2008-01-01

Oxidative stress and DNA damage have been proposed as mechanisms linking pesticide exposure to health effects such as cancer and neurological diseases. A study of pesticide applicators and farmworkers was conducted to examine the relationship between organophosphate pesticide exposure and biomarkers of oxidative stress and DNA damage. Urine samples were analyzed for OP metabolites and 8-hydroxy-2'-deoxyguanosine (8-OH-dG). Lymphocytes were analyzed for oxidative DNA repair activity and DNA damage (Comet assay), and serum was analyzed for lipid peroxides (i.e., malondialdehyde, MDA). Cellular damage in agricultural workers was validated using lymphocyte cell cultures. Urinary OP metabolites were significantly higher in farmworkers and applicators (p < 0.001) when compared to controls. 8-OH-dG levels were 8.5 times and 2.3 times higher in farmworkers or applicators (respectively) than in controls. Serum MDA levels were 4.9 times and 24 times higher in farmworkers or applicators (respectively) than in controls. DNA damage (Comet assay) and oxidative DNA repair were significantly greater in lymphocytes from applicators and farmworkers when compared with controls. Markers of oxidative stress (i.e., increased reactive oxygen species and reduced glutathione levels) and DNA damage were also observed in lymphocyte cell cultures treated with an OP. The findings from these in vivo and in vitro studies indicate that organophosphate pesticides induce oxidative stress and DNA damage in agricultural workers. These biomarkers may be useful for increasing our understanding of the link between pesticides and a number of health effects

Cadmium Chloride Induces DNA Damage and Apoptosis of Human Liver Carcinoma Cells via Oxidative Stress

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Anthony Skipper

2016-01-01

Full Text Available Cadmium is a heavy metal that has been shown to cause its toxicity in humans and animals. Many documented studies have shown that cadmium produces various genotoxic effects such as DNA damage and chromosomal aberrations. Ailments such as bone disease, renal damage, and several forms of cancer are attributed to overexposure to cadmium.  Although there have been numerous studies examining the effects of cadmium in animal models and a few case studies involving communities where cadmium contamination has occurred, its molecular mechanisms of action are not fully elucidated. In this research, we hypothesized that oxidative stress plays a key role in cadmium chloride-induced toxicity, DNA damage, and apoptosis of human liver carcinoma (HepG2 cells. To test our hypothesis, cell viability was determined by MTT assay. Lipid hydroperoxide content stress was estimated by lipid peroxidation assay. Genotoxic damage was tested by the means of alkaline single cell gel electrophoresis (Comet assay. Cell apoptosis was measured by flow cytometry assessment (Annexin-V/PI assay. The result of MTT assay indicated that cadmium chloride induces toxicity to HepG2 cells in a concentration-dependent manner, showing a 48 hr-LD50 of 3.6 µg/mL. Data generated from lipid peroxidation assay resulted in a significant (p < 0.05 increase of hydroperoxide production, specifically at the highest concentration tested. Data obtained from the Comet assay indicated that cadmium chloride causes DNA damage in HepG2 cells in a concentration-dependent manner. A strong concentration-response relationship (p < 0.05 was recorded between annexin V positive cells and cadmium chloride exposure. In summary, these in vitro studies provide clear evidence that cadmium chloride induces oxidative stress, DNA damage, and programmed cell death in human liver carcinoma (HepG2 cells.

Oxidative damage of DNA in subjects occupationally exposed to lead.

Science.gov (United States)

Pawlas, Natalia; Olewińska, Elżbieta; Markiewicz-Górka, Iwona; Kozłowska, Agnieszka; Januszewska, Lidia; Lundh, Thomas; Januszewska, Ewa; Pawlas, Krystyna

2017-09-01

Exposure to lead (Pb) in environmental and occupational settings continues to be a serious public health problem and may pose an elevated risk of genetic damage. The aim of this study was to assess the level of oxidative stress and DNA damage in subjects occupationally exposed to lead. We studied a population of 78 male workers exposed to lead in a lead and zinc smelter and battery recycling plant and 38 men from a control group. Blood lead levels were detected by graphite furnace atomic absorption spectrophotometry and plasma lead levels by inductively coupled plasma-mass spectrometry. The following assays were performed to assess the DNA damage and oxidative stress: comet assay, determination of 8-hydroxy-2'-deoxyguanosine (8-OHdG), lipid peroxidation and total antioxidant status (TAS). The mean concentration of lead in the blood of the exposed group was 392 ± 103 μg/L and was significantly higher than in the control group (30.3 ± 29.4 μg/L, p lead exposure [lead in blood, lead in plasma, zinc protoporphyrin (ZPP)] and urine concentration of 8-OHdG. The level of oxidative damage of DNA was positively correlated with the level of lipid peroxidation (TBARS) and negatively with total anti-oxidative status (TAS). Our study suggests that occupational exposure causes an increase in oxidative damage to DNA, even in subjects with relatively short length of service (average length of about 10 years). 8-OHdG concentration in the urine proved to be a sensitive and non-invasive marker of lead induced genotoxic damage.

A novel SERRS sandwich-hybridization assay to detect specific DNA target.

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Cécile Feuillie

Full Text Available In this study, we have applied Surface Enhanced Resonance Raman Scattering (SERRS technology to the specific detection of DNA. We present an innovative SERRS sandwich-hybridization assay that allows specific DNA detection without any enzymatic amplification, such as is the case with Polymerase Chain Reaction (PCR. In some substrates, such as ancient or processed remains, enzymatic amplification fails due to DNA alteration (degradation, chemical modification or to the presence of inhibitors. Consequently, the development of a non-enzymatic method, allowing specific DNA detection, could avoid long, expensive and inconclusive amplification trials. Here, we report the proof of concept of a SERRS sandwich-hybridization assay that leads to the detection of a specific chamois DNA. This SERRS assay reveals its potential as a non-enzymatic alternative technology to DNA amplification methods (particularly the PCR method with several applications for species detection. As the amount and type of damage highly depend on the preservation conditions, the present SERRS assay would enlarge the range of samples suitable for DNA analysis and ultimately would provide exciting new opportunities for the investigation of ancient DNA in the fields of evolutionary biology and molecular ecology, and of altered DNA in food frauds detection and forensics.

Damage to the DNA of microorganisms from decay of incorporated 125I and the relationship of DNA damage to lethal effects

International Nuclear Information System (INIS)

Krisch, R.E.; Krasin, F.; Sauri, C.J.

1975-01-01

Iodine-125 decays by electron capture and is known to cause severe molecular damage to small organic molecules via vacancy cascades. In an examination of the biological effects of this decay mode we have labelled coliphages T1 and T4, as well as E. coli, with 125 I-5-iododeoxyuridine, which is incorporated into DNA in place of thymidine. Labelled organisms are generally stored in liquid nitrogen at -196 0 C during decay and are periodically assayed for loss of viability and for breakage of DNA, using neutral and alkaline sucrose gradient sedimentation techniques. Briefly, our experiments have demonstrated drastic damage to DNA from the decay of incorporated 125 I, as would be predicted from the data for small molecules. (auth)

Rapid assessment of repair of ultraviolet DNA damage with a modified host-cell reactivation assay using a luciferase reporter gene and correlation with polymorphisms of DNA repair genes in normal human lymphocytes

Energy Technology Data Exchange (ETDEWEB)

Qiao Yawei; Spitz, Margaret R.; Guo Zhaozheng; Hadeyati, Mohammad; Grossman, Lawrence; Kraemer, Kenneth H.; Wei Qingyi

2002-11-30

As DNA repair plays an important role in genetic susceptibility to cancer, assessment of the DNA repair phenotype is critical for molecular epidemiological studies of cancer. In this report, we compared use of the luciferase (luc) reporter gene in a host-cell reactivation (HCR) (LUC) assay of repair of ultraviolet (UV) damage to DNA to use of the chloramphenicol (cat) gene-based HCR (CAT) assay we used previously for case-control studies. We performed both the assays on cryopreserved lymphocytes from 102 healthy non-Hispanic white subjects. There was a close correlation between DNA repair capacity (DRC) as measured by the LUC and CAT assays. Although these two assays had similar variation, the LUC assay was faster and more sensitive. We also analyzed the relationship between DRC and the subjects' previously determined genotypes for four polymorphisms of two nucleotide-excision repair (NER) genes (in intron 9 of xeroderma pigmentosum (XP) C and exons 6, 10 and 23 of XPD) and one polymorphism of a base-excision repair gene in exon 10 of X-ray complementing group 1 (XRCC1). The DRC was significantly lower in subjects homozygous for one or more polymorphisms of the two NER genes than in subjects with other genotypes (P=0.010). In contrast, the polymorphic XRCC1 allele had no significant effect on DRC. These results suggest that the post-UV LUC assay measures NER phenotype and that polymorphisms of XPC and XPD genes modulate DRC. For population studies of the DNA repair phenotype, many samples need to be evaluated, and so the LUC assay has several advantages over the CAT assay: the LUC assay was more sensitive, had less variation, was not radioactive, was easier to perform, and required fewer cryopreserved cells. These features make the LUC-based HCR assay suitable for molecular epidemiological studies.

Repair of human DNA: radiation and chemical damage in normal and xeroderma pigmentosum cells

International Nuclear Information System (INIS)

Regan, J.D.; Setlow, R.B.

1976-01-01

We present the experimental evidence we have gathered, using a particular assay for DNA repair in human cells, the photolysis of bromodeoxyuridine (BrdUrd) incorporated during repair. This assay characterizes the sequence of repair events that occur in human cells after radiation, both ultraviolet and ionizing, and permits an estimation of the size of the average repaired region after these physical insults to DNA. We will discuss chemical insults to DNA and attempt to liken the repair processes after chemical damages of various kinds to those repair processes that occur in human DNA after damage from physical agents. We will also show results indicating that, under certain conditions, repair events resembling those seen after uv-irradiation can be observed in normal human cells after ionizing radiation. Furthermore the XP cells, defective in the repair of uv-induced DNA damage, show defective repair of these uv-like DNA lesions induced by ionizing radiation

Variation of DNA damage levels in peripheral blood mononuclear cells isolated in different laboratories

DEFF Research Database (Denmark)

Godschalk, Roger W L; Ersson, Clara; Stępnik, Maciej

2014-01-01

This study investigated the levels of DNA strand breaks and formamidopyrimidine DNA glycosylase (FPG) sensitive sites, as assessed by the comet assay, in peripheral blood mononuclear cells (PBMC) from healthy women from five different countries in Europe. The laboratory in each country (referred...... to as 'centre') collected and cryopreserved PBMC samples from three donors, using a standardised cell isolation protocol. The samples were analysed in 13 different laboratories for DNA damage, which is measured by the comet assay. The study aim was to assess variation in DNA damage in PBMC samples that were......%) by standardisation of the primary comet assay endpoint with calibration curve samples. The level of DNA strand breaks in the samples from two of the centres (0.56-0.61 lesions/10(6) bp) was significantly higher compared with the other three centres (0.41-0.45 lesions/10(6) bp). In contrast, there was no difference...

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

Directory of Open Access Journals (Sweden)

İsmet Çok

2011-01-01

Full Text Available 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.

DNA damage and repair in human skin in situ

International Nuclear Information System (INIS)

Sutherland, B.M.; Gange, R.W.; Freeman, S.E.; Sutherland, J.C.

1987-01-01

Understanding the molecular and cellular origins of sunlight-induced skin cancers in man requires knowledge of the damages inflicted on human skin during sunlight exposure, as well as the ability of cells in skin to repair or circumvent such damage. Although repair has been studied extensively in procaryotic and eucaryotic cells - including human cells in culture - there are important differences between repair by human skin cells in culture and human skin in situ: quantitative differences in rates of repair, as well as qualitative differences, including the presence or absence of repair mechanisms. Quantitation of DNA damage and repair in human skin required the development of new approaches for measuring damage at low levels in nanogram quantities of non-radioactive DNA. The method allows for analysis of multiple samples and the resulting data should be related to behavior of the DNA molecules by analytic expressions. Furthermore, it should be possible to assay a variety of lesions using the same methodology. The development of new analysis methods, new technology, and new biochemical probes for the study of DNA damage and repair are described. 28 refs., 4 figs

DNA damage and repair in human skin in situ

Energy Technology Data Exchange (ETDEWEB)

Sutherland, B.M.; Gange, R.W.; Freeman, S.E.; Sutherland, J.C.

1987-01-01

Understanding the molecular and cellular origins of sunlight-induced skin cancers in man requires knowledge of the damages inflicted on human skin during sunlight exposure, as well as the ability of cells in skin to repair or circumvent such damage. Although repair has been studied extensively in procaryotic and eucaryotic cells - including human cells in culture - there are important differences between repair by human skin cells in culture and human skin in situ: quantitative differences in rates of repair, as well as qualitative differences, including the presence or absence of repair mechanisms. Quantitation of DNA damage and repair in human skin required the development of new approaches for measuring damage at low levels in nanogram quantities of non-radioactive DNA. The method allows for analysis of multiple samples and the resulting data should be related to behavior of the DNA molecules by analytic expressions. Furthermore, it should be possible to assay a variety of lesions using the same methodology. The development of new analysis methods, new technology, and new biochemical probes for the study of DNA damage and repair are described. 28 refs., 4 figs.

Damage to cellular and isolated DNA induced by a metabolite of aspirin

Energy Technology Data Exchange (ETDEWEB)

Oikawa, Shinji [Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, Mie 514-8507 (Japan)], E-mail: s-oikawa@doc.medic.mie-u.ac.jp; Kobayashi, Hatasu; Tada-Oikawa, Saeko [Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, Mie 514-8507 (Japan); JSPS Research Fellow (Japan); Isono, Yoshiaki [Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, Mie 514-8507 (Japan); Kawanishi, Shosuke [Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, Mie 514-8507 (Japan); Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Suzuka, Mie 513-8670 (Japan)

2009-02-10

Aspirin has been proposed as a possible chemopreventive agent. On the other hand, a recent cohort study showed that aspirin may increase the risk for pancreatic cancer. To clarify whether aspirin is potentially carcinogenic, we investigated the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), which is correlated with the incidence of cancer, in cultured cells treated with 2,3-dihydroxybenzoic acid (2,3-DHBA), a metabolite of aspirin. 2,3-DHBA induced 8-oxodG formation in the PANC-1 human pancreatic cancer cell line. 2,3-DHBA-induced DNA single-strand breaks were also revealed by comet assay using PANC-1 cells. Flow cytometric analyses showed that 2,3-DHBA increased the levels of intracellular reactive oxygen species (ROS) in PANC-1 cells. The 8-oxodG formation and ROS generation were also observed in the HL-60 leukemia cell line, but not in the hydrogen peroxide (H{sub 2}O{sub 2})-resistant clone HP100 cells, suggesting the involvement of H{sub 2}O{sub 2}. In addition, an hprt mutation assay supported the mutagenicity of 2,3-DHBA. We investigated the mechanism underlying the 2,3-DHBA-induced DNA damage using {sup 32}P-labeled DNA fragments of human tumor suppressor genes. 2,3-DHBA induced DNA damage in the presence of Cu(II) and NADH. DNA damage induced by 2,3-DHBA was enhanced by the addition of histone peptide-6 [AKRHRK]. Interestingly, 2,3-DHBA and histone peptide-6 caused base damage in the 5'-ACG-3' and 5'-CCG-3' sequences, hotspots of the p53 gene. Bathocuproine, a Cu(I) chelator, and catalase inhibited the DNA damage. Typical hydroxyl radical scavengers did not inhibit the DNA damage. These results suggest that ROS derived from the reaction of H{sub 2}O{sub 2} with Cu(I) participate in the DNA damage. In conclusion, 2,3-DHBA induces oxidative DNA damage and mutations, which may result in carcinogenesis.

Damage to cellular and isolated DNA induced by a metabolite of aspirin

International Nuclear Information System (INIS)

Oikawa, Shinji; Kobayashi, Hatasu; Tada-Oikawa, Saeko; Isono, Yoshiaki; Kawanishi, Shosuke

2009-01-01

Aspirin has been proposed as a possible chemopreventive agent. On the other hand, a recent cohort study showed that aspirin may increase the risk for pancreatic cancer. To clarify whether aspirin is potentially carcinogenic, we investigated the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), which is correlated with the incidence of cancer, in cultured cells treated with 2,3-dihydroxybenzoic acid (2,3-DHBA), a metabolite of aspirin. 2,3-DHBA induced 8-oxodG formation in the PANC-1 human pancreatic cancer cell line. 2,3-DHBA-induced DNA single-strand breaks were also revealed by comet assay using PANC-1 cells. Flow cytometric analyses showed that 2,3-DHBA increased the levels of intracellular reactive oxygen species (ROS) in PANC-1 cells. The 8-oxodG formation and ROS generation were also observed in the HL-60 leukemia cell line, but not in the hydrogen peroxide (H 2 O 2 )-resistant clone HP100 cells, suggesting the involvement of H 2 O 2 . In addition, an hprt mutation assay supported the mutagenicity of 2,3-DHBA. We investigated the mechanism underlying the 2,3-DHBA-induced DNA damage using 32 P-labeled DNA fragments of human tumor suppressor genes. 2,3-DHBA induced DNA damage in the presence of Cu(II) and NADH. DNA damage induced by 2,3-DHBA was enhanced by the addition of histone peptide-6 [AKRHRK]. Interestingly, 2,3-DHBA and histone peptide-6 caused base damage in the 5'-ACG-3' and 5'-CCG-3' sequences, hotspots of the p53 gene. Bathocuproine, a Cu(I) chelator, and catalase inhibited the DNA damage. Typical hydroxyl radical scavengers did not inhibit the DNA damage. These results suggest that ROS derived from the reaction of H 2 O 2 with Cu(I) participate in the DNA damage. In conclusion, 2,3-DHBA induces oxidative DNA damage and mutations, which may result in carcinogenesis

Improved assay for thymine base damage in E. coli using high performance liquid chromatography

International Nuclear Information System (INIS)

Claycamp, H.G.

1985-01-01

A simple high performance liquid chromatography (HPLC) technique has been established for the simultaneous assay of thymine and thymidine radiation damage products. The HPLC procedure uses an isocratic mobile phase of 4% acetonitrile in 0.2 M ammonium dihydrogen phosphate (pH 5.0), a reversed-phase octadecylsilicate (5 micro-spherical packing) 0.45 x 25 cm column, and a variable wavelength UV detector. This procedure affords much better resolution than other published procedures that use 10 micron columns or separate assays for bases and nucleosides. For example, irradiation of 5 x 10 -3 M thymidine solutions have been performed to calibrate the system for base damage assays in E. coli. This yields up to 15 resolvable residues within 20 minutes. Sensitivity of the system (at 2210 nm) for 5,6- dihydrothymine (DHT) is about 10 -10 moles. Preliminary results show that this translates to about 0.4 DHT residues per 10 6 daltons of E. coli DNA. This is comparable to the sensitivities of monoclonal assays to thymine damage products that have recently been reported by others. Since it is feasible that the sensitivity of this system can be improved by 2-3 times, this HPLC technique should provide a simple and rapid means of detecting E. coli base damage release and base damage in nucleoside hydrolysates of DNA

DNA damage in leukocytes from fanconi anemia patients and heterozygotes induced by mitomycin C and ionizing radiation as assessed by the comet and comet - FISH assay

International Nuclear Information System (INIS)

Mohseni Meybodi, A.; Mozdarani, H.

2009-01-01

Lymphocytes of Fanconi anemia (FA) show an increased sensitivity to the alkylating agents such as mitomycin C (MMC), but their responses to gamma-irradiation is controversial. The extent of DNA damage in leukocytes of FA patients following irradiation and MMC treatment was studied at cellular and single chromosome level. Methods: DNA damage induced by gamma-rays and MMC was measured in leukocytes of FA patients and carriers at whole genome level using the comet assay. Also, at the DNA level of specific chromosome involved in this disease using a modified comet-FISH protocol with whole chromosome painting probes (chromosomes 16 and 13), DNA damage in leukocytes of FA patients and heterozygotes were compared to healthy individuals. Results: Baseline DNA damage in leukocytes of patients and heterozygotes was higher than in controls. Net induced DNA damage by gamma-rays in leukocytes of FA cases was not significantly different from that of healthy donors and heterozygotes. Net induced DNA damage by MMC was statistically higher and significantly different (P<0.05) in patients than other groups. Hybridization of chromosome 16 reveals more signals in the tail but the number of spots in the tail was not significantly higher than the hybridization spots for chromosome 13 in both gamma-irradiated and MMC treated samples. Conclusion: Results indicate that DNA damage induced by MMC could be a better index for diagnosis of FA patients compared to gamma-rays. Results of comet-FISH showed no difference between the sensitivity of chromosome 16 and 13 to MMC and radiation. It may indicate that, although the FA-A gene is located on chromosome 16, this chromosome might have a similar sensitivity as other chromosomes

Evaluation of radio-induced DNA damage and their repair in human lymphocytes by comet assay or single cell gel electrophoresis

International Nuclear Information System (INIS)

Nascimento, Patricia A. do; Suzuki, Miriam F.; Okazaki, Kayo

1997-01-01

The comet assay, also called single cell gel electrophoresis technique, permits to evaluate quantitatively DNA breakage induced by chemical and physical agents at the level of the single cell. The present paper refers to the construction of dose-response curves to DNA damage and repair studies in human peripheral lymphocytes, utilizing the comet assay for the radiosensitivity analysis. So, the blood samples were obtained from healthy donors (40-50 year old), irradiated in a 60 Co source (GAMMACEL 220) with doses of 0.17, 0.25, 0.57, 1.10, 2.12 and 4.22 Gy (0.59 Gy/min.) and processed 1 and 24 hours after the exposition. Results obtained showed a increase in the total lenght of comet (DNA migration) as a function of radiation dose in samples processed 1 and 24 hours after the treatment. The DNA lesion in irradiated lymphocytes with 4.22 Gy (means value of 101.4 μm) were 3.4 times higher than in the untreated lymphocytes (mean value of 30 μm) instead of 24 hours after the irradiation were 1.5 times higher (mean value of 46.3 μm). This reduction on DNA repair occurred in these cells. It was also possible visualized the presence of subpopulations of the cells with different sensitivity and repair capacity to ionizing radiation in these donors. (author). 8 refs., 3 figs

Genome-Wide Requirements for Resistance to Functionally Distinct DNA-Damaging Agents.

Directory of Open Access Journals (Sweden)

2005-08-01

Full Text Available The mechanistic and therapeutic differences in the cellular response to DNA-damaging compounds are not completely understood, despite intense study. To expand our knowledge of DNA damage, we assayed the effects of 12 closely related DNA-damaging agents on the complete pool of ~4,700 barcoded homozygous deletion strains of Saccharomyces cerevisiae. In our protocol, deletion strains are pooled together and grown competitively in the presence of compound. Relative strain sensitivity is determined by hybridization of PCR-amplified barcodes to an oligonucleotide array carrying the barcode complements. These screens identified genes in well-characterized DNA-damage-response pathways as well as genes whose role in the DNA-damage response had not been previously established. High-throughput individual growth analysis was used to independently confirm microarray results. Each compound produced a unique genome-wide profile. Analysis of these data allowed us to determine the relative importance of DNA-repair modules for resistance to each of the 12 profiled compounds. Clustering the data for 12 distinct compounds uncovered both known and novel functional interactions that comprise the DNA-damage response and allowed us to define the genetic determinants required for repair of interstrand cross-links. Further genetic analysis allowed determination of epistasis for one of these functional groups.

DNA damage and polyploidization.

Science.gov (United States)

Chow, Jeremy; Poon, Randy Y C

2010-01-01

A growing body of evidence indicates that polyploidization triggers chromosomal instability and contributes to tumorigenesis. DNA damage is increasingly being recognized for its roles in promoting polyploidization. Although elegant mechanisms known as the DNA damage checkpoints are responsible for halting the cell cycle after DNA damage, agents that uncouple the checkpoints can induce unscheduled entry into mitosis. Likewise, defects of the checkpoints in several disorders permit mitotic entry even in the presence of DNA damage. Forcing cells with damaged DNA into mitosis causes severe chromosome segregation defects, including lagging chromosomes, chromosomal fragments and chromosomal bridges. The presence of these lesions in the cleavage plane is believed to abort cytokinesis. It is postulated that if cytokinesis failure is coupled with defects of the p53-dependent postmitotic checkpoint pathway, cells can enter S phase and become polyploids. Progress in the past several years has unraveled some of the underlying principles of these pathways and underscored the important role of DNA damage in polyploidization. Furthermore, polyploidization per se may also be an important determinant of sensitivity to DNA damage, thereby may offer an opportunity for novel therapies.

Repair of UV-damaged incoming plasmid DNA in Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Keszenman-Pereyra, David

1990-01-01

A whole-cell transformation assay was used for the repair of UV-damaged plasma DNA in highly-transformable haploid strains of Saccharomyces cerevisiae having different repair capabilities. The experiments described demonstrate that three epistasis groups (Friedberg 1988) are involved in the repair of UV-incoming DNA and that the repair processes act less efficiently on incoming DNA than they do on chromosomal DNA. The implications of these findings for UV repair in Saccharomyces cerevisiae are discussed. (author)

Yeast DNA-repair gene RAD14 encodes a zinc metalloprotein with affinity for ultraviolet-damaged DNA

International Nuclear Information System (INIS)

Guzder, S.N.; Sung, P.; Prakash, S.; Prakash, L.

1993-01-01

Xeroderma pigmentosum (XP) patients suffer from a high incidence of skin cancers due to a defect in excision repair of UV light-damaged DNA. Of the seven XP complementation groups, A--G, group A represents a severe and frequent form of the disease. The Saccharomyces cerevisiae RAD14 gene is a homolog of the XP-A correcting (XPAC) gene. Like XP-A cells, rad14-null mutants are defective in the incision step of excision repair of UV-damaged DNA. The authors have purified RAD14 protein to homogeneity from extract of a yeast strain genetically tailored to overexpress RAD14. As determined by atomic emission spectroscopy, RAD14 contains one zinc atom. They also show in vitro that RAD14 binds zinc but does not bind other divalent metal ions. In DNA mobility-shift assays, RAD14 binds specifically to UV-damaged DNA. Removal of cyclobutane pyrimidine dimers from damaged DNA by enzymatic photoreactivation has no effect on binding, strongly suggesting that RAD14 recognizes pyrimidine(6-4)pyrimidone photoproduct sites. These findings indicate that RAD14 functions in damage recognition during excision repair. 37 refs., 4 figs

Evaluation of gamma radiation induced genetic damage in the fish Cyprinus carpio using comet assay

International Nuclear Information System (INIS)

Praveen Kumar, M.K.; Shyama, S.K.; Bhagat, S.S.; Chaubey, R.C.

2013-01-01

Radionuclides released from various sources including the industries, as well as, accidental release during a nuclear disaster can contaminate inland water bodies. Suitable bio-monitoring methods/biomarkers are the need of the day to assess the impact of high/low levels of radiation exposure in aquatic environment. Fishes are very important as a group of ecologically and commercially important non-human biota and are often used as a bioindicators of aquatic pollution. Present work was carried out to assess the genotoxic effect of gamma radiation on fresh water fish Cyprinus carpio (common carp) in vivo using comet assay. Fishes were irradiated with 2, 4, 6, 8 and 10 Gy of gamma rays using a teletherapy machine and comet assay was performed on nucleated erythrocytes after 24, 48 and 72 h of irradiation . A significant increase in % tail DNA was observed at all the doses of gamma radiation as compared to controls indicating radiation induced DNA damage in a dose-dependent manner. Maximum % tail DNA was observed at 24 h which gradually declined till 72 h, in a time-dependent manner. This decrease in damage may indicate repair of the damaged DNA and or loss of heavily damaged cells, over a period of time. The study reveals that the comet assay may be used as a sensitive and rapid method to detect genotoxicity of gamma radiation and other environmental pollutants in sentinel species. (author)

DNA damage in human lymphocytes due to synergistic interaction between ionizing radiation and pesticide

International Nuclear Information System (INIS)

Kim, J. K.; Lee, K. H.; Lee, B. H.; Chun, K. J.

2001-01-01

Biological risks may arise from the possibility of the synergistic interaction between harmful factors such as ionizing radiation and pesticide. The effect of pesticide on radiation-induced DNA damage in human in human blood lymphocytes was evaluated by the single cell gel electrophoresis (SCGE) assay. The lymphocytes, with or without pretreatment of the pesticide, were exposed to 2.0 Gy of gamma ray. Significantly increased tail moment, which was a marker of DNA strand breaks in SCGE assay, showed an excellent dose-response relationship. The present study confirms that the pesticide has the cytotoxic effect on lymphocytes and that it interacts synergistically with ionizing radiationon DNA damage, as well

DNA damage in isolated rat hepatocytes exposed to C.I. pigment orange 5 and C.I. pigment yellow 12 by the alkaline comet assay

DEFF Research Database (Denmark)

Møller, P; Wallin, Håkan; Grunnet, N

1998-01-01

The induction of DNA damage by commonly used printing ink pigments, C.I. pigment orange 5 (C.I. 12075) and C.I. pigment yellow 12 (C.I. 21090), was investigated in freshly isolated rat hepatocytes with the comet assay. C.I. pigment yellow 12 is a 3,3'-dichlorobenzidine-based diarylide pigment...

Variation in the measurement of DNA damage by comet assay measured by the ECVAG dagger inter-laboratory validation trial

DEFF Research Database (Denmark)

Forchhammer, Lykke; Johansson, Clara; Loft, Steffen

2010-01-01

the level of DNA damage in monocyte-derived THP-1 cells by either visual classification or computer-aided image analysis of pre-made slides, coded cryopreserved samples of cells and reference standard cells (calibration curve samples). The reference standard samples were irradiated with ionizing radiation...... by the different laboratories as evidenced by an inter-laboratory coefficient of variation (CV) of 47%. Adjustment of the primary comet assay end points by a calibration curve prepared in each laboratory reduced the CV to 28%, a statistically significant reduction (P test). A large fraction...

DNA damage and repair process in earthworm after in-vivo and in vitro exposure to soils irrigated by wastewaters

International Nuclear Information System (INIS)

Qiao Min; Chen Ying; Wang Chunxia; Wang Zijian; Zhu Yongguan

2007-01-01

In this study, DNA damage to earthworms (Eisenia fetida) after in vivo exposure to contaminated soils was measured by detecting DNA strand breakages (DSBs) and causality was analyzed through fractionation based bioassays. A non-linear dose-response relationship existed between DNA damage and total soil PAHs levels. DNA damage, measured with the comet assay, and its repair process, were observed. To identify the chemical causality, an in vitro comet assay using coelomocytes was subsequently performed on the fractionated organic extracts from soils. The results showed that the PAHs in the soils were responsible for the exerting genotoxic effects on earthworms. When normalized to benzo(a)pyrene toxic equivalent (TEQ BaP ), the saturation dose in the dose-response curve was about 10 ng TEQ BaP g -1 soil (dw). - A non-linear dose-response relationship exists between earthworm DNA damage, measured with comet assay, and total PAHs levels in soils irrigated by wastewaters

DNA damage and repair process in earthworm after in-vivo and in vitro exposure to soils irrigated by wastewaters

Energy Technology Data Exchange (ETDEWEB)

Qiao Min [State Key Laboratory of Environmental Aquatic Chemistry, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085 (China); Chen Ying [State Key Laboratory of Environmental Aquatic Chemistry, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085 (China); Wang Chunxia [State Key Laboratory of Environmental Aquatic Chemistry, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085 (China); Wang Zijian [State Key Laboratory of Environmental Aquatic Chemistry, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085 (China)]. E-mail: wangzj@rcees.ac.cn; Zhu Yongguan [State Key Laboratory of Environmental Aquatic Chemistry, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085 (China)

2007-07-15

In this study, DNA damage to earthworms (Eisenia fetida) after in vivo exposure to contaminated soils was measured by detecting DNA strand breakages (DSBs) and causality was analyzed through fractionation based bioassays. A non-linear dose-response relationship existed between DNA damage and total soil PAHs levels. DNA damage, measured with the comet assay, and its repair process, were observed. To identify the chemical causality, an in vitro comet assay using coelomocytes was subsequently performed on the fractionated organic extracts from soils. The results showed that the PAHs in the soils were responsible for the exerting genotoxic effects on earthworms. When normalized to benzo(a)pyrene toxic equivalent (TEQ{sub BaP}), the saturation dose in the dose-response curve was about 10 ng TEQ{sub BaP} g{sup -1} soil (dw). - A non-linear dose-response relationship exists between earthworm DNA damage, measured with comet assay, and total PAHs levels in soils irrigated by wastewaters.

Assessment of DNA damage induced by terrestrial UV irradiation of dried bloodstains: forensic implications.

Science.gov (United States)

Hall, Ashley; Sims, Lynn M; Ballantyne, Jack

2014-01-01

Few publications have detailed the nature of DNA damage in contemporary (i.e. non-ancient) dried biological stains. The chief concern, from a forensic standpoint, is that the damage can inhibit polymerase-mediated primer extension, ultimately resulting in DNA typing failure. In the work described here, we analyzed the effects of UVA and UVB irradiation on cell-free solubilized DNA, cell-free dehydrated DNA and dehydrated cellular DNA (from bloodstains). After UV exposure ranging from 25 J cm(-2) to 1236 J cm(-2), we assayed for the presence of bipyrimidine photoproducts (BPPPs), oxidative lesions and strand breaks, correlating the damage with the inhibition of STR profiling. Subsequent to irradiation with either UVA and UVB, the incidence of BPPPs, oxidative products and strand breaks were observed in decreasing quantities as follows: cell-free solubilized DNA>cell-free dehydrated DNA>bloodstain DNA. UVA irradiation did not result in even the partial loss of a STR profile in any sample tested. Somewhat different results were observed after genetic analysis of UVB exposed samples, in that the ability to produce a complete STR profile was affected earliest in bloodstain DNA, next in cell-free solubilized DNA and not at all in cell-free dehydrated DNA. Therefore, it is likely that other types of damage contributed to allele-drop-out in these samples but remained undetected by our assays, whereby the endonucleases did not react with the lesions or the presence of the lesions was masked by strand breaks. Under the conditions of the study, strand breaks appeared to be the predominant types of damage that ultimately resulted in DNA typing failure from physiological stains, although some evidence suggested oxidative damage may have played a role as well. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

An extended sequence specificity for UV-induced DNA damage.

Science.gov (United States)

Chung, Long H; Murray, Vincent

2018-01-01

The sequence specificity of UV-induced DNA damage was determined with a higher precision and accuracy than previously reported. UV light induces two major damage adducts: cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6-4)pyrimidone photoproducts (6-4PPs). Employing capillary electrophoresis with laser-induced fluorescence and taking advantages of the distinct properties of the CPDs and 6-4PPs, we studied the sequence specificity of UV-induced DNA damage in a purified DNA sequence using two approaches: end-labelling and a polymerase stop/linear amplification assay. A mitochondrial DNA sequence that contained a random nucleotide composition was employed as the target DNA sequence. With previous methodology, the UV sequence specificity was determined at a dinucleotide or trinucleotide level; however, in this paper, we have extended the UV sequence specificity to a hexanucleotide level. With the end-labelling technique (for 6-4PPs), the consensus sequence was found to be 5'-GCTC*AC (where C* is the breakage site); while with the linear amplification procedure, it was 5'-TCTT*AC. With end-labelling, the dinucleotide frequency of occurrence was highest for 5'-TC*, 5'-TT* and 5'-CC*; whereas it was 5'-TT* for linear amplification. The influence of neighbouring nucleotides on the degree of UV-induced DNA damage was also examined. The core sequences consisted of pyrimidine nucleotides 5'-CTC* and 5'-CTT* while an A at position "1" and C at position "2" enhanced UV-induced DNA damage. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

Increased oxidative DNA damage in mononuclear leukocytes in vitiligo

Energy Technology Data Exchange (ETDEWEB)

Giovannelli, Lisa [Department of Preclinical and Clinical Pharmacology, University of Florence, Viale Pieraccini 6, 50139 Florence (Italy)]. E-mail: lisag@pharm.unifi.it; Bellandi, Serena [Department of Dermatological Sciences, University of Florence, Viale Pieraccini 6, 50139 Florence (Italy); Pitozzi, Vanessa [Department of Preclinical and Clinical Pharmacology, University of Florence, Viale Pieraccini 6, 50139 Florence (Italy); Fabbri, Paolo [Department of Dermatological Sciences, University of Florence, Viale Pieraccini 6, 50139 Florence (Italy); Dolara, Piero [Department of Preclinical and Clinical Pharmacology, University of Florence, Viale Pieraccini 6, 50139 Florence (Italy); Moretti, Silvia [Department of Dermatological Sciences, University of Florence, Viale Pieraccini 6, 50139 Florence (Italy)

2004-11-22

Vitiligo is an acquired pigmentary disorder of the skin of unknown aetiology. The autocytotoxic hypothesis suggests that melanocyte impairment could be related to increased oxidative stress. Evidences have been reported that in vitiligo oxidative stress might also be present systemically. We used the comet assay (single cell alkaline gel electrophoresis) to evaluate DNA strand breaks and DNA base oxidation, measured as formamidopyrimidine DNA glycosylase (FPG)-sensitive sites, in peripheral blood cells from patients with active vitiligo and healthy controls. The basal level of oxidative DNA damage in mononuclear leukocytes was increased in vitiligo compared to normal subjects, whereas DNA strand breaks (SBs) were not changed. This alteration was not accompanied by a different capability to respond to in vitro oxidative challenge. No differences in the basal levels of DNA damage in polymorphonuclear leukocytes were found between patients and healthy subjects. Thus, this study supports the hypothesis that in vitiligo a systemic oxidative stress exists, and demonstrates for the first time the presence of oxidative alterations at the nuclear level. The increase in oxidative DNA damage shown in the mononuclear component of peripheral blood leukocytes from vitiligo patients was not particularly severe. However, these findings support an adjuvant role of antioxidant treatment in vitiligo.

Increased oxidative DNA damage in mononuclear leukocytes in vitiligo

International Nuclear Information System (INIS)

Giovannelli, Lisa; Bellandi, Serena; Pitozzi, Vanessa; Fabbri, Paolo; Dolara, Piero; Moretti, Silvia

2004-01-01

Vitiligo is an acquired pigmentary disorder of the skin of unknown aetiology. The autocytotoxic hypothesis suggests that melanocyte impairment could be related to increased oxidative stress. Evidences have been reported that in vitiligo oxidative stress might also be present systemically. We used the comet assay (single cell alkaline gel electrophoresis) to evaluate DNA strand breaks and DNA base oxidation, measured as formamidopyrimidine DNA glycosylase (FPG)-sensitive sites, in peripheral blood cells from patients with active vitiligo and healthy controls. The basal level of oxidative DNA damage in mononuclear leukocytes was increased in vitiligo compared to normal subjects, whereas DNA strand breaks (SBs) were not changed. This alteration was not accompanied by a different capability to respond to in vitro oxidative challenge. No differences in the basal levels of DNA damage in polymorphonuclear leukocytes were found between patients and healthy subjects. Thus, this study supports the hypothesis that in vitiligo a systemic oxidative stress exists, and demonstrates for the first time the presence of oxidative alterations at the nuclear level. The increase in oxidative DNA damage shown in the mononuclear component of peripheral blood leukocytes from vitiligo patients was not particularly severe. However, these findings support an adjuvant role of antioxidant treatment in vitiligo

Assessment of radiation induced cytogenetic damage in human keratinocytes by comet assay

International Nuclear Information System (INIS)

Joseph, Praveen; Sanjeev Ganesh; Narayana, Y.; Puthali, Abhay; Bhat, N.N.

2010-01-01

In the present study the effect of gamma radiation on normal human keratinocytes (HaCaT) cells has been analyzed using alkaline comet assay and a comparative study over the sensitivity of different comet parameters such as tail length (TL), olive tail moment (OTM) and percentage tail DNA (TDNA) has also been made. Human keratinocytes (HaCaT) cells were grown in Dulbecco's modified essential medium (DMEM) (10% FCS) at 37 °C in a humidified atmosphere containing 5% CO 2 . Cultured cells were harvested with 0.025 % trypsin EDTA. The sample (2 X 10 cells/ml) was exposed to gamma radiation of different dose using a 60 Co gamma source at dose rate of 2 Gy min -1 and the dosimetry has been carried out using Fricke and FBX dosimeters. After irradiation, to quantify the DNA damage the comet assay (single cell gel electrophoresis) was carried out under alkaline conditions, by the methods outlined by Singh et al. The quantification of the DNA strand breaks in each cells were performed using CASP software. The DNA damage quantification can be accomplished by measuring those comet parameters which exhibit a linear dependence on the amount of DNA damage. In the present study, comet parameters such as OTM, TL and TDNA were recorded and the variation of these parameters and their correlation coefficients for different doses of gamma radiation is plotted. The OTM value is normalized with control value and control for TL and TDNA is adjusted to zero to avoid initial variations in different experiments

Application of the DNA comet assay for detection of irradiated meat

International Nuclear Information System (INIS)

Kruszewski, M.; Iwanenko, T.; Wojewodzka, M.; Malec-Czechowska, K.; Dancewicz, A. M.; Szot, Z.

1998-01-01

Radiation induces damage to the DNA. This damage (fragmentation) can be assessed in the irradiated food using Single Cell Gel Electrophoresis (SCGE), known as DNA comet assay. Fragmentation of DNA may also be caused by improper storage of meat and repeated freezing and thawing. This makes identification of irradiated meat by this assay not reliable enough. In order to know the scale of the processes imitating radiation effects in DNA of the comets, their shape and lengths were examined in both irradiated and unirradiated fresh meat (D = 1.5 or 3.0 kGy) stored at 4 o C or frozen (-21 o ) up to 5 months. Comets formed upon SCGE were stained with DAPI or silver and examined in fluorescent or light microscope. They were divided arbitrarily into 4 classes. Comets of IV class were found quite often in fresh meat stored at 4 o C. In meat samples that were irradiated and stored frozen, comets of class I, II and III were observed. The negative comet test is univocal. Positive comet test, however, needs confirmation. The meat should be subjected to further analysis with other validated methods. (author)

Fisetin Protects DNA Against Oxidative Damage and Its Possible Mechanism.

Science.gov (United States)

Wang, Tingting; Lin, Huajuan; Tu, Qian; Liu, Jingjing; Li, Xican

2016-06-01

The paper tries to assess the protective effect of fisetin against •OH-induced DNA damage, then to investigate the possible mechanism. The protective effect was evaluated based on the content of malondialdehyde (MDA). The possible mechanism was analyzed using various antioxidant methods in vitro, including •OH scavenging (deoxyribose degradation), •O2 (-) scavenging (pyrogallol autoxidation), DPPH• scavenging, ABTS•(+) scavenging, and Cu(2+)-reducing power assays. Fisetin increased dose-dependently its protective percentages against •OH-induced DNA damage (IC50 value =1535.00±29.60 µM). It also increased its radical-scavenging percentages in a dose-dependent manner in various antioxidants assays. Its IC50 values in •OH scavenging, •O2(-) scavenging, DPPH• scavenging, ABTS•(+) scavenging, and Cu(2+)-reducing power assays, were 47.41±4.50 µM, 34.05±0.87 µM, 9.69±0.53 µM, 2.43±0.14 µM, and 1.49±0.16 µM, respectively. Fisetin can effectively protect DNA against •OH-induced oxidative damage possibly via reactive oxygen species (ROS) scavenging approach, which is assumed to be hydrogen atom (H•) and/or single electron (e) donation (HAT/SET) pathways. In the HAT pathway, the 3',4'-dihydroxyl moiety in B ring of fisetin is thought to play an important role, because it can be ultimately oxidized to a stable ortho-benzoquinone form.

Oxidative DNA damage during sleep periods among nightshift workers.

Science.gov (United States)

Bhatti, Parveen; Mirick, Dana K; Randolph, Timothy W; Gong, Jicheng; Buchanan, Diana Taibi; Zhang, Junfeng Jim; Davis, Scott

2016-08-01

Oxidative DNA damage may be increased among nightshift workers because of suppression of melatonin, a cellular antioxidant, and/or inflammation related to sleep disruption. However, oxidative DNA damage has received limited attention in previous studies of nightshift work. From two previous cross-sectional studies, urine samples collected during a night sleep period for 217 dayshift workers and during day and night sleep (on their first day off) periods for 223 nightshift workers were assayed for 8-hydroxydeoxyguanosine (8-OH-dG), a marker of oxidative DNA damage, using high-performance liquid chromatography with electrochemical detection. Urinary measures of 6-sulfatoxymelatonin (aMT6s), a marker of circulating melatonin levels, and actigraphy-based sleep quality data were also available. Nightshift workers during their day sleep periods excreted 83% (p=0.2) and 77% (p=0.03) of the 8-OH-dG that dayshift workers and they themselves, respectively, excreted during their night sleep periods. Among nightshift workers, higher aMT6s levels were associated with higher urinary 8-OH-dG levels, and an inverse U-shaped trend was observed between 8-OH-dG levels and sleep efficiency and sleep duration. Reduced excretion of 8-OH-dG among nightshift workers during day sleep may reflect reduced functioning of DNA repair machinery, which could potentially lead to increased cellular levels of oxidative DNA damage. Melatonin disruption among nightshift workers may be responsible for the observed effect, as melatonin is known to enhance repair of oxidative DNA damage. Quality of sleep may similarly impact DNA repair. Cellular levels of DNA damage will need to be evaluated in future studies to help interpret these findings. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Identification of irradiated refrigerated pork with the DNA comet assay

Energy Technology Data Exchange (ETDEWEB)

Araujo, M.M. E-mail: villavic@net.ipen.br; Marin-Huachaca, N.S.; Mancini-Filho, J. E-mail: jmancini@usp.br; Delincee, H.; Villavicencio, A.L.C.H. E-mail: henry.delincee@bfe.uni-karlsruhe.de

2004-10-01

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 {sup 60}Co source, Gammacell 220 (A.E.C.L.) installed in IPEN (Sao Paulo, Brazil). The doses given were 0, 1.5, 3.0 and 4.5 kGy for refrigerated samples. Immediately after irradiation the samples were returned to the refrigerator (6 deg. 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.

Identification of irradiated refrigerated pork with the DNA comet assay

Science.gov (United States)

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.

DNA damage in Populus tremuloides clones exposed to elevated O3

International Nuclear Information System (INIS)

Tai, Helen H.; Percy, Kevin E.; Karnosky, David F.

2010-01-01

The effects of elevated concentrations of atmospheric tropospheric ozone (O 3 ) on DNA damage in five trembling aspen (Populus tremuloides Michx.) clones growing in a free-air enrichment experiment in the presence and absence of elevated concentrations of carbon dioxide (CO 2 ) were examined. Growing season mean hourly O 3 concentrations were 36.3 and 47.3 ppb for ambient and elevated O 3 plots, respectively. The 4th highest daily maximum 8-h ambient and elevated O 3 concentrations were 79 and 89 ppb, respectively. Elevated CO 2 averaged 524 ppm (+150 ppm) over the growing season. Exposure to O 3 and CO 2 in combination with O 3 increased DNA damage levels above background as measured by the comet assay. Ozone-tolerant clones 271 and 8L showed the highest levels of DNA damage under elevated O 3 compared with ambient air; whereas less tolerant clone 216 and sensitive clones 42E and 259 had comparably lower levels of DNA damage with no significant differences between elevated O 3 and ambient air. Clone 8L was demonstrated to have the highest level of excision DNA repair. In addition, clone 271 had the highest level of oxidative damage as measured by lipid peroxidation. The results suggest that variation in cellular responses to DNA damage between aspen clones may contribute to O 3 tolerance or sensitivity. - Ozone tolerant clones and sensitive Populus tremuloides clones show differences in DNA damage and repair.

Modification of lymphocyte DNA damage by carotenoid supplementation in postmenopausal women.

Science.gov (United States)

Zhao, Xianfeng; Aldini, Giancarlo; Johnson, Elizabeth J; Rasmussen, Helen; Kraemer, Klaus; Woolf, Herb; Musaeus, Nina; Krinsky, Norman I; Russell, Robert M; Yeum, Kyung-Jin

2006-01-01

Oxidative stress has been implicated in the pathogenesis of chronic diseases related to aging such as cancer and cardiovascular disease. Carotenoids could be a part of a protective strategy to minimize oxidative damage in vulnerable populations, such as the elderly. Our aim was to determine the protective effect of carotenoids against DNA damage. A randomized, double-blind, placebo-controlled intervention study was conducted. Thirty-seven healthy, nonsmoking postmenopausal women aged 50-70 y were randomly assigned to 1 of 5 groups and were instructed to consume a daily dose of mixed carotenoids (beta-carotene, lutein, and lycopene; 4 mg each), 12 mg of a single carotenoid (beta-carotene, lutein, or lycopene), or placebo for 56 d. Plasma carotenoid concentrations were analyzed by using HPLC, and lymphocyte DNA damage was measured by using a single-cell gel electrophoresis (comet) assay. At day 57, all carotenoid-supplemented groups showed significantly lower endogenous DNA damage than at baseline (P lutein, beta-carotene, and lycopene), an intake that can be achieved by diet, or a larger dose (12 mg) of individual carotenoids exerts protection against DNA damage.

DNA Damage Following Pulmonary Exposure by Instillation to Low Doses of Carbon Black (Printex 90) Nanoparticles in Mice

DEFF Research Database (Denmark)

Kyjovska, Zdenka O.; Jacobsen, Nicklas R.; Saber, Anne T.

2015-01-01

of 0.67, 2, 6, and 162 mu g Printex 90 NPCB and vehicle. Cellular composition and protein concentration was evaluated in BAL fluid as markers of inflammatory response and cell damage. DNA strand breaks in BAL cells, lung, and liver tissue were assessed using the alkaline comet assay. The pulmonary...... the comet assay. We interpret the increased DNA strand breaks occurring following these low exposure doses of NPCB as DNA damage caused by primary genotoxicity in the absence of substantial inflammation, cell damage, and acute phase response. Environ. Mol. Mutagen. 56:41-49, 2015. (c) 2014 The Authors...

Are glutathione S transferases involved in DNA damage signalling? Interactions with DNA damage and repair revealed from molecular epidemiology studies

International Nuclear Information System (INIS)

Dusinska, Maria; Staruchova, Marta; Horska, Alexandra; Smolkova, Bozena; Collins, Andrew; Bonassi, Stefano; Volkovova, Katarina

2012-01-01

Glutathione S-transferases (GSTs) are members of a multigene family of isoenzymes that are important in the control of oxidative stress and in phase II metabolism. Acting non-enzymically, GSTs can modulate signalling pathways of cell proliferation, cell differentiation and apoptosis. Using a molecular epidemiology approach, we have investigated a potential involvement of GSTs in DNA damage processing, specifically the modulation of DNA repair in a group of 388 healthy adult volunteers; 239 with at least 5 years of occupational exposure to asbestos, stone wool or glass fibre, and 149 reference subjects. We measured DNA damage in lymphocytes using the comet assay (alkaline single cell gel electrophoresis): strand breaks (SBs) and alkali-labile sites, oxidised pyrimidines with endonuclease III, and oxidised purines with formamidopyrimidine DNA glycosylase. We also measured GST activity in erythrocytes, and the capacity for base excision repair (BER) in a lymphocyte extract. Polymorphisms in genes encoding three GST isoenzymes were determined, namely deletion of GSTM1 and GSTT1 and single nucleotide polymorphism Ile105Val in GSTP1. Consumption of vegetables and wine correlated negatively with DNA damage and modulated BER. GST activity correlated with oxidised bases and with BER capacity, and differed depending on polymorphisms in GSTP1, GSTT1 and GSTM1. A significantly lower BER rate was associated with the homozygous GSTT1 deletion in all asbestos site subjects and in the corresponding reference group. Multifactorial analysis revealed effects of sex and exposure in GSTP1 Ile/Val heterozygotes but not in Ile/Ile homozygotes. These variants affected also SBs levels, mainly by interactions of GSTP1 genotype with exposure, with sex, and with smoking habit; and by an interaction between sex and smoking. Our results show that GST polymorphisms and GST activity can apparently influence DNA stability and repair of oxidised bases, suggesting a potential new role for these

Are glutathione S transferases involved in DNA damage signalling? Interactions with DNA damage and repair revealed from molecular epidemiology studies

Energy Technology Data Exchange (ETDEWEB)

Dusinska, Maria, E-mail: Maria.DUSINSKA@nilu.no [CEE-Health Effects Group, NILU - Norwegian Institute for Air Research, Kjeller (Norway); Staruchova, Marta; Horska, Alexandra [Department of Experimental and Applied Genetics, Slovak Medical University, Bratislava (Slovakia); Smolkova, Bozena [Laboratory of Cancer Genetics, Cancer Research Institute of the Slovak Academy of Sciences, Bratislava (Slovakia); Collins, Andrew [Department of Nutrition, Faculty of Medicine, University of Oslo (Norway); Bonassi, Stefano [Unit of Clinical and Molecular Epidemiology, IRCCS San Raffaele Pisana, Rome (Italy); Volkovova, Katarina [Department of Experimental and Applied Genetics, Slovak Medical University, Bratislava (Slovakia)

2012-08-01

Glutathione S-transferases (GSTs) are members of a multigene family of isoenzymes that are important in the control of oxidative stress and in phase II metabolism. Acting non-enzymically, GSTs can modulate signalling pathways of cell proliferation, cell differentiation and apoptosis. Using a molecular epidemiology approach, we have investigated a potential involvement of GSTs in DNA damage processing, specifically the modulation of DNA repair in a group of 388 healthy adult volunteers; 239 with at least 5 years of occupational exposure to asbestos, stone wool or glass fibre, and 149 reference subjects. We measured DNA damage in lymphocytes using the comet assay (alkaline single cell gel electrophoresis): strand breaks (SBs) and alkali-labile sites, oxidised pyrimidines with endonuclease III, and oxidised purines with formamidopyrimidine DNA glycosylase. We also measured GST activity in erythrocytes, and the capacity for base excision repair (BER) in a lymphocyte extract. Polymorphisms in genes encoding three GST isoenzymes were determined, namely deletion of GSTM1 and GSTT1 and single nucleotide polymorphism Ile105Val in GSTP1. Consumption of vegetables and wine correlated negatively with DNA damage and modulated BER. GST activity correlated with oxidised bases and with BER capacity, and differed depending on polymorphisms in GSTP1, GSTT1 and GSTM1. A significantly lower BER rate was associated with the homozygous GSTT1 deletion in all asbestos site subjects and in the corresponding reference group. Multifactorial analysis revealed effects of sex and exposure in GSTP1 Ile/Val heterozygotes but not in Ile/Ile homozygotes. These variants affected also SBs levels, mainly by interactions of GSTP1 genotype with exposure, with sex, and with smoking habit; and by an interaction between sex and smoking. Our results show that GST polymorphisms and GST activity can apparently influence DNA stability and repair of oxidised bases, suggesting a potential new role for these

Chronic occupational exposure to hexavalent chromium causes DNA damage in electroplating workers.

Science.gov (United States)

Zhang, Xu-Hui; Zhang, Xuan; Wang, Xu-Chu; Jin, Li-Fen; Yang, Zhang-Ping; Jiang, Cai-Xia; Chen, Qing; Ren, Xiao-Bin; Cao, Jian-Zhong; Wang, Qiang; Zhu, Yi-Min

2011-04-12

Occupational exposure to chromium compounds may result in adverse health effects. This study aims to investigate whether low-level hexavalent chromium (Cr(VI)) exposure can cause DNA damage in electroplating workers. 157 electroplating workers and 93 control subjects with no history of occupational exposure to chromium were recruited in Hangzhou, China. Chromium levels in erythrocytes were determined by graphite furnace atomic absorption spectrophotometer. DNA damage in peripheral lymphocytes was evaluated with the alkaline comet assay by three parameters: Olive tail moment, tail length and percent of DNA in the comet tail (tail DNA%). Urinary 8-OHdG levels were measured by ELISA. Chromium concentration in erythrocytes was about two times higher in electroplating workers (median: 4.41 μg/L) than that in control subjects (1.54 μg/L, P electroplating workers. Low-level occupational chromium exposure induced DNA damage.

The Assessment of DNA Damage in Poultry Spermatozoa after Exposure to Low Doses of Ionising Radiation

International Nuclear Information System (INIS)

Kasuba, V.; Milic, M.; Pejakovic Hlede, J.; Gottstein, Z.; Karadjole, M.; Miljanic, S.

2013-01-01

The existence of dose-related induction of DNA strand breaks in spermatozoa following in vitro exposure to ionising radiation represents sperm DNA integrity as an important parameter in the evaluation of semen functionality. Maintaining of normal sperm becomes even more important when it is known that DNA in semen samples is already fragmentated in certain amount in human and turkey semen and that it lacks DNA repair mechanisms making DNA damage irreversible. The aim of this paper was to provide an insight in the amount of DNA damage detected in chicken spermatozoa (5 cocks, 45 weeks old) of heavy line after radiation with doses of 0.3, 0.5, 1 and 2 Gy gamma radiation and to address the question of the potential ecological consequences of the damage that was measured with comet assay. Scored parameters included tail intensity, tail length and tail moment. Results showed sensitivity of comet assay technique that detected significant DNA damage even after exposure to 0.3 Gy, but also showed no dose-related responses after 0.5, 1 and 2 Gy. Distribution of damaged cells was widely spread for the higher doses, showing the influence of possible adaptive response, but for further conclusions, larger studies are needed to answer that question.(author)

2-Aminopurine hairpin probes for the detection of ultraviolet-induced DNA damage

International Nuclear Information System (INIS)

El-Yazbi, Amira F.; Loppnow, Glen R.

2012-01-01

Highlights: ► Molecular beacon with 2AP bases detects DNA damage in a simple mix-and-read assay. ► Molecular beacons with 2AP bases detect damage at a 17.2 nM limit of detection. ► The 2AP molecular beacon is linear over a 0–3.5 μM concentration range for damage. - Abstract: Nucleic acid exposure to radiation and chemical insults leads to damage and disease. Thus, detection and understanding DNA damage is important for elucidating molecular mechanisms of disease. However, current methods of DNA damage detection are either time-consuming, destroy the sample, or are too specific to be used for generic detection of damage. In this paper, we develop a fluorescence sensor of 2-aminopurine (2AP), a fluorescent analogue of adenine, incorporated in the loop of a hairpin probe for the quantification of ultraviolet (UV) C-induced nucleic acid damage. Our results show that the selectivity of the 2AP hairpin probe to UV-induced nucleic acid damage is comparable to molecular beacon (MB) probes of DNA damage. The calibration curve for the 2AP hairpin probe shows good linearity (R 2 = 0.98) with a limit of detection of 17.2 nM. This probe is a simple, fast and economic fluorescence sensor for the quantification of UV-induced damage in DNA.

An immunochemical approach to the study of DNA damage and repair

International Nuclear Information System (INIS)

Wallace, S.S.

1991-08-01

The overall objective of this project is to produce antibodies to unique modified DNA bases and develop immunochemical assays to quantitate these lesions in damaged DNA. During this past year we have characterized antibodies to 8-oxopurines, produced novel antibodies to 5-hydroxyuracil and developed new methodologies to increase our level of sensitivity of detection. 7 refs., 5 figs

DNA damage in neurodegenerative diseases

Energy Technology Data Exchange (ETDEWEB)

Coppedè, Fabio, E-mail: fabio.coppede@med.unipi.it; Migliore, Lucia, E-mail: lucia.migliore@med.unipi.it

2015-06-15

Highlights: • Oxidative DNA damage is one of the earliest detectable events in the neurodegenerative process. • The mitochondrial DNA is more vulnerable to oxidative attack than the nuclear DNA. • Cytogenetic damage has been largely documented in Alzheimer's disease patients. • The question of whether DNA damage is cause or consequence of neurodegeneration is still open. • Increasing evidence links DNA damage and repair with epigenetic phenomena. - Abstract: Following the observation of increased oxidative DNA damage in nuclear and mitochondrial DNA extracted from post-mortem brain regions of patients affected by neurodegenerative diseases, the last years of the previous century and the first decade of the present one have been largely dedicated to the search of markers of DNA damage in neuronal samples and peripheral tissues of patients in early, intermediate or late stages of neurodegeneration. Those studies allowed to demonstrate that oxidative DNA damage is one of the earliest detectable events in neurodegeneration, but also revealed cytogenetic damage in neurodegenerative conditions, such as for example a tendency towards chromosome 21 malsegregation in Alzheimer's disease. As it happens for many neurodegenerative risk factors the question of whether DNA damage is cause or consequence of the neurodegenerative process is still open, and probably both is true. The research interest in markers of oxidative stress was shifted, in recent years, towards the search of epigenetic biomarkers of neurodegenerative disorders, following the accumulating evidence of a substantial contribution of epigenetic mechanisms to learning, memory processes, behavioural disorders and neurodegeneration. Increasing evidence is however linking DNA damage and repair with epigenetic phenomena, thereby opening the way to a very attractive and timely research topic in neurodegenerative diseases. We will address those issues in the context of Alzheimer's disease

DNA damage in neurodegenerative diseases

International Nuclear Information System (INIS)

Coppedè, Fabio; Migliore, Lucia

2015-01-01

Highlights: • Oxidative DNA damage is one of the earliest detectable events in the neurodegenerative process. • The mitochondrial DNA is more vulnerable to oxidative attack than the nuclear DNA. • Cytogenetic damage has been largely documented in Alzheimer's disease patients. • The question of whether DNA damage is cause or consequence of neurodegeneration is still open. • Increasing evidence links DNA damage and repair with epigenetic phenomena. - Abstract: Following the observation of increased oxidative DNA damage in nuclear and mitochondrial DNA extracted from post-mortem brain regions of patients affected by neurodegenerative diseases, the last years of the previous century and the first decade of the present one have been largely dedicated to the search of markers of DNA damage in neuronal samples and peripheral tissues of patients in early, intermediate or late stages of neurodegeneration. Those studies allowed to demonstrate that oxidative DNA damage is one of the earliest detectable events in neurodegeneration, but also revealed cytogenetic damage in neurodegenerative conditions, such as for example a tendency towards chromosome 21 malsegregation in Alzheimer's disease. As it happens for many neurodegenerative risk factors the question of whether DNA damage is cause or consequence of the neurodegenerative process is still open, and probably both is true. The research interest in markers of oxidative stress was shifted, in recent years, towards the search of epigenetic biomarkers of neurodegenerative disorders, following the accumulating evidence of a substantial contribution of epigenetic mechanisms to learning, memory processes, behavioural disorders and neurodegeneration. Increasing evidence is however linking DNA damage and repair with epigenetic phenomena, thereby opening the way to a very attractive and timely research topic in neurodegenerative diseases. We will address those issues in the context of Alzheimer's disease

DNA damage in the gill cells of the marine scallop Mizuhopecten yessoensis during anoxic stress and aerobic recovery

Science.gov (United States)

Slobodskova, Valentina V.; Zhukovskaya, Avianna F.; Chelomin, Victor P.

2012-06-01

Anoxia-induced DNA damage in the gill cells of the marine scallop Mizuhopecten yessoensis was assessed with the alkaline comet assay (single-cell gel electrophoresis). The alkaline comet assay method for detecting DNA strand breaks and alkali labile sites in individual cells. DNA damage was determened in the scallops ( M. yessoensis) gill cells. The scallops were exposed to air for 8 h showing a clear increase in the levels of DNA damage. After the air exposure, M. yessoensis were re-submersed for a period of 12 h, leading values to return to a pre-aerial exposure level. Control animals were kept immersed during the whole period. The resulting data demonstrate that natural influences, such as oxygen depletion (anoxia) in seawater, can be responsible for the induction of DNA damage. If the scallops were re-immersed in oxic conditions, the anoxically induced breaks were repaired. The main mechanisms influencing the integrity of the DNA structure are discussed in this paper.

Evaluation of the neutral comet assay for detection of alpha-particle induced DNA-double-strand-breaks

International Nuclear Information System (INIS)

Hofbauer, Daniela

2010-01-01

Aim of this study was to differentiate DNA-double-strand-breaks from DNA-single-strand-breaks on a single cell level, using the comet assay after α- and γ-irradiation. Americium-241 was used as a alpha-irradiation-source, Caesium-137 was used for γ-irradiation. Because of technical problems with both the neutral and alkaline comet assay after irradiation of gastric cancer cells and human lymphocytes, no definite differentiation of DNA-damage was possible.

Assessment of DNA damage in radiation workers by using single cell gel electrophoresis

International Nuclear Information System (INIS)

Jia Lili; Zhang Tao; Yang Yonghua; Wang Yan; Du Liqing; Cao Jia; Wang Hong; Liu Qiang; Fan Feiyue

2010-01-01

Objective: To assess the DNA damage of radiation workers in different grade hospitals, and to explore the correlation between the types of work or work time and the levels of DNA damage. Methods: DNA single strand break were detected by using alkaline single cell gel electrophoresis (SCGE), and the comet was analyzed with CASP (Comet Assay Software Project). TDNA%, TL, TM and OTM were calculated. Results: The parameters of SCGE in the radiation group were higher than those of control group (F=3.93, P<0.01). The significant difference was found not only among the different types of work or different work time, but also among the different grade hospitals (F=1.83, 1.91, P<0.05). Conclusions: Various levels of DNA damage could be detected in the radiation workers of the two hospitals. DNA damage of radiation workers is less serious in the higher-grade hospital than the lower grade one. Different types of work or work time might affect the DNA damage level. (authors)

Prototype Systems Containing Human Cytochrome P450 for High-Throughput Real-Time Detection of DNA Damage by Compounds That Form DNA-Reactive Metabolites.

Science.gov (United States)

Brito Palma, Bernardo; Fisher, Charles W; Rueff, José; Kranendonk, Michel

2016-05-16

The formation of reactive metabolites through biotransformation is the suspected cause of many adverse drug reactions. Testing for the propensity of a drug to form reactive metabolites has increasingly become an integral part of lead-optimization strategy in drug discovery. DNA reactivity is one undesirable facet of a drug or its metabolites and can lead to increased risk of cancer and reproductive toxicity. Many drugs are metabolized by cytochromes P450 in the liver and other tissues, and these reactions can generate hard electrophiles. These hard electrophilic reactive metabolites may react with DNA and may be detected in standard in vitro genotoxicity assays; however, the majority of these assays fall short due to the use of animal-derived organ extracts that inadequately represent human metabolism. The current study describes the development of bacterial systems that efficiently detect DNA-damaging electrophilic reactive metabolites generated by human P450 biotransformation. These assays use a GFP reporter system that detects DNA damage through induction of the SOS response and a GFP reporter to control for cytotoxicity. Two human CYP1A2-competent prototypes presented here have appropriate characteristics for the detection of DNA-damaging reactive metabolites in a high-throughput manner. The advantages of this approach include a short assay time (120-180 min) with real-time measurement, sensitivity to small amounts of compound, and adaptability to a microplate format. These systems are suitable for high-throughput assays and can serve as prototypes for the development of future enhanced versions.

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

Science.gov (United States)

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

DNA damage by carbonyl stress in human skin cells

International Nuclear Information System (INIS)

Roberts, Michael J.; Wondrak, Georg T.; Laurean, Daniel Cervantes; Jacobson, Myron K.; Jacobson, Elaine L.

2003-01-01

Reactive carbonyl species (RCS) are potent mediators of cellular carbonyl stress originating from endogenous chemical processes such as lipid peroxidation and glycation. Skin deterioration as observed in photoaging and diabetes has been linked to accumulative protein damage from glycation, but the effects of carbonyl stress on skin cell genomic integrity are ill defined. In this study, the genotoxic effects of acute carbonyl stress on HaCaT keratinocytes and CF3 fibroblasts were assessed. Administration of the α-dicarbonyl compounds glyoxal and methylglyoxal as physiologically relevant RCS inhibited skin cell proliferation, led to intra-cellular protein glycation as evidenced by the accumulation of N ε -(carboxymethyl)-L-lysine (CML) in histones, and caused extensive DNA strand cleavage as assessed by the comet assay. These effects were prevented by treatment with the carbonyl scavenger D-penicillamine. Both glyoxal and methylglyoxal damaged DNA in intact cells. Glyoxal caused DNA strand breaks while methylglyoxal produced extensive DNA-protein cross-linking as evidenced by pronounced nuclear condensation and total suppression of comet formation. Glycation by glyoxal and methylglyoxal resulted in histone cross-linking in vitro and induced oxygen-dependent cleavage of plasmid DNA, which was partly suppressed by the hydroxyl scavenger mannitol. We suggest that a chemical mechanism of cellular DNA damage by carbonyl stress occurs in which histone glycoxidation is followed by reactive oxygen induced DNA stand breaks. The genotoxic potential of RCS in cultured skin cells and its suppression by a carbonyl scavenger as described in this study have implications for skin damage and carcinogenesis and its prevention by agents selective for carbonyl stress

DNA damage by carbonyl stress in human skin cells

Energy Technology Data Exchange (ETDEWEB)

Roberts, Michael J.; Wondrak, Georg T.; Laurean, Daniel Cervantes; Jacobson, Myron K.; Jacobson, Elaine L

2003-01-28

Reactive carbonyl species (RCS) are potent mediators of cellular carbonyl stress originating from endogenous chemical processes such as lipid peroxidation and glycation. Skin deterioration as observed in photoaging and diabetes has been linked to accumulative protein damage from glycation, but the effects of carbonyl stress on skin cell genomic integrity are ill defined. In this study, the genotoxic effects of acute carbonyl stress on HaCaT keratinocytes and CF3 fibroblasts were assessed. Administration of the {alpha}-dicarbonyl compounds glyoxal and methylglyoxal as physiologically relevant RCS inhibited skin cell proliferation, led to intra-cellular protein glycation as evidenced by the accumulation of N{sup {epsilon}}-(carboxymethyl)-L-lysine (CML) in histones, and caused extensive DNA strand cleavage as assessed by the comet assay. These effects were prevented by treatment with the carbonyl scavenger D-penicillamine. Both glyoxal and methylglyoxal damaged DNA in intact cells. Glyoxal caused DNA strand breaks while methylglyoxal produced extensive DNA-protein cross-linking as evidenced by pronounced nuclear condensation and total suppression of comet formation. Glycation by glyoxal and methylglyoxal resulted in histone cross-linking in vitro and induced oxygen-dependent cleavage of plasmid DNA, which was partly suppressed by the hydroxyl scavenger mannitol. We suggest that a chemical mechanism of cellular DNA damage by carbonyl stress occurs in which histone glycoxidation is followed by reactive oxygen induced DNA stand breaks. The genotoxic potential of RCS in cultured skin cells and its suppression by a carbonyl scavenger as described in this study have implications for skin damage and carcinogenesis and its prevention by agents selective for carbonyl stress.

Close encounters for the first time: Helicase interactions with DNA damage.

Science.gov (United States)

Khan, Irfan; Sommers, Joshua A; Brosh, Robert M

2015-09-01

DNA helicases are molecular motors that harness the energy of nucleoside triphosphate hydrolysis to unwinding structured DNA molecules that must be resolved during cellular replication, DNA repair, recombination, and transcription. In vivo, DNA helicases are expected to encounter a wide spectrum of covalent DNA modifications to the sugar phosphate backbone or the nitrogenous bases; these modifications can be induced by endogenous biochemical processes or exposure to environmental agents. The frequency of lesion abundance can vary depending on the lesion type. Certain adducts such as oxidative base modifications can be quite numerous, and their effects can be helix-distorting or subtle perturbations to DNA structure. Helicase encounters with specific DNA lesions and more novel forms of DNA damage will be discussed. We will also review the battery of assays that have been used to characterize helicase-catalyzed unwinding of damaged DNA substrates. Characterization of the effects of specific DNA adducts on unwinding by various DNA repair and replication helicases has proven to be insightful for understanding mechanistic and biological aspects of helicase function in cellular DNA metabolism. Published by Elsevier B.V.

ATM-activated autotaxin (ATX) propagates inflammation and DNA damage in lung epithelial cells: a new mode of action for silica-induced DNA damage?

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Zheng, Huiyuan; Högberg, Johan; Stenius, Ulla

2017-12-07

Silica exposure is a common risk factor for lung cancer. It has been claimed that key elements in cancer development are activation of inflammatory cells that indirectly induce DNA damage and proliferative stimuli in respiratory epithelial cells. We studied DNA damage induced by silica particles in respiratory epithelial cells and focused the role of the signaling enzyme autotaxin (ATX). A549 and 16 bronchial epithelial cells (16HBE) lung epithelial cells were exposed to silica particles. Reactive oxygen species (ROS), NOD-like receptor family pyrin domain containing-3 (NLRP3) inflammasome activation, ATX, ataxia telangiectasia mutated (ATM), and DNA damage (γH2AX, pCHK1, pCHK2, comet assay) were end points. Low doses of silica induced NLRP3 activation, DNA damage accumulation, and ATM phosphorylation. A novel finding was that ATM induced ATX generation and secretion. Not only silica but also rotenone, camptothecin and H2O2 activated ATX via ATM, suggesting that ATX is part of a generalized ATM response to double-strand breaks (DSBs). Surprisingly, ATX inhibition mitigated DNA damage accumulation at later time points (6-16 h), and ATX transfection caused NLRP3 activation and DNA damage. Furthermore, the product of ATX enzymatic activity, lysophosphatidic acid, recapitulated the effects of ATX transfection. These data indicate an ATM-ATX-dependent loop that propagates inflammation and DSB accumulation, making low doses of silica effective inducers of DSBs in epithelial cells. We conclude that an ATM-ATX axis interconnects DSBs with silica-induced inflammation and propagates these effects in epithelial cells. Further studies of this adverse outcome pathway may give an accurate assessment of the lowest doses of silica that causes cancer. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Micronutrients intake associated with DNA damage assessed by in a human biomonitoring study

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Carina Ladeira

2015-05-01

Retinol was positively correlated with oxidative DNA damage in controls. The study by van Helden et al. (2009 demonstrated that vitamin A enhances OH radical formation in the Fenton reaction, showing that vitamin A can act as pro-oxidant or antioxidant, depending on the type of radicals involved, and may lead to DNA oxidative damage (Alakhras et al., 2011. Azqueta & Collins (2012 clearly distinguished between effects of vitamin A, pro-vitamin A carotenoids, and non-vitamin A carotenoids; being the latter group almost invariably reported to protect against DNA damage, whether endogenous or induced by exogenous agents, the pro-vitamin A carotenoids show a wider spectrum of effects, sometimes protecting and sometimes enhancing DNA damage. Vitamin E was found to be positively correlated with % DNA in tail. Watters et al. (2007 also found a positive association of vitamin E and oxidative DNA damage in a healthy, non-smoking population of young adults. A possible explanation for this result stems from some evidence that in the presence of copper or in smokers with a fat rich diet, vitamin E can act as a strong pro-oxidant, nevertheless it remains an unexpected result. Results found a positive correlation between iron and % DNA in tail, meaning that higher intake of iron associates with higher DNA damage. Oxidative lesions, and more specifically 8-OHdG, is one of the most prevalent lesions induced by iron containing substances (Prá et al., 2012, however the FPG biomarker was not statistically associated with iron. There is sound evidence that iron deficiency increases genome instability, among other mechanisms, by impairing enzymes involved in antioxidant and nuclei acid metabolism (Prá et al., 2012. Results presented herein found that the amount of calories ingested was negatively correlated with both biomarkers assessed by comet assay. This was somewhat unexpected, as calories restriction reduces metabolic rate and oxidative stress, meaning that lower calories

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

Science.gov (United States)

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

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

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Qiang Liu

2013-11-01

Full Text Available 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.

Investigations of antioxidant-mediated protection and mitigation of radiation-induced DNA damage and lipid peroxidation in murine skin.

Science.gov (United States)

Jelveh, Salomeh; Kaspler, Pavel; Bhogal, Nirmal; Mahmood, Javed; Lindsay, Patricia E; Okunieff, Paul; Doctrow, Susan R; Bristow, Robert G; Hill, Richard P

2013-08-01

Radioprotection and mitigation effects of the antioxidants, Eukarion (EUK)-207, curcumin, and the curcumin analogs D12 and D68, on radiation-induced DNA damage or lipid peroxidation in murine skin were investigated. These antioxidants were studied because they have been previously reported to protect or mitigate against radiation-induced skin reactions. DNA damage was assessed using two different assays. A cytokinesis-blocked micronucleus (MN) assay was performed on primary skin fibroblasts harvested from the skin of C3H/HeJ male mice 1 day, 1 week and 4 weeks after 5 Gy or 10 Gy irradiation. Local skin or whole body irradiation (100 kVp X-rays or caesium (Cs)-137 γ-rays respectively) was performed. DNA damage was further quantified in keratinocytes by immunofluorescence staining of γ-histone 2AX (γ-H2AX) foci in formalin-fixed skin harvested 1 hour or 1 day post-whole body irradiation. Radiation-induced lipid peroxidation in the skin was investigated at the same time points as the MN assay by measuring malondialdehyde (MDA) with a Thiobarbituric acid reactive substances (TBARS) assay. None of the studied antioxidants showed significant mitigation of skin DNA damage induced by local irradiation. However, when EUK-207 or curcumin were delivered before irradiation they provided some protection against DNA damage. In contrast, all the studied antioxidants demonstrated significant mitigating and protecting effects on radiation-induced lipid peroxidation at one or more of the three time points after local skin irradiation. Our results show no evidence for mitigation of DNA damage by the antioxidants studied in contrast to mitigation of lipid peroxidation. Since these agents have been reported to mitigate skin reactions following irradiation, the data suggest that changes in lipid peroxidation levels in skin may reflect developing skin reactions better than residual post-irradiation DNA damage in skin cells. Further direct comparison studies are required to confirm

Chronic occupational exposure to hexavalent chromium causes DNA damage in electroplating workers

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Ren Xiao-Bin

2011-04-01

Full Text Available Abstract Background Occupational exposure to chromium compounds may result in adverse health effects. This study aims to investigate whether low-level hexavalent chromium (Cr(VI exposure can cause DNA damage in electroplating workers. Methods 157 electroplating workers and 93 control subjects with no history of occupational exposure to chromium were recruited in Hangzhou, China. Chromium levels in erythrocytes were determined by graphite furnace atomic absorption spectrophotometer. DNA damage in peripheral lymphocytes was evaluated with the alkaline comet assay by three parameters: Olive tail moment, tail length and percent of DNA in the comet tail (tail DNA%. Urinary 8-OHdG levels were measured by ELISA. Results Chromium concentration in erythrocytes was about two times higher in electroplating workers (median: 4.41 μg/L than that in control subjects (1.54 μg/L, P P P P Conclusion The findings in this study indicated that there was detectable chromium exposure in electroplating workers. Low-level occupational chromium exposure induced DNA damage.

Comparison between sensitivity of a viscometric method and sensitivity of the alkaline elution assay for the determination of DNA damage induced by dimethylsulfate in vitro.

Science.gov (United States)

Parodi, S; Balbi, C; Taningher, M; Abelmoschi, M L; Pala, M; Parodi, G; Santi, L

1982-03-01

DNA damage induced by dimethylsulfate (DMS) was measured with a new oscillating crucible viscometer, having a U-shaped circular channel. Rat liver nuclei were treated in vitro. Viscosity was measured by lysing nuclei in an aklaline lysing solution (pH 12.5; 25 degrees C). Nuclei were lysed immediately in the viscometer and released DNA started to uncoil. In control samples the viscosity increased very slowly with time, reaching a maximum only after about 8 h. A progressively more rapid increase in viscosity was seen with increasing concentrations of DMS. The time of DNA disentanglement was sensitive to about 30 times less breaks than the alkaline elution assay.

Hepatocytes, rather than leukocytes reverse DNA damage in vivo induced by whole body y-irradiation of mice, as shown by the alkaline comet assay

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JUANA PINCHEIRA

2008-01-01

Full Text Available DNA damage repair was assessed in quiescent (G0 leukocytes and in hepatocytes of mice, after 1 and 2 hours recovery from a single whole body y-irradiation with 0.5, 1 or 2 Gy. Evaluation of single-strand breaks (SSB and alkali-labile sites together were carried out by a single-cell electrophoresis at pH>13.0 (alkaline comet assay. In non-irradiated (control mice, the constitutive, endogenous DNA damage (basal was around 1.5 times higher in leukocytes than in hepatocytes. Irradiation immediately increased SSB frequency in both cell types, in a dose-dependent manner. Two sequential phases took place during the in vivo repair of the radio-induced DNA lesions. The earliest one, present in both hepatocytes and leukocytes, further increased the SSB frequency, making evident the processing of some primary lesions in DNA bases into the SSB repair intermediates. In a second phase, SSB frequency decreased because of their removal. In hepatocytes, such a frequency regressed to the constitutive basal level after 2 hours recovery from either 0.5 orí Gy. On the other hand, the SSB repair phase was specifically abrogated in leukocytes, at the doses and recovery times analyzed. Thus, the efficiency of in vivo repair of radio-induced DNA damage in dormant cells (lymphocytes is quite different from that in hepatocytes whose low proliferation activity accounts only for cell renewal.

Mesotrione herbicide promotes biochemical changes and DNA damage in two fish species

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L.D.S. Piancini

2015-01-01

Full Text Available Mesotrione is one of the new herbicides that have emerged as an alternative after the ban of atrazine in the European Union. To our knowledge, any work using genetic or biochemical biomarkers was performed in any kind of fish evaluating the toxicity of this compound. The impact of acute (96 h exposure to environmentally relevant mesotrione concentrations (1.8, 7, 30, 115 e 460 μg L−1 were evaluated on the liver of Oreochorimis niloticus and Geophagus brasiliensis by assessing the activity of superoxide dismutase (SOD, glutathione peroxidase (GPx and glutathione-S- transferase (GST, the levels of reduced glutathione (GSH, carbonyl assays (PCO and lipid peroxide (LPO as well as the DNA damage to erithrocytes, liver and gills through the comet assay. We observed an increase in the concentration of GSH and the GPx activity in O. niloticus, and the GST and SOD activity in G. brasiliensis. We found significant increase in DNA damage in all tissues in both species. The results indicated that the acute exposure to mesotrione can induce oxidative stress and DNA damage in both species.

DNA damage response in monozygotic twins discordant for smoking habits.

Science.gov (United States)

Marcon, Francesca; Carotti, Daniela; Andreoli, Cristina; Siniscalchi, Ester; Leopardi, Paola; Caiola, Stefania; Biffoni, Mauro; Zijno, Andrea; Medda, Emanuela; Nisticò, Lorenza; Rossi, Sabrina; Crebelli, Riccardo

2013-03-01

Previous studies in twins indicate that non-shared environment, beyond genetic factors, contributes substantially to individual variation in mutagen sensitivity; however, the role of specific causative factors (e.g. tobacco smoke, diet) was not elucidated. In this investigation, a population of 22 couples of monozygotic twins with discordant smoking habits was selected with the aim of evaluating the influence of tobacco smoke on individual response to DNA damage. The study design virtually eliminated the contribution of genetic heterogeneity to the intra-pair variation in DNA damage response, and thus any difference in the end-points investigated could directly be attributed to the non-shared environment experienced by co-twins, which included as main factor cigarette smoke exposure. Peripheral lymphocytes of study subjects were challenged ex vivo with γ-rays, and the induction, processing, fixation of DNA damage evaluated through multiple approaches. Folate status of study subjects was considered significant covariate since it is affected by smoking habits and can influence radiosensitivity. Similar responses were elicited by γ-rays in co-twins for all the end-points analysed, despite their discordant smoking habits. Folate status did not modify DNA damage response, even though a combined effect of smoking habits, low-plasma folic acid level, and ionising radiation was observed on apoptosis. A possible modulation of DNA damage response by duration and intensity of tobacco smoke exposure was suggested by Comet assay and micronucleus data, but the effect was quantitatively limited. Overall, the results obtained indicate that differences in smoking habits do not contribute to a large extent to inter-individual variability in the response to radiation-induced DNA damage observed in healthy human populations.

Radiation-induced DNA damage in tumors and normal tissues. II. Influence of dose, residual DNA damage and physiological factors in oxygenated cells

International Nuclear Information System (INIS)

Zhang, H.; Wheeler, K.T.

1994-01-01

Detection and quantification of hypoxic cells in solid tumors is important for many experimental and clinical situations. Several laboratories, including ours, have suggested that assays which measure radiation-induced DNA strand breaks and DNA-protein crosslinks (DPCs) might be used to detect or quantify hypoxic cells in tumors and normal tissues. Recently, we demonstrated the feasibility of using an alkaline elution assay that measures strand breaks and DPCs to detect and/or quantify hypoxic cells in tissues. For this approach to be valid, DPCs must not be formed to any great extent in irradiated oxygenated cells, and the formation and repair of strand breaks and DPCs in oxygenated cells must not be modified appreciably by physiological factors (e.g., temperature, pH and nutrient depletion) that are often found in solid tumors. To address these issues, two sets of experiments were performed. In one set of experiments, oxygenated 9L cells in tissue culture, subcutaneous 9L tumors and rat cerebella were irradiated with doses of 15 or 50 Gy and allowed to repair until the residual strand break damage was low enough to detect DPCs. In another set of experiments, oxygenated exponentially growing or plateau-phase 9L cells in tissue culture were irradiated with a dose of 15 Gy at 37 or 20 degrees C, while the cells were maintained at a pH of either 6.6 or 7.3. DNA-protein crosslinks were formed in oxygenated cells about 100 times less efficiently than in hypoxic cells. In addition, temperature, pH, nutrient depletion and growth phase did not appreciably alter the formation and repair of strand breaks or the formation of DPCs in oxygenated 9L cells. These results support the use of this DNA damage assay for the detection and quantification of hypoxic cells in solid tumors. 27 refs., 5 tabs

Fertilization capacity with rainbow trout DNA-damaged sperm and embryo developmental success.

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Pérez-Cerezales, S; Martínez-Páramo, S; Beirão, J; Herráez, M P

2010-06-01

Mammalian spermatozoa undergo a strong selection process along the female tract to guarantee fertilization by good quality cells, but risks of fertilization with DNA-damaged spermatozoa have been reported. In contrast, most external fertilizers such as fish seem to have weaker selection procedures. This fact, together with their high prolificacy and external embryo development, indicates that fish could be useful for the study of the effects of sperm DNA damage on embryo development. We cryopreserved sperm from rainbow trout using egg yolk and low-density lipoprotein as additives to promote different rates of DNA damage. DNA fragmentation and oxidization were analyzed using comet assay with and without digestion with restriction enzymes, and fertilization trials were performed. Some embryo batches were treated with 3-aminobenzamide (3AB) to inhibit DNA repair by the poly (ADP-ribose) polymerase, which is an enzyme of the base excision repair pathway. Results showed that all the spermatozoa cryopreserved with egg yolk carried more than 10% fragmented DNA, maintaining fertilization rates of 61.1+/-2.3 but a high rate of abortions, especially during gastrulation, and only 14.5+/-4.4 hatching success. Furthermore, after 3AB treatment, hatching dropped to 3.2+/-2.2, showing that at least 10% DNA fragmentation was repaired. We conclude that trout sperm maintains its ability to fertilize in spite of having DNA damage, but that embryo survival is affected. Damage is partially repaired by the oocyte during the first cleavage. Important advantages of using rainbow trout for the study of processes related to DNA damage and repair during development have been reported.

Antioxidant and DNA damage protective properties of anthocyanin-rich extracts from Hibiscus and Ocimum: a comparative study.

Science.gov (United States)

Sarkar, Biswatrish; Kumar, Dhananjay; Sasmal, Dinakar; Mukhopadhyay, Kunal

2014-01-01

Anthocyanin extracts (AEs) from Ocimum tenuiflorum (leaf), Hibiscus rosa-sinensis (petal) and Hibiscus sabdariffa (calyx) were investigated and compared for in vitro antioxidant activity and DNA damage protective property. Total phenolic content (TPC) and total anthocyanin content (TAC) of the AEs were determined and the major anthocyanins were characterised. In vitro antioxidant activities were assessed by ferric-reducing antioxidant power (FRAP) assay, 2,2-diphenyl-1-picryl hydrazyl (DPPH) radical-scavenging activity, 2-deoxy-D-ribose degradation assay and lipid peroxidation assay. The protective property of the AEs was also examined against oxidative DNA damage by H2O2 and UV using pUC19 plasmid. All the AEs particularly those from O. tenuiflorum demonstrated efficient antioxidant activity and protected DNA from damage. Strong correlation between antioxidant capacity and TPC and TAC was observed. Significant correlation between antioxidant capacity and TPC and TAC ascertained that phenolics and anthocyanins were the major contributors of antioxidant activity.

Regenerative capacity of old muscle stem cells declines without significant accumulation of DNA damage.

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Wendy Cousin

Full Text Available The performance of adult stem cells is crucial for tissue homeostasis but their regenerative capacity declines with age, leading to failure of multiple organs. In skeletal muscle this failure is manifested by the loss of functional tissue, the accumulation of fibrosis, and reduced satellite cell-mediated myogenesis in response to injury. While recent studies have shown that changes in the composition of the satellite cell niche are at least in part responsible for the impaired function observed with aging, little is known about the effects of aging on the intrinsic properties of satellite cells. For instance, their ability to repair DNA damage and the effects of a potential accumulation of DNA double strand breaks (DSBs on their regenerative performance remain unclear. This work demonstrates that old muscle stem cells display no significant accumulation of DNA DSBs when compared to those of young, as assayed after cell isolation and in tissue sections, either in uninjured muscle or at multiple time points after injury. Additionally, there is no significant difference in the expression of DNA DSB repair proteins or globally assayed DNA damage response genes, suggesting that not only DNA DSBs, but also other types of DNA damage, do not significantly mark aged muscle stem cells. Satellite cells from DNA DSB-repair-deficient SCID mice do have an unsurprisingly higher level of innate DNA DSBs and a weakened recovery from gamma-radiation-induced DNA damage. Interestingly, they are as myogenic in vitro and in vivo as satellite cells from young wild type mice, suggesting that the inefficiency in DNA DSB repair does not directly correlate with the ability to regenerate muscle after injury. Overall, our findings suggest that a DNA DSB-repair deficiency is unlikely to be a key factor in the decline in muscle regeneration observed upon aging.

Umbelliferone suppresses radiation induced DNA damage and apoptosis in hematopoietic cells of mice

International Nuclear Information System (INIS)

Jayakumar, S.; Bhilwade, H.N.; Chaubey, R.C.

2012-01-01

Radiotherapy is one of the major modes of treatment for different types of cancers. But the success of radiotherapy is limited by injury to the normal cells. Protection of the normal cells from radiation damage by radioprotectors can increase therapeutic efficiency. These radioprotectors can also be used during nuclear emergency situations. Umbelliferone (UMB) is a wide spread natural product of the coumarin family. It occurs in many plants from the Apiaceae family. In the present study radioprotective effect of UMB was investigated in vitro and in vivo. Anti genotoxic effect of Umbelliferone was tested by treating the splenic lymphocytes with various doses of UMB (6.5 μM - 50 μM) prior to radiation (6Gy) exposure. After the radiation exposure, extent of DNA damage was assessed by comet assay at 5 mm and two hours after radiation exposure. At both the time points, it was observed that the pretreatment of UMB reduced the radiation induced DNA damage to a significant extent in comparison to radiation control. UMB pretreatment also significantly reduced the radiation induced apoptosis enumerated by propidium iodide staining assay. Results of clonogenic survival assay using intestinal cell line showed that pretreatment with UMB significantly protected against radiation induced loss of colony forming units. To assess the anti genotoxic role of umbelliferone in vivo two different doses of UMB (20 mg/Kg and 40 mg/Kg of body weight) were injected into Swiss mice or with vehicle and exposed to radiation. Thirty minutes after the radiation comet assay was performed in peripheral leukocytes. Frequency of micro nucleated erythrocytes was scored in bone marrow cells. It was observed that UMB alone did not cause any significant increase in DNA damage in comparison to control. Animals which are exposed to radiation alone showed significant increase in DNA damage and micronuclei frequency. But animals treated with UMB prior to the radiation exposure showed significant decrease

A high sensitivity, high throughput, automated single-cell gel electrophoresis ('Comet') DNA damage assay

International Nuclear Information System (INIS)

Vojnovic, B.; Barber, P.R.; Johnston, P.J.; Gregory, H.C.; Locke, R.J.

2003-01-01

A fully automated microscopy machine vision image capture and analysis system for the collection of data from slides of 'comets' has been developed. The novel image processing algorithms employed in delineating the 'comet head' from the 'comet tail' allow us to determine accurately very low levels of damage. In conjunction with calibrated and automated image capture methods, we are able to eliminate operator subjectivity and analyse large numbers of cells (>2500) in a short time (<1 hour). The image processing algorithm is designed to handle particularly difficult nuclei containing a high degree of structure, due to DNA clumping. We also present techniques used to extend the assay's dynamic range by removing interfering background fluorescence and to define a region of interest. If subtle biological variations are to be quantified (e.g. cell cycle dependant damage), then the use of large cell populations is dictated. Under those circumstances, the use of a fully automated system is particularly advantageous providing that the manner in which data is extracted does not introduce any inadvertent bias. In practice, it is essential that the image processing steps are geared towards the correct recognition of an acceptable cell nucleus, i.e. comet 'head'. We acknowledge the financial support of CRUK, Programme Grant C133/A1812 - SP 2195-01/02 and the US Department of Energy Low Dose Radiation Research Program grant DE-FG07-99ER62878

DNA damage detected by the alkaline comet assay in the liver of mice after oral administration of tetrachloroethylene

DEFF Research Database (Denmark)

Cederberg, H.; Henriksson, J.; Binderup, Mona-Lise

2010-01-01

in hepatocytes, indicating that tetrachloroethylene induced DNA damage in the liver. No effect on DNA damage was observed in the kidney. The results are in agreement with carcinogenicity data in mice, in which tetrachloroethylene induced tumours in the liver but not in the kidney, and support that a genotoxic...... mode of action might be involved in liver carcinogenicity in mice. An alternative interpretation of the results conveyed by the Study director at the test facility, involving that tetrachloroethylene did not induce DNA damage in the liver and kidney of mice, is also presented and discussed....

Association between age and repair of oxidatively damaged DNA in human peripheral blood mononuclear cells

DEFF Research Database (Denmark)

Løhr, Mille; Jensen, Annie; Eriksen, Louise

2015-01-01

damaged DNA in peripheral blood mononuclear cells (PBMCs). We isolated PBMCs from subjects aged 18-83 years, as part of a health survey of the Danish population that focussed on lifestyle factors. The level of DNA repair activity was measured as incisions on potassium bromate-damaged DNA by the comet...... assay. There was an inverse association between age and DNA repair activity with a 0.65% decline in activity per year from age 18 to 83 (95% confidence interval: 0.16-1.14% per year). Univariate regression analysis also indicated inverse associations between DNA repair activity and waist-hip ratio (P...

Sperm DNA damage in relation to lipid peroxidation following freezing-thawing of boar semen

OpenAIRE

Fraser, L.; Strzeżek, J.; Wasilewska, K.; Pareek, C.S.

2017-01-01

This study investigated the relationships between lipid peroxidation (LPO) and sperm DNA damage following freezing-thawing of boar semen in different extenders. The comet assay was used to measure the extent of sperm DNA damage in a cryoprotectant-free extender or in cryoprotectant-based extenders after single and repeated freezing and thawing. As well as an analysis of sperm motion characteristics, mitochondrial function, membrane integrity, and lipid peroxidation (LPO) were assessed simulta...

Inactivating UBE2M impacts the DNA damage response and genome integrity involving multiple cullin ligases.

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Scott Cukras

Full Text Available Protein neddylation is involved in a wide variety of cellular processes. Here we show that the DNA damage response is perturbed in cells inactivated with an E2 Nedd8 conjugating enzyme UBE2M, measured by RAD51 foci formation kinetics and cell based DNA repair assays. UBE2M knockdown increases DNA breakages and cellular sensitivity to DNA damaging agents, further suggesting heightened genomic instability and defective DNA repair activity. Investigating the downstream Cullin targets of UBE2M revealed that silencing of Cullin 1, 2, and 4 ligases incurred significant DNA damage. In particular, UBE2M knockdown, or defective neddylation of Cullin 2, leads to a blockade in the G1 to S progression and is associated with delayed S-phase dependent DNA damage response. Cullin 4 inactivation leads to an aberrantly high DNA damage response that is associated with increased DNA breakages and sensitivity of cells to DNA damaging agents, suggesting a DNA repair defect is associated. siRNA interrogation of key Cullin substrates show that CDT1, p21, and Claspin are involved in elevated DNA damage in the UBE2M knockdown cells. Therefore, UBE2M is required to maintain genome integrity by activating multiple Cullin ligases throughout the cell cycle.

Inactivating UBE2M impacts the DNA damage response and genome integrity involving multiple cullin ligases.

Science.gov (United States)

Cukras, Scott; Morffy, Nicholas; Ohn, Takbum; Kee, Younghoon

2014-01-01

Protein neddylation is involved in a wide variety of cellular processes. Here we show that the DNA damage response is perturbed in cells inactivated with an E2 Nedd8 conjugating enzyme UBE2M, measured by RAD51 foci formation kinetics and cell based DNA repair assays. UBE2M knockdown increases DNA breakages and cellular sensitivity to DNA damaging agents, further suggesting heightened genomic instability and defective DNA repair activity. Investigating the downstream Cullin targets of UBE2M revealed that silencing of Cullin 1, 2, and 4 ligases incurred significant DNA damage. In particular, UBE2M knockdown, or defective neddylation of Cullin 2, leads to a blockade in the G1 to S progression and is associated with delayed S-phase dependent DNA damage response. Cullin 4 inactivation leads to an aberrantly high DNA damage response that is associated with increased DNA breakages and sensitivity of cells to DNA damaging agents, suggesting a DNA repair defect is associated. siRNA interrogation of key Cullin substrates show that CDT1, p21, and Claspin are involved in elevated DNA damage in the UBE2M knockdown cells. Therefore, UBE2M is required to maintain genome integrity by activating multiple Cullin ligases throughout the cell cycle.

Attenuated DNA damage repair by trichostatin A through BRCA1 suppression.

Science.gov (United States)

Zhang, Yin; Carr, Theresa; Dimtchev, Alexandre; Zaer, Naghmeh; Dritschilo, Anatoly; Jung, Mira

2007-07-01

Recent studies have demonstrated that some histone deacetylase (HDAC) inhibitors enhance cellular radiation sensitivity. However, the underlying mechanism for such a radiosensitizing effect remains unexplored. Here we show evidence that treatment with the HDAC inhibitor trichostatin A (TSA) impairs radiation-induced repair of DNA damage. The effect of TSA on the kinetics of DNA damage repair was measured by performing the comet assay and gamma-H2AX focus analysis in radioresistant human squamous carcinoma cells (SQ-20B). TSA exposure increased the amount of radiation-induced DNA damage and slowed the repair kinetics. Gene expression profiling also revealed that a majority of the genes that control cell cycle, DNA replication and damage repair processes were down-regulated after TSA exposure, including BRCA1. The involvement of BRCA1 was further demonstrated by expressing ectopic wild-type BRCA1 in a BRCA1 null cell line (HCC-1937). TSA treatment enhanced radiation sensitivity of HCC-1937/wtBRCA1 clonal cells, which restored cellular radiosensitivity (D(0) = 1.63 Gy), to the control level (D(0) = 1.03 Gy). However, TSA had no effect on the level of radiosensitivity of BRCA1 null cells. Our data demonstrate for the first time that TSA treatment modulates the radiation-induced DNA damage repair process, in part by suppressing BRCA1 gene expression, suggesting that BRCA1 is one of molecular targets of TSA.

Study of genetic damage in the Japanese oyster induced by an environmentally-relevant exposure to diuron: evidence of vertical transmission of DNA damage.

Science.gov (United States)

Barranger, A; Akcha, F; Rouxel, J; Brizard, R; Maurouard, E; Pallud, M; Menard, D; Tapie, N; Budzinski, H; Burgeot, T; Benabdelmouna, A

2014-01-01

Pesticides represent a major proportion of the chemical pollutants detected in French coastal waters and hence a significant environmental risk with regards to marine organisms. Commercially-raised bivalves are particularly exposed to pollutants, among them pesticides, as shellfish farming zones are subject to considerable pressure from agricultural activities on the mainland. The aims of this study were to determine (1) the genotoxic effects of diuron exposure on oyster genitors and (2) the possible transmission of damaged DNA to offspring and its repercussions on oyster fitness. To investigate these points, oysters were exposed to concentrations of diuron close to those detected in the Marennes-Oleron Basin (two 7-day exposure pulses at 0.4 and 0.6 μg L(-1)) during the gametogenesis period. Genomic abnormalities were characterized using two complementary approaches. The Comet assay was applied for the measurement of early and reversible primary DNA damage, whereas flow cytometry was used to assess the clastogenic and aneugenic effect of diuron exposure. Polar Organic Chemical Integrative Samplers (POCIS) were used in exposed and assay tanks to confirm the waterborne concentration of diuron reached during the experiment. The results obtained by the Comet assay clearly showed a higher level of DNA strand breaks in both the hemocytes and spermatozoa of diuron-exposed genitors. The transmission of damaged genetic material to gamete cells could be responsible for the genetic damage measured in offspring. Indeed, flow cytometry analyses showed the presence of DNA breakage and a significant decrease in DNA content in spat from diuron-exposed genitors. The transmission of DNA damage to the offspring could be involved in the negative effects observed on offspring development (decrease in hatching rate, higher level of larval abnormalities, delay in metamorphosis) and growth. In this study, the vertical transmission of DNA damage was so highlighted by subjecting oyster

Evaluation of DNA-damaging marine natural product with potential anticancer activity

International Nuclear Information System (INIS)

Nisa, M.; Amjad, S.; Chaudhary, M.I.; Sualah, R.; Khan, S.H.

2002-01-01

The treatment for the dreadful disease cancer require a continued development of novel and improved chemo preventive and chemotherapeutic agents. An exploitable feature of tumor cell is that it has defect in its ability to repair damage to DNA as compared with normal cell, suggesting that agent with selective toxicity towards DNA repair deficient cell might be potential anticancer agent. In a recently developed mechanism based approach discovery. DNA repair a recombination-deficient mutants of the yeast Saccharomyces cerevisiae were utilized, as yeast and bacteria are the popular genetically engineered microorganisms. We have scanned organic solvent extracts of about thirty five different species of marine flora and fauna under DNA-damaging activity assays. Marine plants showed no activity towards this bioassay, whereas marine animals tested under this bioassay showed good activity. Detail results of our studies will be discussed in this paper. (author)

The alkaline comet assay used in evaluation of genotoxic damage of drinking water disinfection by-products (bromoform and chloroform

Directory of Open Access Journals (Sweden)

Messaouda Khallef

2015-06-01

Full Text Available The alkaline comet assay (pH 12.3 is a useful method for monitoring genotoxic effects of environmental pollutants in the root nuclei of Allium cepa and various plants; it allows the detection of single- and double-strand breaks, incomplete excision-repair sites and cross-links. It has been introduced to detect even small changes in DNA structure. It is a technically simple, highly sensitive, fast and economic test which detects in vitro and in vivo genotoxicity (DNA integrity and packing mode in any cell types examined, and requires just a few cells for its execution (Liman et al., 2011; Yıldız et al., 2009. Chloroform and bromoform are the most important trihalomethanes found in drinking water. Different concentrations of bromoform (25, 50, 75and 100µg/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 using one-way analysis of variance (ANOVA were employed and p<0.05 was accepted as the test of significance. Comet assay results showed that DNA damage was significant at p <0.05 for the different concentrations of chloroform and bromoform compared to the negative control which has a damage rate equal to 3.5 ± 0.7 and the positive control which has damage rate equal to 13.5 ± 2.12. The exposure of root tip cells to these disinfection by-products increases DNA damage. All concentrations examined in this study of bromoform and chloroform cause significant harm, which could be due to DNA damage induced by oxidative stress. The measurement of DNA damage in the nuclei of higher plant tissues is a new area of study with SCGE. This assay could be incorporated into in situ monitoring of atmosphere, water and soil: the comet assay allows a fast detection without

DNA damage by Auger emitters

International Nuclear Information System (INIS)

Martin, R.F.; d'Cunha, Glenn; Gibbs, Richard; Murray, Vincent; Pardee, Marshall; Allen, B.J.

1988-01-01

125 I atoms can be introduced at specific locations along a defined DNA target molecule, either by site-directed incorporation of an 125 I-labelled deoxynucleotide or by binding of an 125 I-labelled sequence-selective DNA ligand. After allowing accumulation of 125 I decay-induced damage to the DNA, application of DNA sequencing techniques enables positions of strand breaks to be located relative to the site of decay, at a resolution corresponding to the distance between adjacent nucleotides [0.34 nm]. Thus, DNA provides a molecular framework to analyse the extent of damage following [averaged] individual decay events. Results can be compared with energy deposition data generated by computer-simulation methods developed by Charlton et al. The DNA sequencing technique also provides information about the chemical nature of the termini of the DNA chains produced following Auger decay-induced damage. In addition to reviewing the application of this approach to the analysis of 125 I decay induced DNA damage, some more recent results obtained by using 67 Ga are also presented. (author)

DNA Damage, Repair, and Cancer Metabolism

Science.gov (United States)

Turgeon, Marc-Olivier; Perry, Nicholas J. S.; Poulogiannis, George

2018-01-01

Although there has been a renewed interest in the field of cancer metabolism in the last decade, the link between metabolism and DNA damage/DNA repair in cancer has yet to be appreciably explored. In this review, we examine the evidence connecting DNA damage and repair mechanisms with cell metabolism through three principal links. (1) Regulation of methyl- and acetyl-group donors through different metabolic pathways can impact DNA folding and remodeling, an essential part of accurate double strand break repair. (2) Glutamine, aspartate, and other nutrients are essential for de novo nucleotide synthesis, which dictates the availability of the nucleotide pool, and thereby influences DNA repair and replication. (3) Reactive oxygen species, which can increase oxidative DNA damage and hence the load of the DNA-repair machinery, are regulated through different metabolic pathways. Interestingly, while metabolism affects DNA repair, DNA damage can also induce metabolic rewiring. Activation of the DNA damage response (DDR) triggers an increase in nucleotide synthesis and anabolic glucose metabolism, while also reducing glutamine anaplerosis. Furthermore, mutations in genes involved in the DDR and DNA repair also lead to metabolic rewiring. Links between cancer metabolism and DNA damage/DNA repair are increasingly apparent, yielding opportunities to investigate the mechanistic basis behind potential metabolic vulnerabilities of a substantial fraction of tumors. PMID:29459886

Biomonitoring of genotoxic risk in radar facility workers: comparison of the comet assay with micronucleus assay and chromatid breakage assay

International Nuclear Information System (INIS)

Garaj-Vrhovac, V.; Kopjar, N.

2003-01-01

Genotoxic risks of occupational exposure in a radar facility were evaluated by using alkaline comet assay, micronucleus assay and chromatid breakage assay on peripheral blood leukocytes in exposed subjects and corresponding controls. Results show that occupational exposure to microwave radiation correlates with an increase of genome damage in somatic cells. The levels of DNA damage in exposed subjects determined by using alkaline comet assay were increased compared to control and showed interindividual variations. Incidence of micronuclei was also significantly increased compared to baseline control values. After short exposure of cultured lymphocytes to bleomycin, cells of occupationally exposed subjects responded with high numbers of chromatid breaks. Although the level of chromosome damage generated by bleomycin varied greatly between individuals, in exposed subjects a significantly elevated number of chromatid breaks was observed. Our results support data reported in literature indicating that microwave radiation represents a potential DNA-damaging hazard. Alkaline comet assay is confirmed as a sensitive and highly reproducible technique for detection of primary DNA damage inflicted in somatic cells. Micronucleus assay was confirmed as reliable bio-markers of effect and chromatid breakage assay as sensitive bio-marker of individual cancer susceptibility. The results obtained also confirm the necessity to improve measures and to perform accurate health surveillance of individuals occupationally exposed to microwave radiation

The DNA damage response during mitosis

International Nuclear Information System (INIS)

Heijink, Anne Margriet; Krajewska, Małgorzata; Vugt, Marcel A.T.M. van

2013-01-01

Cells are equipped with a cell-intrinsic signaling network called the DNA damage response (DDR). This signaling network recognizes DNA lesions and initiates various downstream pathways to coordinate a cell cycle arrest with the repair of the damaged DNA. Alternatively, the DDR can mediate clearance of affected cells that are beyond repair through apoptosis or senescence. The DDR can be activated in response to DNA damage throughout the cell cycle, although the extent of DDR signaling is different in each cell cycle phase. Especially in response to DNA double strand breaks, only a very marginal response was observed during mitosis. Early on it was recognized that cells which are irradiated during mitosis continued division without repairing broken chromosomes. Although these initial observations indicated diminished DNA repair and lack of an acute DNA damage-induced cell cycle arrest, insight into the mechanistic re-wiring of DDR signaling during mitosis was only recently provided. Different mechanisms appear to be at play to inactivate specific signaling axes of the DDR network in mitosis. Importantly, mitotic cells not simply inactivate the entire DDR, but appear to mark their DNA damage for repair after mitotic exit. Since the treatment of cancer frequently involves agents that induce DNA damage as well as agents that block mitotic progression, it is clinically relevant to obtain a better understanding of how cancer cells deal with DNA damage during interphase versus mitosis. In this review, the molecular details concerning DDR signaling during mitosis as well as the consequences of encountering DNA damage during mitosis for cellular fate are discussed

The DNA damage response during mitosis

Energy Technology Data Exchange (ETDEWEB)

Heijink, Anne Margriet; Krajewska, Małgorzata; Vugt, Marcel A.T.M. van, E-mail: m.vugt@umcg.nl

2013-10-15

Cells are equipped with a cell-intrinsic signaling network called the DNA damage response (DDR). This signaling network recognizes DNA lesions and initiates various downstream pathways to coordinate a cell cycle arrest with the repair of the damaged DNA. Alternatively, the DDR can mediate clearance of affected cells that are beyond repair through apoptosis or senescence. The DDR can be activated in response to DNA damage throughout the cell cycle, although the extent of DDR signaling is different in each cell cycle phase. Especially in response to DNA double strand breaks, only a very marginal response was observed during mitosis. Early on it was recognized that cells which are irradiated during mitosis continued division without repairing broken chromosomes. Although these initial observations indicated diminished DNA repair and lack of an acute DNA damage-induced cell cycle arrest, insight into the mechanistic re-wiring of DDR signaling during mitosis was only recently provided. Different mechanisms appear to be at play to inactivate specific signaling axes of the DDR network in mitosis. Importantly, mitotic cells not simply inactivate the entire DDR, but appear to mark their DNA damage for repair after mitotic exit. Since the treatment of cancer frequently involves agents that induce DNA damage as well as agents that block mitotic progression, it is clinically relevant to obtain a better understanding of how cancer cells deal with DNA damage during interphase versus mitosis. In this review, the molecular details concerning DDR signaling during mitosis as well as the consequences of encountering DNA damage during mitosis for cellular fate are discussed.

Environmental ozone exposure and oxidative DNA damage in adult residents of Florence, Italy

Energy Technology Data Exchange (ETDEWEB)

Palli, Domenico, E-mail: d.palli@ispo.toscana.i [Molecular and Nutritional Epidemiology Unit, Cancer Prevention and Research Institute (ISPO), Via Cosimo il Vecchio 2, 50139 Florence (Italy); Sera, Francesco, E-mail: f.sera@ispo.toscana.i [Molecular and Nutritional Epidemiology Unit, Cancer Prevention and Research Institute (ISPO), Via Cosimo il Vecchio 2, 50139 Florence (Italy); Giovannelli, Lisa, E-mail: lisag@pharm.unifi.i [Department of Pharmacology, University of Florence, Viale G.Pieraccini 6, 50139 Florence (Italy); Masala, Giovanna, E-mail: g.masala@ispo.toscana.i [Molecular and Nutritional Epidemiology Unit, Cancer Prevention and Research Institute (ISPO), Via Cosimo il Vecchio 2, 50139 Florence (Italy); Grechi, Daniele [Regional Environmental Protection Agency of Tuscany (ARPAT), Via Porpora 22, 50144 Florence (Italy); Bendinelli, Benedetta, E-mail: b.bendinelli@ispo.toscana.i [Molecular and Nutritional Epidemiology Unit, Cancer Prevention and Research Institute (ISPO), Via Cosimo il Vecchio 2, 50139 Florence (Italy); Caini, Saverio, E-mail: s.caini@ispo.toscana.i [Molecular and Nutritional Epidemiology Unit, Cancer Prevention and Research Institute (ISPO), Via Cosimo il Vecchio 2, 50139 Florence (Italy); Dolara, Piero, E-mail: piero.dolara@unifi.i [Department of Pharmacology, University of Florence, Viale G.Pieraccini 6, 50139 Florence (Italy); Saieva, Calogero, E-mail: c.saieva@ispo.toscana.i [Molecular and Nutritional Epidemiology Unit, Cancer Prevention and Research Institute (ISPO), Via Cosimo il Vecchio 2, 50139 Florence (Italy)

2009-05-15

In 71 adults residing in Florence, Italy, enrolled in a prospective study, we investigated the correlation between individual levels of oxidative DNA damage detected by the Comet assay in circulating lymphocytes, and a specific ozone exposure score calculated in 10 different time-windows (0-5 to 0-90 days) before blood drawing, based on daily measurements provided by the local environmental monitoring system. Overall, statistically significant positive correlations between average ozone concentrations and DNA damage emerged in almost all time-windows considered; correlations were more evident among males, non-smokers, and traffic-exposed workers. Multivariate regression analyses taking into account selected individual characteristics, showed an independent effect on DNA damage of average ozone concentrations in the last 60-90 days before blood drawing. Local residents showed a divergent pattern with correlations restricted to shorter time-windows. Our results suggest that ozone concentrations at ground levels modulate oxidative DNA damage in circulating lymphocytes of residents of polluted areas. - Ozone concentrations over the 60-90 days before blood drawing correlated with DNA damage in circulating lymphocytes of adults living in the metropolitan area of Florence, Italy.

Environmental ozone exposure and oxidative DNA damage in adult residents of Florence, Italy

International Nuclear Information System (INIS)

Palli, Domenico; Sera, Francesco; Giovannelli, Lisa; Masala, Giovanna; Grechi, Daniele; Bendinelli, Benedetta; Caini, Saverio; Dolara, Piero; Saieva, Calogero

2009-01-01

In 71 adults residing in Florence, Italy, enrolled in a prospective study, we investigated the correlation between individual levels of oxidative DNA damage detected by the Comet assay in circulating lymphocytes, and a specific ozone exposure score calculated in 10 different time-windows (0-5 to 0-90 days) before blood drawing, based on daily measurements provided by the local environmental monitoring system. Overall, statistically significant positive correlations between average ozone concentrations and DNA damage emerged in almost all time-windows considered; correlations were more evident among males, non-smokers, and traffic-exposed workers. Multivariate regression analyses taking into account selected individual characteristics, showed an independent effect on DNA damage of average ozone concentrations in the last 60-90 days before blood drawing. Local residents showed a divergent pattern with correlations restricted to shorter time-windows. Our results suggest that ozone concentrations at ground levels modulate oxidative DNA damage in circulating lymphocytes of residents of polluted areas. - Ozone concentrations over the 60-90 days before blood drawing correlated with DNA damage in circulating lymphocytes of adults living in the metropolitan area of Florence, Italy.

Involvement of DNA polymerase beta in repair of ionizing radiation damage as measured by in vitro plasmid assays.

NARCIS (Netherlands)

Vens, C.; Hofland, I.; Begg, A.C.

2007-01-01

Characteristic of damage introduced in DNA by ionizing radiation is the induction of a wide range of lesions. Single-strand breaks (SSBs) and base damages outnumber double-strand breaks (DSBs). If unrepaired, these lesions can lead to DSBs and increased mutagenesis. XRCC1 and DNA polymerase beta

Radiation-induced DNA damage as a function of DNA hydration

International Nuclear Information System (INIS)

Swarts, S.G.; Miao, L.; Wheeler, K.T.; Sevilla, M.D.; Becker, D.

1995-01-01

Radiation-induced DNA damage is produced from the sum of the radicals generated by the direct ionization of the DNA (direct effect) and by the reactions of the DNA with free radicals formed in the surrounding environment (indirect effect). The indirect effect has been believed to be the predominant contributor to radiation-induced intracellular DNA damage, mainly as the result of reactions of bulk water radicals (e.g., OH·) with DNA. However, recent evidence suggests that DNA damage, derived from the irradiation of water molecules that are tightly bound in the hydration layer, may occur as the result of the transfer of electron-loss centers (e.g. holes) and electrons from these water molecules to the DNA. Since this mechanism for damaging DNA more closely parallels that of the direct effect, the irradiation of these tightly bound water molecules may contribute to a quasi-direct effect. These water molecules comprise a large fraction of the water surrounding intracellular DNA and could account for a significant proportion of intracellular radiation-induced DNA damage. Consequently, the authors have attempted to characterize this quasi-direct effect to determine: (1) the extent of the DNA hydration layer that is involved with this effect, and (2) what influence this effect has on the types and quantities of radiation-induced DNA damage

DNA Comet Assay. A simple screening technique for identification of some irradiated foods

International Nuclear Information System (INIS)

Khan, A.A.; Khan, H.M.

2008-01-01

DNA Comet Assay method was carried out to detect irradiation treatment of some foods like meat, spices, beans and lentils. The fresh meat of cow and duck were irradiated up to radiation doses of 3 kGy, the spices (cardamoms and cumin black) were irradiated to radiation doses of 5, 10, 15 and 20 kGy while the beans (black beans and white beans) and lentils (red and green lentils) were irradiated to 0.5 and 1 kGy. All the foods were then analyzed for radiation treatment using simple microgel electrophoresis of single cells or nuclei (DNA Comet Assay). Sedimentation, lysis and staining times were adjusted to get optimized conditions for correct and easy analysis of each food. Using these optimized conditions, it was found out that radiation damaged DNA showed comets in case of irradiated food samples, whereas in non-treated food samples, round or conical spots of stained DNA were visible. Shape, length and intensity of these comets were also radiation dose dependent. Screening of unirradiated and irradiated samples by Comet Assay was successful in the case of all the foods under consideration under the optimized conditions of assay. Therefore, for different kinds of irradiated foods studied in the present study, the DNA Comet Assay can be used as a rapid, simple and inexpensive screening test. (author)

Assessment of okadaic acid effects on cytotoxicity, DNA damage and DNA repair in human cells.

Science.gov (United States)

Valdiglesias, Vanessa; Méndez, Josefina; Pásaro, Eduardo; Cemeli, Eduardo; Anderson, Diana; Laffon, Blanca

2010-07-07

Okadaic acid (OA) is a phycotoxin produced by several types of dinoflagellates causing diarrheic shellfish poisoning (DSP) in humans. Symptoms induced by DSP toxins are mainly gastrointestinal, but the intoxication does not appear to be fatal. Despite this, this toxin presents a potential threat to human health even at concentrations too low to induce acute toxicity, since previous animal studies have shown that OA has very potent tumour promoting activity. However, its concrete action mechanism has not been described yet and the results reported with regard to OA cytotoxicity and genotoxicity are often contradictory. In the present study, the genotoxic and cytotoxic effects of OA on three different types of human cells (peripheral blood leukocytes, HepG2 hepatoma cells, and SHSY5Y neuroblastoma cells) were evaluated. Cells were treated with a range of OA concentrations in the presence and absence of S9 fraction, and MTT test and Comet assay were performed in order to evaluate cytotoxicity and genotoxicity, respectively. The possible effects of OA on DNA repair were also studied by means of the DNA repair competence assay, using bleomycin as DNA damage inductor. Treatment with OA in absence of S9 fraction induced not statistically significant decrease in cell viability and significant increase in DNA damage in all cell types at the highest concentrations investigated. However, only SHSY5Y cells showed OA induced genotoxic and cytotoxic effects in presence of S9 fraction. Furthermore, we found that OA can induce modulations in DNA repair processes when exposure was performed prior to BLM treatment, in co-exposure, or during the subsequent DNA repair process. Copyright 2010 Elsevier B.V. All rights reserved.

The DNA damage response during mitosis.

Science.gov (United States)

Heijink, Anne Margriet; Krajewska, Małgorzata; van Vugt, Marcel A T M

2013-10-01

Cells are equipped with a cell-intrinsic signaling network called the DNA damage response (DDR). This signaling network recognizes DNA lesions and initiates various downstream pathways to coordinate a cell cycle arrest with the repair of the damaged DNA. Alternatively, the DDR can mediate clearance of affected cells that are beyond repair through apoptosis or senescence. The DDR can be activated in response to DNA damage throughout the cell cycle, although the extent of DDR signaling is different in each cell cycle phase. Especially in response to DNA double strand breaks, only a very marginal response was observed during mitosis. Early on it was recognized that cells which are irradiated during mitosis continued division without repairing broken chromosomes. Although these initial observations indicated diminished DNA repair and lack of an acute DNA damage-induced cell cycle arrest, insight into the mechanistic re-wiring of DDR signaling during mitosis was only recently provided. Different mechanisms appear to be at play to inactivate specific signaling axes of the DDR network in mitosis. Importantly, mitotic cells not simply inactivate the entire DDR, but appear to mark their DNA damage for repair after mitotic exit. Since the treatment of cancer frequently involves agents that induce DNA damage as well as agents that block mitotic progression, it is clinically relevant to obtain a better understanding of how cancer cells deal with DNA damage during interphase versus mitosis. In this review, the molecular details concerning DDR signaling during mitosis as well as the consequences of encountering DNA damage during mitosis for cellular fate are discussed. Copyright © 2013 Elsevier B.V. All rights reserved.

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

Directory of Open Access Journals (Sweden)

J Strzezek

2004-03-01

Full Text Available 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

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

Science.gov (United States)

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

DNA Damage, Mutagenesis and Cancer

Directory of Open Access Journals (Sweden)

Ashis K. Basu

2018-03-01

Full Text Available A large number of chemicals and several physical agents, such as UV light and γ-radiation, have been associated with the etiology of human cancer. Generation of DNA damage (also known as DNA adducts or lesions induced by these agents is an important first step in the process of carcinogenesis. Evolutionary processes gave rise to DNA repair tools that are efficient in repairing damaged DNA; yet replication of damaged DNA may take place prior to repair, particularly when they are induced at a high frequency. Damaged DNA replication may lead to gene mutations, which in turn may give rise to altered proteins. Mutations in an oncogene, a tumor-suppressor gene, or a gene that controls the cell cycle can generate a clonal cell population with a distinct advantage in proliferation. Many such events, broadly divided into the stages of initiation, promotion, and progression, which may occur over a long period of time and transpire in the context of chronic exposure to carcinogens, can lead to the induction of human cancer. This is exemplified in the long-term use of tobacco being responsible for an increased risk of lung cancer. This mini-review attempts to summarize this wide area that centers on DNA damage as it relates to the development of human cancer.

Monitoring of DNA and cytogenetic damage in lymphocytes in patients with skin cancer disease

International Nuclear Information System (INIS)

Cebulska-Wasilewska, A.; Dyga, W.; Krasnowolski, S.; Wierzewska, A.; Budzanowska, E.

1999-01-01

One major oncogenesis process is activation of proto-oncogenes by point mutations or chromosomal translocations. There is substantial evidence that indicates that human carcinogenesis involves loss of heterozygosity of certain chromosomes. Our study aimed at searching the possible association between cancer and DNA and cytogenetic abnormalities induced in lymphocytes of people with various categories of skin cancer cells. Fresh blood was collected by venepuncture from 25 individuals (including nine prior to cancer treatment). All patients were nonsmoking males, however 42.3% of them were former smokers. Blood samples were divided into two parts; in the first part of samples cytogenetic studies were performed immediately, while lymphocytes from the other part were isolated and stored at -70 0C for further studies in vitro. A single cell gel electrophoresis assay (SCGE), known as a Comet assay, was performed on them to study individual susceptibility to the induction of DNA damage by UV or radiation and to estimate variability in cellular repair capabilities. On average 220 good metaphase spreads per sample in the first mitotic division, and 100 spreads per sample in the second division were accepted for analysis of the cytogenetic damage. Chromosome and chromatid type aberrations were scored in the cells in the first mitosis, and expressed as total aberration frequency including and excluding gaps. Sister chromatid exchanges , high frequency cells and proliferating rate index were screened and evaluated in the second mitosis. Each patient showed a level exceeding (in at least one of the cytogenetic biomarker) the biomarker level in a reference group. In order to estimate susceptibility of people to environmentally induced damage, the isolated lymphocytes were irradiated with 2 Gy dose of X-rays or 6 J/m 2 of UV radiation, and the single cell gel electrophoresis (SCGE assay) was performed. To compare various individual capabilities to repair the induced damage

Genetic polymorphisms in homologous recombination repair genes in healthy Slovenian population and their influence on DNA damage

International Nuclear Information System (INIS)

Goricar, Katja; Erculj, Nina; Zadel, Maja; Dolzan, Vita

2012-01-01

Homologous recombination (HR) repair is an important mechanism involved in repairing double-strand breaks in DNA and for maintaining genomic stability. Polymorphisms in genes coding for enzymes involved in this pathway may influence the capacity for DNA repair. The aim of this study was to select tag single nucleotide polymorphisms (SNPs) in specific genes involved in HR repair, to determine their allele frequencies in a healthy Slovenian population and their influence on DNA damage detected with comet assay. In total 373 individuals were genotyped for nine tag SNPs in three genes: XRCC3 722C>T, XRCC3 -316A>G, RAD51 -98G>C, RAD51 -61G>T, RAD51 1522T>G, NBS1 553G>C, NBS1 1197A>G, NBS1 37117C>T and NBS1 3474A>C using competitive allele-specific amplification (KASPar assay). Comet assay was performed in a subgroup of 26 individuals to determine the influence of selected SNPs on DNA damage. We observed that age significantly affected genotype frequencies distribution of XRCC3 -316A>G (P = 0.039) in healthy male blood donors. XRCC3 722C>T (P = 0.005), RAD51 -61G>T (P = 0.023) and NBS1 553G>C (P = 0.008) had a statistically significant influence on DNA damage. XRCC3 722C>T, RAD51 -61G>T and NBS1 553G>C polymorphisms significantly affect the repair of damaged DNA and may be of clinical importance as they are common in Slovenian population

Low levels of methylmercury induce DNA damage in rats: protective effects of selenium

Energy Technology Data Exchange (ETDEWEB)

Grotto, Denise; Barcelos, Gustavo R.M.; Antunes, Lusania M.G.; Barbosa, Fernando [Universidade de Sao Paulo, Departamento de Analises Clinicas, Toxicologicas e Bromatologicas, Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, Ribeirao Preto, Sao Paulo (Brazil); Valentini, Juliana [Universidade de Sao Paulo, Departamento de Analises Clinicas, Toxicologicas e Bromatologicas, Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, Ribeirao Preto, Sao Paulo (Brazil); Universidade Federal de Santa Maria, Departamento de Analises Clinicas e Toxicologicas, Santa Maria, Rio Grande do Sul (Brazil); Angeli, Jose Pedro F. [Universidade de Sao Paulo, Departamento de Bioquimica, Instituto de Quimica, Sao Paulo (Brazil); Garcia, Solange C. [Universidade Federal de Santa Maria, Departamento de Analises Clinicas e Toxicologicas, Santa Maria, Rio Grande do Sul (Brazil)

2009-03-15

In this study we examined the possible antigenotoxic effect of selenium (Se) in rats chronically exposed to low levels of methylmercury (MeHg) and the association between glutathione peroxidase (GSH-Px) activity and DNA lesions (via comet assay) in the same exposed animals. Rats were divided into six groups as follows: (Group I) received water; (Group II) received MeHg (100 {mu}g/day); (Group III) received Se (2 mg/L drinking water); (Group IV) received Se (6 mg/L drinking water); (Group V) received MeHg (100 {mu}g/day) and Se (2 mg/L drinking water); (Group VI) received MeHg (100 {mu}g/day) and Se (6 mg/L drinking water). Total treatment time was 100 days. GSH-Px activity was determined spectrophotometrically and DNA damage was determined by comet assay. Mean GSH-Px activity in groups I, II, III, IV, V and VI were, respectively: 40.19{+-}17.21; 23.63{+-}6.04; 42.64{+-}5.70; 38.50{+-}7.15; 34.54{+-}6.18 and 41.39{+-}11.67 nmolNADPH/min/gHb. DNA damage was represented by a mean score from 0 to 300; the results for groups I, II, III, IV, V and VI were, respectively: 6.87{+-}3.27; 124.12{+-}13.74; 10.62{+-}3.81; 13.25{+-}1.76; 86.87{+-}11.95 and 76.25{+-}7.48. There was a significant inhibition of GSH-Px activity in group II compared with group I (P<0.05). Groups V and VI did not show a difference in enzyme activity compared with groups III and IV, showing the possible protective action of Se. Comet assay presented a significant difference in DNA migration between group II and group I (P<0.0001). Groups V and VI showed a significant reduction in MeHg-induced genotoxicity (P < 0.001) when compared with group II. A negative correlation (r = -0.559, P<0.05) was found between GSH-Px activity and DNA lesion, showing that the greater the DNA damage, the lower the GSH-Px activity. Our findings demonstrated the oxidative and genotoxic properties of MeHg, even at low doses. Moreover, Se co-administration reestablished GSH-Px activity and reduced DNA damage. (orig.)

DNA damage in plant herbarium tissue.

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Staats, Martijn; Cuenca, Argelia; Richardson, James E; Vrielink-van Ginkel, Ria; Petersen, Gitte; Seberg, Ole; Bakker, Freek T

2011-01-01

Dried plant herbarium specimens are potentially a valuable source of DNA. Efforts to obtain genetic information from this source are often hindered by an inability to obtain amplifiable DNA as herbarium DNA is typically highly degraded. DNA post-mortem damage may not only reduce the number of amplifiable template molecules, but may also lead to the generation of erroneous sequence information. A qualitative and quantitative assessment of DNA post-mortem damage is essential to determine the accuracy of molecular data from herbarium specimens. In this study we present an assessment of DNA damage as miscoding lesions in herbarium specimens using 454-sequencing of amplicons derived from plastid, mitochondrial, and nuclear DNA. In addition, we assess DNA degradation as a result of strand breaks and other types of polymerase non-bypassable damage by quantitative real-time PCR. Comparing four pairs of fresh and herbarium specimens of the same individuals we quantitatively assess post-mortem DNA damage, directly after specimen preparation, as well as after long-term herbarium storage. After specimen preparation we estimate the proportion of gene copy numbers of plastid, mitochondrial, and nuclear DNA to be 2.4-3.8% of fresh control DNA and 1.0-1.3% after long-term herbarium storage, indicating that nearly all DNA damage occurs on specimen preparation. In addition, there is no evidence of preferential degradation of organelle versus nuclear genomes. Increased levels of C→T/G→A transitions were observed in old herbarium plastid DNA, representing 21.8% of observed miscoding lesions. We interpret this type of post-mortem DNA damage-derived modification to have arisen from the hydrolytic deamination of cytosine during long-term herbarium storage. Our results suggest that reliable sequence data can be obtained from herbarium specimens.

Correlation of binding efficacies of DNA to flavonoids and their induced cellular damage.

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Das, Asmita; Majumder, Debashis; Saha, Chabita

2017-05-01

Flavonoids are dietary intakes which are bestowed with several health benefits. The most studied property of flavonoids is their antioxidant efficacy. Among the chosen flavonoids Quercetin, Kaempferol and Myricetin is catagorized as flavonols whereas Apigenin and Luteolin belong to the flavone group. In the present study anti-cancer properties of flavonoids are investigated on the basis of their binding efficacy to ct-DNA and their ability to induce cytotoxicity in K562 leukaemic cells. The binding affinities of the flavonoids with calf thymus DNA (ct-DNA) are in the order Quercetin>Myricetin>Luteolin>Kaempferol>Apigenin. Quercetin with fewer OH than myricetin has higher affinity towards DNA suggesting that the number and position of OH influence the binding efficacies of flavonoids to ct-DNA. CD spectra and EtBr displacement studies evidence myricetin and apigenin to be stronger intercalators of DNA compared to quercetin. From comet assay results it is observed that quercetin and myricetin when used in combination induce higher DNA damage in K562 leukemic cells than when tested individually. Higher binding efficacy has been recorded for quercetin to DNA at lower pH, which is the micro environment of cancerous cells, and hence quercetin can act as a potential anti-cancer agent. Presence of Cu also increases cellular damage as recorded by comet assay. Copyright © 2017. Published by Elsevier B.V.

Characterization of ionizing radiation damage in DNA. Progress report, May 1, 1975--April 30, 1976

International Nuclear Information System (INIS)

Hawkins, R.B.

1976-01-01

The objective of this research is the characterization and quantitative assay of ionizing radiation-induced damage in DNA and nucleoprotein. Two lines of investigation have been pursued. The first is aimed at detection and assay of DNA to protein covalent cross linkage in coliphage T7. Protein and DNA are labeled with 14 C and 32 P, respectively. Cross linkage is assessed from the amount of labeled protein distributing like DNA and labeled DNA distributing like protein on a phenol-water countercurrent distribution system. It has been found that damage involving cross linkage occurs by two modes of radiation action in phage irradiated with 60 Co γ rays in .001M histidine medium. Indirect effects play a large role in one mode and direct effects play a large role in the other. In the second line of investigation, the hydrodynamic and viscoelastic properties of DNA from irradiated phage and cells will be examined to determine the extent to which DNA to DNA cross linkage and points of altered flexibility are introduced by radiation. An instrument for viscoelastic measurements has been constructed in preparation for these studies

Endogenous melatonin and oxidatively damaged guanine in DNA

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Poulsen Henrik E

2009-10-01

Full Text Available Abstract Background A significant body of literature indicates that melatonin, a hormone primarily produced nocturnally by the pineal gland, is an important scavenger of hydroxyl radicals and other reactive oxygen species. Melatonin may also lower the rate of DNA base damage resulting from hydroxyl radical attack and increase the rate of repair of that damage. This paper reports the results of a study relating the level of overnight melatonin production to the overnight excretion of the two primary urinary metabolites of the repair of oxidatively damaged guanine in DNA. Methods Mother-father-daughter(s families (n = 55 were recruited and provided complete overnight urine samples. Total overnight creatinine-adjusted 6-sulphatoxymelatonin (aMT6s/Cr has been shown to be highly correlated with total overnight melatonin production. Urinary 8-oxo-7,8-dihydro-guanine (8-oxoGua results from the repair of DNA or RNA guanine via the nucleobase excision repair pathway, while urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG may possibly result from the repair of DNA guanine via the nucleotide excision repair pathway. Total overnight urinary levels of 8-oxodG and 8-oxoGua are therefore a measure of total overnight guanine DNA damage. 8-oxodG and 8-oxoGua were measured using a high-performance liquid chromatography-electrospray ionization tandem mass spectrometry assay. The mother, father, and oldest sampled daughter were used for these analyses. Comparisons between the mothers, fathers, and daughters were calculated for aMT6s/Cr, 8-oxodG, and 8-oxoGua. Regression analyses of 8-oxodG and 8-oxoGua on aMT6s/Cr were conducted for mothers, fathers, and daughters separately, adjusting for age and BMI (or weight. Results Among the mothers, age range 42-80, lower melatonin production (as measured by aMT6s/CR was associated with significantly higher levels of 8-oxodG (p Conclusion Low levels of endogenous melatonin production among older individuals may lead to

DNA damage and repair in plants

International Nuclear Information System (INIS)

Britt, A.B.

1996-01-01

The biological impact of any DNA damaging agent is a combined function of the chemical nature of the induced lesions and the efficiency and accuracy of their repair. Although much has been learned frommicrobes and mammals about both the repair of DNA damage and the biological effects of the persistence of these lesions, much remains to be learned about the mechanism and tissue-specificity of repair in plants. This review focuses on recent work on the induction and repair of DNA damage in higher plants, with special emphasis on UV-induced DNA damage products. (author)

A constitutive damage specific DNA-binding protein is synthesized at higher levels in UV-irradiated primate cells

International Nuclear Information System (INIS)

Hirschfeld, S.; Levine, A.S.; Ozato, K.; Protic, M.

1990-01-01

Using a DNA band shift assay, we have identified a DNA-binding protein complex in primate cells which is present constitutively and has a high affinity for UV-irradiated, double-stranded DNA. Cells pretreated with UV light, mitomycin C, or aphidicolin have higher levels of this damage-specific DNA-binding protein complex, suggesting that the signal for induction can either be damage to the DNA or interference with cellular DNA replication. Physiochemical modifications of the DNA and competition analysis with defined substrates suggest that the most probable target site for the damage-specific DNA-binding protein complex is a 6-4'-(pyrimidine-2'-one)-pyrimidine dimer: specific binding could not be detected with probes which contain -TT- cyclobutane dimers, and damage-specific DNA binding did not decrease after photoreactivation of UV-irradiated DNA. This damage-specific DNA-binding protein complex is the first such inducible protein complex identified in primate cells. Cells from patients with the sun-sensitive cancer-prone disease, xeroderma pigmentosum (group E), are lacking both the constitutive and the induced damage-specific DNA-binding activities. These findings suggest a possible role for this DNA-binding protein complex in lesion recognition and DNA repair of UV-light-induced photoproducts

Effects of acute and chronic administration of fenproporex on DNA damage parameters in young and adult rats.

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Gonçalves, Cinara L; Rezin, Gislaine T; Ferreira, Gabriela K; Jeremias, Isabela C; Cardoso, Mariane R; Valvassori, Samira S; Munhoz, Bruna J P; Borges, Gabriela D; Bristot, Bruno N; Leffa, Daniela D; Andrade, Vanessa M; Quevedo, João; Streck, Emilio L

2013-08-01

Obesity is a chronic and multifactorial disease, whose prevalence is increasing in many countries. Pharmaceutical strategies for the treatment of obesity include drugs that regulate food intake, thermogenesis, fat absorption, and fat metabolism. Fenproporex is the second most commonly consumed amphetamine-based anorectic worldwide; this drug is rapidly converted in vivo into amphetamine, which is associated with neurotoxicity. In this context, the present study evaluated DNA damage parameters in the peripheral blood of young and adult rats submitted to an acute administration and chronic administration of fenproporex. In the acute administration, both young and adult rats received a single injection of fenproporex (6.25, 12.5 or 25 mg/kg i.p.) or vehicle. In the chronic administration, both young and adult rats received one daily injection of fenproporex (6.25, 12.5, or 25 mg/kg i.p.) or Tween for 14 days. 2 h after the last injection, the rats were killed by decapitation and their peripheral blood removed for evaluation of DNA damage parameters by alkaline comet assay. Our study showed that acute administration of fenproporex in young and adult rats presented higher levels of damage index and frequency in the DNA. However, chronic administration of fenproporex in young and adult rats did not alter the levels of DNA damage in both parameters of comet assay. The present findings showed that acute administration of fenproporex promoted damage in DNA, in both young and adult rats. Our results are consistent with other reports which showed that other amphetamine-derived drugs also caused DNA damage. We suggest that the activation of an efficient DNA repair mechanism may occur after chronic exposition to fenproporex. Our results are consistent with other reports that showed some amphetamine-derived drugs also caused DNA damage.

Electromagnetic noise inhibits radiofrequency radiation-induced DNA damage and reactive oxygen species increase in human lens epithelial cells

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Wu, Wei; Wang, KaiJun; Ni, Shuang; Ye, PanPan; Yu, YiBo; Ye, Juan; Sun, LiXia

2008-01-01

Purpose The goal of this study was to investigate whether superposing of electromagnetic noise could block or attenuate DNA damage and intracellular reactive oxygen species (ROS) increase of cultured human lens epithelial cells (HLECs) induced by acute exposure to 1.8 GHz radiofrequency field (RF) of the Global System for Mobile Communications (GSM). Methods An sXc-1800 RF exposure system was used to produce a GSM signal at 1.8 GHz (217 Hz amplitude-modulated) with the specific absorption rate (SAR) of 1, 2, 3, and 4 W/kg. After 2 h of intermittent exposure, the ROS level was assessed by the fluorescent probe, 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). DNA damage to HLECs was examined by alkaline comet assay and the phosphorylated form of histone variant H2AX (γH2AX) foci formation assay. Results After exposure to 1.8 GHz RF for 2 h, HLECs exhibited significant intracellular ROS increase in the 2, 3, and 4 W/kg groups. RF radiation at the SAR of 3 W/kg and 4 W/kg could induce significant DNA damage, examined by alkaline comet assay, which was used to detect mainly single strand breaks (SSBs), while no statistical difference in double strand breaks (DSBs), evaluated by γH2AX foci, was found between RF exposure (SAR: 3 and 4 W/kg) and sham exposure groups. When RF was superposed with 2 μT electromagnetic noise could block RF-induced ROS increase and DNA damage. Conclusions DNA damage induced by 1.8 GHz radiofrequency field for 2 h, which was mainly SSBs, may be associated with the increased ROS production. Electromagnetic noise could block RF-induced ROS formation and DNA damage. PMID:18509546

Is lack of sleep capable of inducing DNA damage in aged skin?

Science.gov (United States)

Kahan, V; Ribeiro, D A; Egydio, F; Barros, L A; Tomimori, J; Tufik, S; Andersen, M L

2014-01-01

Skin naturally changes with age, becoming more fragile. Various stimuli can alter skin integrity. The aim of this study was to evaluate whether sleep deprivation affects the integrity of DNA in skin and exacerbates the effects of aging. Fifteen-month old female Hairless mice underwent 72 h of paradoxical sleep deprivation or 15 days of chronic sleep restriction. Punch biopsies of the skin were taken to evaluate DNA damage by single cell gel (comet) assay. Neither paradoxical sleep deprivation nor sleep restriction increased genetic damage, measured by tail movement and tail intensity values. Taken together, the findings are consistent with the notion that aging overrides the effect of sleep loss on the genetic damage in elderly mice. © 2014 S. Karger AG, Basel.

Mechanisms for radiation damage in DNA. Final report, June 1, 1986--August 31, 1996

International Nuclear Information System (INIS)

Sevilla, M.D.

1996-08-01

Over the last 10 years significant advances have been made impacting the understanding of radiation damage to DNA. The principal objective of this work was the elucidation of the fundamental mechanisms of radiation damage to DNA through the direct and indirect effects. Recently the work concentrated on the direct effect of radiation damage on DNA. The objective was to elucidate the ultimate radiation chemical damage to DNA arising from the direct effect. In this effort the focus was on the application of three techniques. ESR spectroscopic measurement of initial radicals formed in DNA and its hydration layer at low temperatures. Ab initio molecular orbital calculations were employed to give highly accurate theoretical predictions of early events such as electron and hole localization sites which serve to test and to clarify the experimental observations. HPLC and GC-mass spectroscopic assays of DNA base products formation provide the ultimate chemical outcome of the initial radiation events. The bridge between the early ion radical species and the non-radical products is made in ESR studies which follow the chemistry of the early species as they react with water and or other DNA bases. The use of these techniques has resulted in a new and fundamental understanding of the radiation damage to DNA on a molecular scale. From this work, a working model for DNA damage from the initial ionization event to the eventual formation of molecular base damage products and strand breaks has been formulated. Results over the past several years which have led to the formulation of this model are described

Evaluation of DNA damage and mutagenicity induced by lead in tobacco plants.

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Gichner, Tomás; Znidar, Irena; Száková, Jirina

2008-04-30

Tobacco (Nicotiana tabacum L. var. xanthi) seedlings were treated with aqueous solutions of lead nitrate (Pb2+) at concentrations ranging from 0.4 mM to 2.4 mM for 24 h and from 25 microM to 200 microM for 7 days. The DNA damage measured by the comet assay was high in the root nuclei, but in the leaf nuclei a slight but significant increase in DNA damage could be demonstrated only after a 7-day treatment with 200 microM Pb2+. In tobacco plants growing for 6 weeks in soil polluted with Pb2+ severe toxic effects, expressed by the decrease in leaf area, and a slight but significant increase in DNA damage were observed. The tobacco plants with increased levels of DNA damage were severely injured and showed stunted growth, distorted leaves and brown root tips. The frequency of somatic mutations in tobacco plants growing in the Pb2+-polluted soil did not significantly increase. Analytical studies by inductively coupled plasma optical emission spectrometry demonstrate that after a 24-h treatment of tobacco with 2.4 mM Pb2+, the accumulation of the heavy metal is 40-fold higher in the roots than in the above-ground biomass. Low Pb2+ accumulation in the above-ground parts may explain the lower levels or the absence of Pb2+-induced DNA damage in leaves.

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

Science.gov (United States)

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.

Mechanisms of mutagenesis: DNA replication in the presence of DNA damage.

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Liu, Binyan; Xue, Qizhen; Tang, Yong; Cao, Jia; Guengerich, F Peter; Zhang, Huidong

2016-01-01

Environmental mutagens cause DNA damage that disturbs replication and produces mutations, leading to cancer and other diseases. We discuss mechanisms of mutagenesis resulting from DNA damage, from the level of DNA replication by a single polymerase to the complex DNA replisome of some typical model organisms (including bacteriophage T7, T4, Sulfolobus solfataricus, Escherichia coli, yeast and human). For a single DNA polymerase, DNA damage can affect replication in three major ways: reducing replication fidelity, causing frameshift mutations, and blocking replication. For the DNA replisome, protein interactions and the functions of accessory proteins can yield rather different results even with a single DNA polymerase. The mechanism of mutation during replication performed by the DNA replisome is a long-standing question. Using new methods and techniques, the replisomes of certain organisms and human cell extracts can now be investigated with regard to the bypass of DNA damage. In this review, we consider the molecular mechanism of mutagenesis resulting from DNA damage in replication at the levels of single DNA polymerases and complex DNA replisomes, including translesion DNA synthesis. Copyright © 2016 Elsevier B.V. All rights reserved.

Characterization of environmental chemicals with potential for DNA damage using isogenic DNA repair-deficient chicken DT40 cell lines.

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Yamamoto, Kimiyo N; Hirota, Kouji; Kono, Koichi; Takeda, Shunichi; Sakamuru, Srilatha; Xia, Menghang; Huang, Ruili; Austin, Christopher P; Witt, Kristine L; Tice, Raymond R

2011-08-01

Included among the quantitative high throughput screens (qHTS) conducted in support of the US Tox21 program are those being evaluated for the detection of genotoxic compounds. One such screen is based on the induction of increased cytotoxicity in seven isogenic chicken DT40 cell lines deficient in DNA repair pathways compared to the parental DNA repair-proficient cell line. To characterize the utility of this approach for detecting genotoxic compounds and identifying the type(s) of DNA damage induced, we evaluated nine of 42 compounds identified as positive for differential cytotoxicity in qHTS (actinomycin D, adriamycin, alachlor, benzotrichloride, diglycidyl resorcinol ether, lovastatin, melphalan, trans-1,4-dichloro-2-butene, tris(2,3-epoxypropyl)isocyanurate) and one non-cytotoxic genotoxic compound (2-aminothiamine) for (1) clastogenicity in mutant and wild-type cells; (2) the comparative induction of γH2AX positive foci by melphalan; (3) the extent to which a 72-hr exposure duration increased assay sensitivity or specificity; (4) the use of 10 additional DT40 DNA repair-deficient cell lines to better analyze the type(s) of DNA damage induced; and (5) the involvement of reactive oxygen species in the induction of DNA damage. All compounds but lovastatin and 2-aminothiamine were more clastogenic in at least one DNA repair-deficient cell line than the wild-type cells. The differential responses across the various DNA repair-deficient cell lines provided information on the type(s) of DNA damage induced. The results demonstrate the utility of this DT40 screen for detecting genotoxic compounds, for characterizing the nature of the DNA damage, and potentially for analyzing mechanisms of mutagenesis. Copyright © 2011 Wiley-Liss, Inc.

Measurement of spontaneous DNA damage and DNA repair capacity in healthy adult individuals from high and normal level natural radiation areas of Kerala, India

International Nuclear Information System (INIS)

Vivek Kumar, P.R.; Jaikrishan, G.; Das, Birajalaxmi

2014-01-01

Inhabitants of the south west coastal areas of Kerala receive high level natural radiation due to the presence of monazite ( 232 Th) in the beach sand. This provides a unique opportunity to investigate the biological effects of high level natural radiation on humans. This study evaluate basal DNA damage in 149 healthy adult male subjects (104 from high level natural radiation areas (HLNRA, > 1mSv year -1 ) and 45 from normal level natural radiation area (NLNRA, d''1mSv year -1 ) by the alkaline comet assay. Oxidative DNA damage (ENDO III, FPG and hOGG1-sensitive sites) was measured by the enzyme modified comet assay. Induction and rejoining of DNA strand breaks was measured after irradiating peripheral blood lymphocytes with 2 Gy or 4 Gy gamma radiation. Basal damage due to age and residential area of the donors showed significant interaction (P=0.001), when all subjects were analyzed using a general linear model (GLM). In subgroup analysis, basal damage increased with age in subjects from the NLNRA (P=0.007), while a significant negative correlation (P=0.01) was observed in subjects from HLNRA. Oxidative DNA damage was not influenced by age, smoking habit or residential area in the entire sample. Subjects of high dose group from HLNRA (>5.75 mSv/y, N=34) showed a significant reduction in the induction of DNA damage after 2 Gy (P=0.03) and 4 Gy (P=0.05) compared to subjects form NLNRA. The study showed increased rejoining of DNA strand breaks in subjects from HLNRA when measured at 7 minutes after irradiation (P=0.04). In this pilot study, a low basal damage in elderly subjects from HLNRA and a reduced induction of DNA damage after 2 Gy and 4 Gy irradiation in high dose group subjects from HLNRA might suggest a possible role of chronic low dose natural radiation on the induction of an in vivo radio adaptive response. However, our findings need more validation in a larger study population. (author)

Characterization of coal fly ash nanoparticles and induced oxidative DNA damage in human peripheral blood mononuclear cells

International Nuclear Information System (INIS)

Dwivedi, Sourabh; Saquib, Quaiser; Al-Khedhairy, Abdulaziz A.; Ali, Al-Yousef Sulaiman; Musarrat, Javed

2012-01-01

The nano-sized particles present in coal fly ash (CFA) were characterized through the X-ray diffraction (XRD), transmission and scanning electron microscopy (TEM, SEM), atomic force microscopy (AFM) and Fourier transform infrared spectroscopy (FTIR) analyses. The XRD data revealed the average crystallite size of the CFA nanoparticles (CFA-NPs) as 14 nm. TEM and SEM imaging demonstrated predominantly spherical and some polymorphic structures in the size range of 11 to 25 nm. The amount of heavy metal associated with CFA particles (μg/g) were determined as Fe (34160.0 ± 1.38), Ni (150.8 ± 0.78), Cu (99.3 ± 0.56) and Cr (64.0 ± 0.86). However, the bioavailability of heavy metals in terms of percent release was in the order as Cr > Ni > Cu > Fe in CFA-dimethyl sulfoxide (DMSO) extract. The comet and cytokinesis blocked micronucleus (CBMN) assays revealed substantial genomic DNA damage in peripheral blood mononuclear (PBMN) cells treated with CFA-NPs in Aq and DMSO extracts. About 1.8 and 3.6 strand breaks per unit of DNA were estimated through alkaline unwinding assay at 1:100 DNA nucleotide/CFA ppm ratios with the Aq and DMSO extracts, respectively. The DNA and mitochondrial damage was invariably greater with CFA-DMSO extract vis-à-vis -Aq extract. Generation of superoxide anions (O 2 • − ) and intracellular reactive oxygen species (ROS) through metal redox-cycling, alteration in mitochondrial potential and 8-oxodG production elucidated CFA-NPs induced oxidative stress as a plausible mechanism for CFA-induced genotoxicity. -- Highlights: ► CFA consists of spherical crystalline nanoparticles in size range of 11–25 nm. ► Alkaline unwinding assay revealed single-strandedness in CFA treated ctDNA. ► CFA nanoparticles exhibited the ability to induce ROS and oxidative DNA damage. ► Comet and CBMN assays revealed DNA and chromosomal breakage in PBMN cells. ► CFA-NPs resulted in mitochondrial membrane damage in PBMN cells.

Characterization of coal fly ash nanoparticles and induced oxidative DNA damage in human peripheral blood mononuclear cells

Energy Technology Data Exchange (ETDEWEB)

Dwivedi, Sourabh; Saquib, Quaiser; Al-Khedhairy, Abdulaziz A. [Department of Zoology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451 (Saudi Arabia); Ali, Al-Yousef Sulaiman [Department of Medical Laboratory Sciences, College of Applied Medical Science, University of Dammam, P.O. Box 1683, Hafr Al Batin-31991 (Saudi Arabia); Musarrat, Javed, E-mail: musarratj1@yahoo.com [Department of Zoology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451 (Saudi Arabia); Department of Agricultural Microbiology, Faculty of Agricultural Sciences, AMU, Aligarh202002 (India)

2012-10-15

The nano-sized particles present in coal fly ash (CFA) were characterized through the X-ray diffraction (XRD), transmission and scanning electron microscopy (TEM, SEM), atomic force microscopy (AFM) and Fourier transform infrared spectroscopy (FTIR) analyses. The XRD data revealed the average crystallite size of the CFA nanoparticles (CFA-NPs) as 14 nm. TEM and SEM imaging demonstrated predominantly spherical and some polymorphic structures in the size range of 11 to 25 nm. The amount of heavy metal associated with CFA particles ({mu}g/g) were determined as Fe (34160.0 {+-} 1.38), Ni (150.8 {+-} 0.78), Cu (99.3 {+-} 0.56) and Cr (64.0 {+-} 0.86). However, the bioavailability of heavy metals in terms of percent release was in the order as Cr > Ni > Cu > Fe in CFA-dimethyl sulfoxide (DMSO) extract. The comet and cytokinesis blocked micronucleus (CBMN) assays revealed substantial genomic DNA damage in peripheral blood mononuclear (PBMN) cells treated with CFA-NPs in Aq and DMSO extracts. About 1.8 and 3.6 strand breaks per unit of DNA were estimated through alkaline unwinding assay at 1:100 DNA nucleotide/CFA ppm ratios with the Aq and DMSO extracts, respectively. The DNA and mitochondrial damage was invariably greater with CFA-DMSO extract vis-a-vis -Aq extract. Generation of superoxide anions (O{sub 2} Bullet {sup -}) and intracellular reactive oxygen species (ROS) through metal redox-cycling, alteration in mitochondrial potential and 8-oxodG production elucidated CFA-NPs induced oxidative stress as a plausible mechanism for CFA-induced genotoxicity. -- Highlights: Black-Right-Pointing-Pointer CFA consists of spherical crystalline nanoparticles in size range of 11-25 nm. Black-Right-Pointing-Pointer Alkaline unwinding assay revealed single-strandedness in CFA treated ctDNA. Black-Right-Pointing-Pointer CFA nanoparticles exhibited the ability to induce ROS and oxidative DNA damage. Black-Right-Pointing-Pointer Comet and CBMN assays revealed DNA and chromosomal

Endogenous DNA Damage and Risk of Testicular Germ Cell Tumors

Energy Technology Data Exchange (ETDEWEB)

Cook, M B; Sigurdson, A J; Jones, I M; Thomas, C B; Graubard, B I; Korde, L; Greene, M H; McGlynn, K A

2008-01-18

Testicular germ cell tumors (TGCT) are comprised of two histologic groups, seminomas and nonseminomas. We postulated that the possible divergent pathogeneses of these histologies may be partially explained by variable endogenous DNA damage. To assess our hypothesis, we conducted a case-case analysis of seminomas and nonseminomas using the alkaline comet assay to quantify single-strand DNA breaks and alkali-labile sites. The Familial Testicular Cancer study and the U.S. Radiologic Technologists cohort provided 112 TGCT cases (51 seminomas & 61 nonseminomas). A lymphoblastoid cell line was cultured for each patient and the alkaline comet assay was used to determine four parameters: tail DNA, tail length, comet distributed moment (CDM) and Olive tail moment (OTM). Odds ratios (OR) and 95% confidence intervals (95%CI) were estimated using logistic regression. Values for tail length, tail DNA, CDM and OTM were modeled as categorical variables using the 50th and 75th percentiles of the seminoma group. Tail DNA was significantly associated with nonseminoma compared to seminoma (OR{sub 50th percentile} = 3.31, 95%CI: 1.00, 10.98; OR{sub 75th percentile} = 3.71, 95%CI: 1.04, 13.20; p for trend=0.039). OTM exhibited similar, albeit statistically non-significant, risk estimates (OR{sub 50th percentile} = 2.27, 95%CI: 0.75, 6.87; OR{sub 75th percentile} = 2.40, 95%CI: 0.75, 7.71; p for trend=0.12) whereas tail length and CDM showed no association. In conclusion, the results for tail DNA and OTM indicate that endogenous DNA damage levels are higher in patients who develop nonseminoma compared with seminoma. This may partly explain the more aggressive biology and younger age-of-onset of this histologic subgroup compared with the relatively less aggressive, later-onset seminoma.

[DNA damage in human pleural mesothelial cells induced by exposure to carbon nanotubes].

Science.gov (United States)

Ogasawara, Yuki; Umezu, Noriaki; Ishii, Kazuyuki

2012-01-01

Nanomaterials are currently used in electronics, industrial materials, cosmetics, and medicine because they have useful physicochemical properties, such as strength, conductivity, durability, and chemical stability. As these materials have become widespread, many questions have arisen regarding their effects on health and the environment. In particular, recent studies have demonstrated that carbon nanotubes (CNTs) cause significant inflammation and mesothelioma in vivo. In this study, we investigated the potential risk posed by singlewalled carbon nanotube (SWCNT) and multiwalled carbon nanotube (MWCNT) exposure in human pleural mesothelial cells. CNT cytotoxicity was determined by a trypan blue exclusion assay, and DNA damage was detected by an alkaline comet assay. The concentration of 8-oxodeoxyguanosine (8-OHdG) in DNA was measured by high perhormance liquid chromatography with electrochemical detection. The expression of base excision repair enzymes in the cell was estimated by immunoblot analysis. We observed inhibitory effects on cell proliferation and the induction of DNA damage following exposure of cells to purified CNTs that were suspended in dispersion medium. However, accumulation of 8-OHdG in DNA was not found. In addition, the expression levels of base excision enzymes that are involved in hOGG1, hMTH1, and MYH in MeT-5A cells remained unchanged for 24 h after carbon nanotube exposure. CNTs significantly inhibit cell proliferation and decrease DNA damage in human pleural mesothelial cells. Our results indicate that the mechanism of CNT-induced genotoxicity is different from that following exposure to reactive oxygen species, which causes oxidative DNA modifications and 8-OHdG production. Further investigation is required to characterize the specific DNA mutations that occur following CNT exposure.

Monitoring of DNA and cytogenetic damage in lymphocytes from persons with skin cancer diseases

International Nuclear Information System (INIS)

Cebulska-Wasilewska, A.; Dyga, W.; Krasnowolski, S.; Wierzewska, A.; Budzanowska, E.

1999-01-01

There is a lot of interest in the studies that would help to understand whether there is a casual association between cancer and various types of molecular or cytogenetic damage detected in human cells. One major oncogenesis process is activation of proto-oncogenes by point mutations or chromosomal translocation. There are substantial evidence that indicates that the loss of heterozygosity of certain chromosomes is involved in human cancerogenesis. Our study aimed to elicit the possible association between cancer and DNA and cytogenetic abnormalities induced in lymphocytes of people bearing various categories of skin cancer cells. Fresh blood was collected by venipuncture from 25 individuals (including nine prior to cancer treatment). All patients were nonsmoking males, however 42.3 % of them were former smokers. Blood samples were divided into two parts and in the first part of samples cytogenetic studies were performed immediately, while from the second part lymphocytes were isolated and stored at -70 o C for further studies in vitro. In the later one a single cell gel electrophoresis assay (SCGE) known as a Comet assay was performed to study individual susceptibility to the induction of DNA damage by UV or radiation and to estimate variability in cellular repair capabilities. An average of 220 per sample of good metaphase spreads in the first mitotic division, and 100 per sample in the second division, were accepted for analysis of cytogenetic damage. Chromosome and chromatid type aberrations were scored in the cells in the first mitosis and expressed as total aberration frequency including gaps and excluding gaps. Sister chromatid exchanges, high frequency cells and proliferative rate index were screened and evaluated in the second mitosis. Each of the patient revealed exceeding in at least one of the cytogenetic biomarkers level from the biomarker's level detected in a reference group. In order to estimate susceptibility of people to environmentally induced

Intermittent hypoxia reduces microglia proliferation and induces DNA damage in vitro

Directory of Open Access Journals (Sweden)

Song Liu

2016-05-01

Full Text Available Objective(s:Intermittent hypoxia (IH, caused by obstructive sleep apnea (OSA, could cause hippocampus or neuron damage through multiple signaling pathways, while the underlying mechanisms are still unclear. Thus, the present study aimed to explore the effect of IH on the biological functions of microglia cells. Materials and Methods:Cell proliferation of BV2 cells after exposure to IH were observed by MTT assay and then DNA damage was detected by comet assay. RNA-sequencing assay was performed in cells under IH condition and normal conditions to find out the differentially expressed genes, which were further confirmed by reverse transcriptase polymerase chain reaction (RT-PCR and Western blot assay. Results:As results, IH inhibited the proliferation of BV2 cells, as well as caused DNA damage. RNA-sequencing assay revealed 4 differentially expressed genes (p21, Cyclin D1, Cyclin E2, and Gadd45α which were associated with the network of P53 signaling pathways in BV2 cells, among which, p21 and Gadd45α were dramatically increased while Cyclin D1 and Cyclin E2 were both decreased significantly. Moreover, inflammatory factors including IL-6, TNF-α and iNOS were significantly up-regulated in microglia cells under IH conditions for 8 hr. Conclusion:Our results indicated that IH could inhibit cyclin D1 and cyclin E2 expression via initiating multiple P53 pathways, which further blocked cell cycle transition and attenuated proliferative capability of BV2 cells. Meanwhile, IH activated inflammation reactions in BV2 cells. Present study elaborate the effects of IH on biological functions of microglia and provide theoretical foundation for further study on new therapy methods for OSA.

Development of fluorescent methods for DNA methyltransferase assay

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Li, Yueying; Zou, Xiaoran; Ma, Fei; Tang, Bo; Zhang, Chun-yang

2017-03-01

DNA methylation modified by DNA methyltransferase (MTase) plays an important role in regulating gene transcription, cell growth and proliferation. The aberrant DNA MTase activity may lead to a variety of human diseases including cancers. Therefore, accurate and sensitive detection of DNA MTase activity is crucial to biomedical research, clinical diagnostics and therapy. However, conventional DNA MTase assays often suffer from labor-intensive operations and time-consuming procedures. Alternatively, fluorescent methods have significant advantages of simplicity and high sensitivity, and have been widely applied for DNA MTase assay. In this review, we summarize the recent advances in the development of fluorescent methods for DNA MTase assay. These emerging methods include amplification-free and the amplification-assisted assays. Moreover, we discuss the challenges and future directions of this area.

Cellular responses of Saccharomyces cerevisiae to DNA damage

International Nuclear Information System (INIS)

Ciesla, Z.; Sledziewska-Gojska, E.; Nowicka, A.; Mieczkowski, P.; Fikus, M.U.; Koprowski, P.

1998-01-01

with MMS or exposed to UV light. Northern RNA analysis indicates that DIN8 is induced in response to DNA damage at the transcriptional level. DIN8 was cloned and the phenotype of cells with disruption of the gene is under study. POL2-MEC1-RAD53-DUN1-signal transducing pathway has recently been postulated to be involved in the regulation of response of S. cerevisiae cells to DNA-damaging agents. We analyzed the expression of a known damage inducible DNA-repair gene, MAG1, encoding 3-methyladenine glycosylase, in S. cerevisiae strains carrying MAG1 ::lacZ fusion and deficient in either POL2, MEC1, RAD53 or DUN1 function. β-galactosidase activity was assayed in cycling cells exposed to MMS or UV light. It was found that, in contrast to model DNA damage inducible RNR genes, neither mutation in t he sensory C-terminal part of polymerase ε (pol2-11) nor the in the Mec1, Sad1/Rad53 or Dun1 cellular kinases blocks the induction of MAG1 in response to MMS or UV light in cycling yeasts. (author)

Bioactivation of carboxylic acid compounds by UDP-Glucuronosyltransferases to DNA-damaging intermediates: role of glycoxidation and oxidative stress in genotoxicity.

Science.gov (United States)

Sallustio, Benedetta C; Degraaf, Yvette C; Weekley, Josephine S; Burcham, Philip C

2006-05-01

Nonenzymatic modification of proteins by acyl glucuronides is well documented; however, little is known about their potential to damage DNA. We have previously reported that clofibric acid undergoes glucuronidation-dependent bioactivation to DNA-damaging species in cultured mouse hepatocytes. The aim of this study was to investigate the mechanisms underlying such DNA damage, and to screen chemically diverse carboxylic acid drugs for their DNA-damaging potential in glucuronidation proficient murine hepatocytes. Cells were incubated with each aglycone for 18 h, followed by assessment of compound cytotoxicity using the MTT assay and evaluation of DNA damage using the Comet assay. Relative cytotoxic potencies were ketoprofen > diclofenac, benoxaprofen, nafenopin > gemfibrozil, probenecid > bezafibrate > clofibric acid. At a noncytotoxic (0.1 mM) concentration, only benoxaprofen, nafenopin, clofibric acid, and probenecid significantly increased Comet moments (P Clofibric acid and probenecid exhibited the greatest DNA-damaging potency, producing significant DNA damage at 0.01 mM concentrations. The two drugs produced maximal increases in Comet moment of 4.51 x and 2.57 x control, respectively. The glucuronidation inhibitor borneol (1 mM) abolished the induction of DNA damage by 0.5 mM concentrations of clofibric acid and probenecid. In an in vitro cell-free system, clofibric acid glucuronide was 10 x more potent than glucuronic acid in causing DNA strand-nicking, although both compounds showed similar rates of autoxidation to generate hydroxyl radicals. In cultured hepatocytes, the glycation inhibitor, aminoguanidine, and the iron chelator, desferrioxamine mesylate, inhibited DNA damage by clofibric acid, whereas the free radical scavengers Trolox and butylated hydroxytoluene, and the superoxide dismutase mimetic bis-3,5-diisopropylsalicylate had no effect. In conclusion, clinically relevant concentrations of two structurally unrelated carboxylic acids, probenecid and

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

Science.gov (United States)

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

2012-01-05

3-methyladenine DNA glycosylase (AlkD) belongs to a new family of DNA glycosylases; it initiates repair of cytotoxic and promutagenic alkylated bases (its main substrates being 3-methyladenine and 7-methylguanine). The modification of the comet assay (single cell gel electrophoresis) using AlkD enzyme thus allows assessment of specific DNA alkylation lesions. The resulting baseless sugars are alkali-labile, and under the conditions of the alkaline comet assay they appear as DNA strand breaks. The alkylating agent methyl methanesulfonate (MMS) was used to induce alkylation lesions and to optimize conditions for the modified comet assay method with AlkD on human lymphoblastoid (TK6) cells. We also studied cellular and in vitro DNA repair of alkylated bases in DNA in TK6 cells after treatment with MMS. Results from cellular repair indicate that 50% of DNA alkylation is repaired in the first 60 min. The in vitro repair assay shows that while AlkD recognises most alkylation lesions after 60 min, a cell extract from TK6 cells recognises most of the MMS-induced DNA adducts already in the first 15 min of incubation, with maximum detection of lesions after 60 min' incubation. Additionally, we tested the in vitro repair capacity of human lymphocyte extracts from 5 individuals and found them to be able to incise DNA alkylations in the same range as AlkD. The modification of the comet assay with AlkD can be useful for in vitro and in vivo genotoxicity studies to detect alkylation damage and repair and also for human biomonitoring and molecular epidemiology studies. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Dihydropyridines decrease X-ray-induced DNA base damage in mammalian cells

Energy Technology Data Exchange (ETDEWEB)

Wojewodzka, M., E-mail: marylaw@ichtj.waw.pl [Center of Radiobiology and Biological Dosimetry, Institute of Nuclear Chemistry and Technology, Warszawa (Poland); Gradzka, I.; Buraczewska, I.; Brzoska, K.; Sochanowicz, B. [Center of Radiobiology and Biological Dosimetry, Institute of Nuclear Chemistry and Technology, Warszawa (Poland); Goncharova, R.; Kuzhir, T. [Institute of Genetics and Cytology, Belarussian National Academy of Sciences, Minsk (Belarus); Szumiel, I. [Center of Radiobiology and Biological Dosimetry, Institute of Nuclear Chemistry and Technology, Warszawa (Poland)

2009-12-01

Compounds with the structural motif of 1,4-dihydropyridine display a broad spectrum of biological activities, often defined as bioprotective. Among them are L-type calcium channel blockers, however, also derivatives which do not block calcium channels exert various effects at the cellular and organismal levels. We examined the effect of sodium 3,5-bis-ethoxycarbonyl-2,6-dimethyl-1,4-dihydropyridine-4-carboxylate (denoted here as DHP and previously also as AV-153) on X-ray-induced DNA damage and mutation frequency at the HGPRT (hypoxanthine-guanine phosphoribosyl transferase) locus in Chinese hamster ovary CHO-K1 cells. Using formamido-pyrimidine glycosylase (FPG) comet assay, we found that 1-h DHP (10 nM) treatment before X-irradiation considerably reduced the initial level of FPG-recognized DNA base damage, which was consistent with decreased 8-oxo-7,8-dihydro-2'-deoxyguanosine content and mutation frequency lowered by about 40%. No effect on single strand break rejoining or on cell survival was observed. Similar base damage-protective effect was observed for two calcium channel blockers: nifedipine (structurally similar to DHP) or verapamil (structurally unrelated). So far, the specificity of the DHP-caused reduction in DNA damage - practically limited to base damage - has no satisfactory explanation.

ZRBA1, a Mixed EGFR/DNA Targeting Molecule, Potentiates Radiation Response Through Delayed DNA Damage Repair Process in a Triple Negative Breast Cancer Model

Energy Technology Data Exchange (ETDEWEB)

Heravi, Mitra [Department of Human Genetics, McGill University, Montreal (Canada); Department of Radiation Oncology, McGill University, Montreal (Canada); Segal Cancer Center, Jewish General Hospital, Montreal (Canada); Kumala, Slawomir [Department of Radiation Oncology, McGill University, Montreal (Canada); Segal Cancer Center, Jewish General Hospital, Montreal (Canada); Rachid, Zakaria; Jean-Claude, Bertrand J. [Cancer Drug Research Laboratory, McGill University Health Center, Montreal (Canada); Radzioch, Danuta [Department of Human Genetics, McGill University, Montreal (Canada); Muanza, Thierry M., E-mail: tmuanza@yahoo.com [Department of Radiation Oncology, McGill University, Montreal (Canada); Segal Cancer Center, Jewish General Hospital, Montreal (Canada)

2015-06-01

Purpose: ZRBA1 is a combi-molecule designed to induce DNA alkylating lesions and to block epidermal growth factor receptor (EGFR) TK domain. Inasmuch as ZRBA1 downregulates the EGFR TK-mediated antisurvival signaling and induces DNA damage, we postulated that it might be a radiosensitizer. The aim of this study was to further investigate the potentiating effect of ZRBA1 in combination with radiation and to elucidate the possible mechanisms of interaction between these 2 treatment modalities. Methods and Materials: The triple negative human breast MDA-MB-468 cancer cell line and mouse mammary cancer 4T1 cell line were used in this study. Clonogenic assay, Western blot analysis, and DNA damage analysis were performed at multiple time points after treatment. To confirm our in vitro findings, in vivo tumor growth delay assay was performed. Results: Our results show that a combination of ZRBA1 and radiation increases the radiation sensitivity of both cell lines significantly with a dose enhancement factor of 1.56, induces significant numbers of DNA strand breaks, prolongs higher DNA damage up to 24 hours after treatment, and significantly increases tumor growth delay in a syngeneic mouse model. Conclusions: Our data suggest that the higher efficacy of this combination could be partially due to increased DNA damage and delayed DNA repair process and to the inhibition of EGFR. The encouraging results of this combination demonstrated a significant improvement in treatment efficiency and therefore could be applicable in early clinical trial settings.

The time course of repair of ultraviolet-induced DNA damage; implications for the structural organization of repair

International Nuclear Information System (INIS)

Collins, A.; Squires, S.

1986-01-01

Alternative molecular mechanisms can be envisaged for the cellular repair of UV-damaged DNA. In the 'random collision' model, DNA damage distributed throughout the genome is recognised and repaired by a process of random collision between DNA damage and repair enzymes. The other model assumes a 'processive' mechanism, whereby DNA is scanned for damage by a repair complex moving steadily along its length. Random collision should result in a declining rate of repair with time as the concentration of lesions in the DNA falls; but the processive model predicts a constant rate until scanning is complete. The authors have examined the time course of DNA repair in human fibroblasts given low doses of UV light. Using 3 distinct assays, the authors find no sign of a constant repair rate after 4 J/m 2 or less, even when the first few hours after irradiation are examined. Thus DNA repair is likely to depend on random collision. (Auth.)

Radiation damage in DNA

International Nuclear Information System (INIS)

Lafleur, V.

1978-01-01

A number of experiments are described with the purpose to obtain a better insight in the chemical nature and the biological significance of radiation-induced damage in DNA, with some emphasis on the significance of alkali-labile sites. It is shown that not only reactions of OH radicals but also of H radicals introduce breaks and other inactivating damage in single-standed phiX174 DNA. It is found that phosphate buffer is very suitable for the study of the reactions of H radicals with DNA, as the H 2 PO 4 - ions convert the hydrated electrons into H radicals. The hydrated electron, which does react with DNA, does not cause a detectable inactivation. (Auth.)

The DNA damage response in mammalian oocytes

Directory of Open Access Journals (Sweden)

John eCarroll

2013-06-01

Full Text Available DNA damage is one of the most common insults that challenge all cells. To cope, an elaborate molecular and cellular response has evolved to sense, respond to and correct the damage. This allows the maintenance of DNA fidelity essential for normal cell viability and the prevention of genomic instability that can lead to tumour formation. In the context of oocytes, the impact of DNA damage is not one of tumour formation but of the maintenance of fertility. Mammalian oocytes are particularly vulnerable to DNA damage because physiologically they may lie dormant in the ovary for many years (>40 in humans until they receive the stimulus to grow and acquire the competence to become fertilized. The implication of this is that in some organisms, such as humans, oocytes face the danger of cumulative genetic damage for decades. Thus, the ability to detect and repair DNA damage is essential to maintain the supply of oocytes necessary for reproduction. Therefore, failure to confront DNA damage in oocytes could cause serious anomalies in the embryo that may be propagated in the form of mutations to the next generation allowing the appearance of hereditary disease. Despite the potential impact of DNA damage on reproductive capacity and genetic fidelity of embryos, the mechanisms available to the oocyte for monitoring and repairing such insults have remained largely unexplored until recently. Here, we review the different aspects of the response to DNA damage in mammalian oocytes. Specifically, we address the oocyte DNA damage response from embryonic life to adulthood and throughout oocyte development.

Oxidative DNA damage & repair: An introduction.

Science.gov (United States)

Cadet, Jean; Davies, Kelvin J A

2017-06-01

This introductory article should be viewed as a prologue to the Free Radical Biology & Medicine Special Issue devoted to the important topic of Oxidatively Damaged DNA and its Repair. This special issue is dedicated to Professor Tomas Lindahl, co-winner of the 2015 Nobel Prize in Chemistry for his seminal discoveries in the area repair of oxidatively damaged DNA. In the past several years it has become abundantly clear that DNA oxidation is a major consequence of life in an oxygen-rich environment. Concomitantly, survival in the presence of oxygen, with the constant threat of deleterious DNA mutations and deletions, has largely been made possible through the evolution of a vast array of DNA repair enzymes. The articles in this Oxidatively Damaged DNA & Repair special issue detail the reactions by which intracellular DNA is oxidatively damaged, and the enzymatic reactions and pathways by which living organisms survive such assaults by repair processes. Copyright © 2017 Elsevier Inc. All rights reserved.

Targeted detection of in vivo endogenous DNA base damage reveals preferential base excision repair in the transcribed strand.

Science.gov (United States)

Reis, António M C; Mills, Wilbur K; Ramachandran, Ilangovan; Friedberg, Errol C; Thompson, David; Queimado, Lurdes

2012-01-01

Endogenous DNA damage is removed mainly via base excision repair (BER), however, whether there is preferential strand repair of endogenous DNA damage is still under intense debate. We developed a highly sensitive primer-anchored DNA damage detection assay (PADDA) to map and quantify in vivo endogenous DNA damage. Using PADDA, we documented significantly higher levels of endogenous damage in Saccharomyces cerevisiae cells in stationary phase than in exponential phase. We also documented that yeast BER-defective cells have significantly higher levels of endogenous DNA damage than isogenic wild-type cells at any phase of growth. PADDA provided detailed fingerprint analysis at the single-nucleotide level, documenting for the first time that persistent endogenous nucleotide damage in CAN1 co-localizes with previously reported spontaneous CAN1 mutations. To quickly and reliably quantify endogenous strand-specific DNA damage in the constitutively expressed CAN1 gene, we used PADDA on a real-time PCR setting. We demonstrate that wild-type cells repair endogenous damage preferentially on the CAN1 transcribed strand. In contrast, yeast BER-defective cells accumulate endogenous damage preferentially on the CAN1 transcribed strand. These data provide the first direct evidence for preferential strand repair of endogenous DNA damage and documents the major role of BER in this process.

Progress on clustered DNA damage in radiation research

International Nuclear Information System (INIS)

Yang Li'na; Zhang Hong; Di Cuixia; Zhang Qiuning; Wang Xiaohu

2012-01-01

Clustered DNA damage which caused by high LET heavy ion radiation can lead to mutation, tumorigenesis and apoptosis. Promoting apoptosis of cancer cells is always the basis of cancer treatment. Clustered DNA damage has been the hot topic in radiobiology. The detect method is diversity, but there is not a detail and complete protocol to analyze clustered DNA damage. In order to provide reference for clustered DNA damage in the radiotherapy study, the clustered DNA damage characteristics, the latest progresses on clustered DNA damage and the detecting methods are reviewed and discussed in detail in this paper. (authors)

MicroRNAs, the DNA damage response and cancer

International Nuclear Information System (INIS)

Wouters, Maikel D.; Gent, Dik C. van; Hoeijmakers, Jan H.J.; Pothof, Joris

2011-01-01

Many carcinogenic agents such as ultra-violet light from the sun and various natural and man-made chemicals act by damaging the DNA. To deal with these potentially detrimental effects of DNA damage, cells induce a complex DNA damage response (DDR) that includes DNA repair, cell cycle checkpoints, damage tolerance systems and apoptosis. This DDR is a potent barrier against carcinogenesis and defects within this response are observed in many, if not all, human tumors. DDR defects fuel the evolution of precancerous cells to malignant tumors, but can also induce sensitivity to DNA damaging agents in cancer cells, which can be therapeutically exploited by the use of DNA damaging treatment modalities. Regulation of and coordination between sub-pathways within the DDR is important for maintaining genome stability. Although regulation of the DDR has been extensively studied at the transcriptional and post-translational level, less is known about post-transcriptional gene regulation by microRNAs, the topic of this review. More specifically, we highlight current knowledge about DNA damage responsive microRNAs and microRNAs that regulate DNA damage response genes. We end by discussing the role of DNA damage response microRNAs in cancer etiology and sensitivity to ionizing radiation and other DNA damaging therapeutic agents.

Oxidative Stress, DNA Damage and DNA Repair in Female Patients with Diabetes Mellitus Type 2.

Directory of Open Access Journals (Sweden)

Annemarie Grindel

Full Text Available Diabetes mellitus type 2 (T2DM is associated with oxidative stress which in turn can lead to DNA damage. The aim of the present study was to analyze oxidative stress, DNA damage and DNA repair in regard to hyperglycemic state and diabetes duration.Female T2DM patients (n = 146 were enrolled in the MIKRODIAB study and allocated in two groups regarding their glycated hemoglobin (HbA1c level (HbA1c≤7.5%, n = 74; HbA1c>7.5%, n = 72. In addition, tertiles according to diabetes duration (DD were created (DDI = 6.94±3.1 y, n = 49; DDII = 13.35±1.1 y, n = 48; DDIII = 22.90±7.3 y, n = 49. Oxidative stress parameters, including ferric reducing ability potential, malondialdehyde, oxidized and reduced glutathione, reduced thiols, oxidized LDL and F2-Isoprostane as well as the activity of antioxidant enzymes superoxide dismutase, catalase and glutathione peroxidase were measured. Damage to DNA was analyzed in peripheral blood mononuclear cells and whole blood with single cell gel electrophoresis. DNA base excision repair capacity was tested with the modified comet repair assay. Additionally, mRNA expressions of nine genes related to base excision repair were analyzed in a subset of 46 matched individuals.No significant differences in oxidative stress parameters, antioxidant enzyme activities, damage to DNA and base excision repair capacity, neither between a HbA1c cut off />7.5%, nor between diabetes duration was found. A significant up-regulation in mRNA expression was found for APEX1, LIG3 and XRCC1 in patients with >7.5% HbA1c. Additionally, we observed higher total cholesterol, LDL-cholesterol, LDL/HDL-cholesterol, triglycerides, Framingham risk score, systolic blood pressure, BMI and lower HDL-cholesterol in the hyperglycemic group.BMI, blood pressure and blood lipid status were worse in hyperglycemic individuals. However, no major disparities regarding oxidative stress, damage to DNA and DNA repair were present which might be due to good medical

Sea urchin coelomocytes are resistant to a variety of DNA damaging agents

International Nuclear Information System (INIS)

Loram, Jeannette; Raudonis, Renee; Chapman, Jecar; Lortie, Mae; Bodnar, Andrea

2012-01-01

Increasing anthropogenic activities are creating environmental pressures that threaten marine ecosystems. Effective environmental health assessment requires the development of rapid, sensitive, and cost-effective tools to predict negative impacts at the individual and ecosystem levels. To this end, a number of biological assays using a variety of cells and organisms measuring different end points have been developed for biomonitoring programs. The sea urchin fertilization/development test has been useful for evaluating environmental toxicology and it has been proposed that sea urchin coelomocytes represent a novel cellular biosensor of environmental stress. In this study we investigated the sensitivity of coelomocytes from the sea urchin Lytechinus variegatus to a variety of DNA-damaging agents including ultraviolet (UV) radiation, hydrogen peroxide (H 2 O 2 ), methylmethane sulfonate (MMS) and benzo[a]pyrene (BaP). LD 50 values determined for coelomocytes after 24 h of exposure to these DNA damaging agents indicated a high level of resistance to all treatments. Significant increases in the formation of apurinic/apyrimidinic (AP or abasic) sites in DNA were only detected using high doses of H 2 O 2 , MMS and UV radiation. Comparison of sea urchin coelomocytes with hemocytes from the gastropod mollusk Aplysia dactylomela and the decapod crustacean Panulirus argus indicated that sensitivity to different DNA damaging agents varies between species. The high level of resistance to genotoxic agents suggests that DNA damage may not be an informative end point for environmental health assessment using sea urchin coelomocytes however, natural resistance to DNA damaging agents may have implications for the occurrence of neoplastic disease in these animals.

The DNA damage response during mitosis

NARCIS (Netherlands)

Heijink, Anne Margriet; Krajewska, Malgorzata; van Vugt, Marcel A. T. M.

2013-01-01

Cells are equipped with a cell-intrinsic signaling network called the DNA damage response (DDR). This signaling network recognizes DNA lesions and initiates various downstream pathways to coordinate a cell cycle arrest with the repair of the damaged DNA. Alternatively, the DDR can mediate clearance

Early models of DNA damage formation

International Nuclear Information System (INIS)

Śmiałek, Małgorzata A

2012-01-01

Quantification of DNA damage, induced by various types of incident radiation as well as chemical agents, has been the subject of many theoretical and experimental studies, supporting the development of modern cancer therapy. The primary observations showed that many factors can lead to damage of DNA molecules. It became clear that the development of experimental techniques for exploring this phenomenon is required. Another problem was simultaneously dealt with, anticipating on how the damage is distributed within the double helix of the DNA molecule and how the single strand break formation and accumulation can influence the lethal double strand break formation. In this work the most important probabilistic models for DNA strand breakage and damage propagation are summarized and compared.

Personal samplers of bioavailable pesticides integrated with a hair follicle assay of DNA damage to assess environmental exposures and their associated risks in children.

Science.gov (United States)

Vidi, Pierre-Alexandre; Anderson, Kim A; Chen, Haiying; Anderson, Rebecca; Salvador-Moreno, Naike; Mora, Dana C; Poutasse, Carolyn; Laurienti, Paul J; Daniel, Stephanie S; Arcury, Thomas A

2017-10-01

Agriculture in the United States employs youth ages ten and older in work environments with high pesticide levels. Younger children in rural areas may also be affected by indirect pesticide exposures. The long-term effects of pesticides on health and development are difficult to assess and poorly understood. Yet, epidemiologic studies suggest associations with cancer as well as cognitive deficits. We report a practical and cost-effective approach to assess environmental pesticide exposures and their biological consequences in children. Our approach combines silicone wristband personal samplers and DNA damage quantification from hair follicles, and was tested as part of a community-based participatory research (CBPR) project involving ten Latino children from farmworker households in North Carolina. Our study documents high acceptance among Latino children and their caregivers of these noninvasive sampling methods. The personal samplers detected organophosphates, organochlorines, and pyrethroids in the majority of the participants (70%, 90%, 80%, respectively). Pesticides were detected in all participant samplers, with an average of 6.2±2.4 detections/participant sampler. DNA damage in epithelial cells from the sheath and bulb of plucked hairs follicles was quantified by immunostaining 53BP1-labled DNA repair foci. This method is sensitive, as shown by dose response analyses to γ radiations where the lowest dose tested (0.1Gy) led to significant increased 53BP1 foci density. Immunolabeling of DNA repair foci has significant advantages over the comet assay in that specific regions of the follicles can be analyzed. In this cohort of child participants, significant association was found between the number of pesticide detections and DNA damage in the papilla region of the hairs. We anticipate that this monitoring approach of bioavailable pesticides and genotoxicity will enhance our knowledge of the biological effects of pesticides to guide education programs and

New Application of the Comet Assay

Science.gov (United States)

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

DNA Damage Induction and Repair Evaluated in Human Lymphocytes Irradiated with X-Rays an Neutrons

International Nuclear Information System (INIS)

Niedzwiedz, W.; Cebulska-Wasilewska, A.

2000-12-01

The objective of this study was to evaluate the kinetic of the DNA damage induction and their subsequent repair in human lymphocytes exposed to various types of radiation. PBLs cells were isolated from the whole blood of two young healthy male subjects and one skin cancer patient, and than exposed to various doses of low LET X-rays and high LET neutrons from 252 Cf source. To evaluate the DNA damage we have applied the single cell get electrophoresis technique (SCGE) also known as the comet assay. In order to estimate the repair efficiency, cells, which had been irradiated with a certain dose, were incubated at 37 o C for various periods of time (0 to 60 min). The kinetic of DNA damage recovery was investigated by an estimation of residual DNA damage persisted at cells after various times of post-irradiation incubation (5, 10, 15, 30 and 60 min). We observed an increase of the DNA damage (reported as a Tail DNA and Tail moment parameters) in linear and linear-quadratic manner, with increasing doses of X-rays and 252 Cf neutrons, respectively. Moreover, for skin cancer patient (Code 3) at whole studied dose ranges the higher level of the DNA damage was observed comparing to health subjects (Code 1 and 2), however statistically insignificant (for Tail DNA p=0.056; for Tail moment p=0.065). In case of the efficiency of the DNA damage repair it was observed that after 1 h of post-irradiation incubation the DNA damage induced with both, neutrons and X-rays had been significantly reduced (from 65% to 100 %). Furthermore, in case of skin cancer patient we observed lover repair efficiency of X-rays induced DNA damage. After irradiation with neutrons within first 30 min, the Tail DNA and Tail moment decreased of about 50%. One hour after irradiation, almost 70% of residual and new formed DNA damage was still observed. In this case, the level of unrepaired DNA damage may represent the fraction of the double strand breaks as well as more complex DNA damage (i.e.-DNA or DNA

Exposure to non-ionizing electromagnetic fields emitted from mobile phones induced DNA damage in human ear canal hair follicle cells.

Science.gov (United States)

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.

DNA-damage effect of polycyclic aromatic hydrocarbons from urban area, evaluated in lung fibroblast cultures

International Nuclear Information System (INIS)

Calesso Teixeira, Elba; Pra, Daniel; Idalgo, Daniele; Henriques, João Antonio Pêgas; Wiegand, Flavio

2012-01-01

This study was designed to biomonitor the effect of PAH extracts from urban areas on the DNA of lung cell cultures. The analyses of the polycyclic aromatic hydrocarbons (PAHs) were performed in atmospheric PM 2.5 and PM 10 collected at three sampling sites with heavy traffic located in the Metropolitan Area of Porto Alegre (MAPA) (Brazil). The concentrations of 16 major PAHs were determined according to EPA. Comet assay on V79 hamster lung cells was chosen for genotoxicity evaluation. Temperature, humidity, and wind speed were recorded. With regard to the damage index, higher levels were reported in the extract of particulate matter samples from the MAPA during the summer. High molecular weight compounds showed correlation with DNA damage frequency and their respective carcinogenicity. - Highlights: ► Cell line V79 was used to assess the effect of PAHs in PM 2.5 and PM 10 from urban area. ► Temperature showed a significant seasonal variation with the level of DNA damage. ► PAHs with higher molecular weight contributed to higher DNA damage levels. - DNA-damage effect of polycyclic aromatic hydrocarbons from urban area, showed difference according to season

Are endogenous sex hormones related to DNA damage in paradoxically sleep-deprived female rats?

Science.gov (United States)

Andersen, Monica L; Ribeiro, Daniel A; Alvarenga, Tathiana A; Silva, Andressa; Araujo, Paula; Zager, Adriano; Tenorio, Neuli M; Tufik, Sergio

2010-02-01

The aim of this investigation was to evaluate overall DNA damage induced by experimental paradoxical sleep deprivation (PSD) in estrous-cycling and ovariectomized female rats to examine possible hormonal involvement during DNA damage. Intact rats in different phases of the estrous cycle (proestrus, estrus, and diestrus) or ovariectomized female Wistar rats were subjected to PSD by the single platform technique for 96 h or were maintained for the equivalent period as controls in home-cages. After this period, peripheral blood and tissues (brain, liver, and heart) were collected to evaluate genetic damage using the single cell gel (comet) assay. The results showed that PSD caused extensive genotoxic effects in brain cells, as evident by increased DNA migration rates in rats exposed to PSD for 96 h when compared to negative control. This was observed for all phases of the estrous cycle indistinctly. In ovariectomized rats, PSD also led to DNA damage in brain cells. No significant statistically differences were detected in peripheral blood, the liver or heart for all groups analyzed. In conclusion, our data are consistent with the notion that genetic damage in the form of DNA breakage in brain cells induced by sleep deprivation overrides the effects related to endogenous female sex hormones. Copyright 2009 Elsevier Inc. All rights reserved.

Is Lack of Sleep Capable of Inducing DNA Damage in Aged Skin?

OpenAIRE

Kahan, Vanessa [UNIFESP; Ribeiro, Daniel Araki [UNIFESP; Egydio, Flavia [UNIFESP; Barros, L. A. [UNIFESP; Tomimori, Jane [UNIFESP; Tufik, Sergio [UNIFESP; Andersen, Monica Levy [UNIFESP

2014-01-01

Skin naturally changes with age, becoming more fragile. Various stimuli can alter skin integrity. the aim of this study was to evaluate whether sleep deprivation affects the integrity of DNA in skin and exacerbates the effects of aging. Fifteen-month old female Hairless mice underwent 72 h of paradoxical sleep deprivation or 15 days of chronic sleep restriction. Punch biopsies of the skin were taken to evaluate DNA damage by single cell gel (comet) assay. Neither paradoxical sleep deprivation...

Quantitative PCR analysis of diepoxybutane and epihalohydrin damage to nuclear versus mitochondrial DNA

Energy Technology Data Exchange (ETDEWEB)

LaRiviere, Frederick J. [Department of Chemistry, Washington and Lee University, Lexington, VA 24450 (United States); Newman, Adam G.; Watts, Megan L.; Bradley, Sharonda Q.; Juskewitch, Justin E. [Department of Chemistry, Colby College, 5757 Mayflower Hill Drive, Waterville, ME 04901 (United States); Greenwood, Paul G. [Department of Biology, Colby College, Waterville, ME 04901 (United States); Millard, Julie T., E-mail: jtmillar@colby.edu [Department of Chemistry, Colby College, 5757 Mayflower Hill Drive, Waterville, ME 04901 (United States)

2009-05-12

The bifunctional alkylating agents diepoxybutane (DEB) and epichlorohydrin (ECH) are linked to the elevated incidence of certain cancers among workers in the synthetic polymer industry. Both compounds form interstrand cross-links within duplex DNA, an activity suggested to contribute to their cytotoxicity. To assess the DNA targeting of these compounds in vivo, we assayed for damage within chicken erythro-progenitor cells at three different sites: one within mitochondrial DNA, one within expressed nuclear DNA, and one within unexpressed nuclear DNA. We determined the degree of damage at each site via a quantitative polymerase chain reaction, which compares amplification of control, untreated DNA to that from cells exposed to the agent in question. We found that ECH and the related compound epibromohydrin preferentially target nuclear DNA relative to mitochondrial DNA, whereas DEB reacts similarly with the two genomes. Decreased reactivity of the mitochondrial genome could contribute to the reduced apoptotic potential of ECH relative to DEB. Additionally, formation of lesions by all agents occurred at comparable levels for unexpressed and expressed nuclear loci, suggesting that alkylation is unaffected by the degree of chromatin condensation.

Quantitative PCR analysis of diepoxybutane and epihalohydrin damage to nuclear versus mitochondrial DNA

International Nuclear Information System (INIS)

LaRiviere, Frederick J.; Newman, Adam G.; Watts, Megan L.; Bradley, Sharonda Q.; Juskewitch, Justin E.; Greenwood, Paul G.; Millard, Julie T.

2009-01-01

The bifunctional alkylating agents diepoxybutane (DEB) and epichlorohydrin (ECH) are linked to the elevated incidence of certain cancers among workers in the synthetic polymer industry. Both compounds form interstrand cross-links within duplex DNA, an activity suggested to contribute to their cytotoxicity. To assess the DNA targeting of these compounds in vivo, we assayed for damage within chicken erythro-progenitor cells at three different sites: one within mitochondrial DNA, one within expressed nuclear DNA, and one within unexpressed nuclear DNA. We determined the degree of damage at each site via a quantitative polymerase chain reaction, which compares amplification of control, untreated DNA to that from cells exposed to the agent in question. We found that ECH and the related compound epibromohydrin preferentially target nuclear DNA relative to mitochondrial DNA, whereas DEB reacts similarly with the two genomes. Decreased reactivity of the mitochondrial genome could contribute to the reduced apoptotic potential of ECH relative to DEB. Additionally, formation of lesions by all agents occurred at comparable levels for unexpressed and expressed nuclear loci, suggesting that alkylation is unaffected by the degree of chromatin condensation.

Physical association of pyrimidine dimer DNA glycosylase and apurinic/apyrimidinic DNA endonuclease essential for repair of ultraviolet-damaged DNA

International Nuclear Information System (INIS)

Nakabeppu, Y.; Sekiguchi, M.

1981-01-01

T4 endonuclease, which is involved in repair of uv-damaged DNA, has been purified to apparent physical homogeneity. Incubation of uv-irradiated poly(dA).poly(dT) with the purified enzyme preparations resulted in production of alkali-labile apyrimidinic sites, followed by formation of nicks in the polymer. By performing a limited reaction with T4 endonuclease V at pH 8.5, irradiated polymer was converted to an intermediate form that carried a large number of alkali-labile sites but only a few nicks. The intermediate was used as substrate for the assay of apurinic/apyrimidinic DNA endonuclease activity. The two activities, a pyrimidine dimer DNA glycosylase and an apurinic/apyrimidinic DNA endonuclease, were copurified and found in enzyme preparations that contained only a 16,000-dalton polypeptide. These results strongly suggested that a DNA glycosylase specific for pyrimidine dimers and an apurinic/apyrimidinic DNA endonuclease reside in a single polypeptide chain coded by the denV gene of bacteriophage T4

DNA-repair measurements by use of the modified comet assay

DEFF Research Database (Denmark)

Godschalk, Roger W L; Ersson, Clara; Riso, Patrizia

2013-01-01

The measurement of DNA-repair activity by extracts from cells or tissues by means of the single-cell gel electrophoresis (comet) assay has a high potential to become widely used in biomonitoring studies. We assessed the inter-laboratory variation in reported values of DNA-repair activity...... on substrate cells that had been incubated with Ro19-8022 plus light to generate oxidatively damaged DNA. Eight laboratories assessed the DNA-repair activity of three cell lines (i.e. one epithelial and two fibroblast cell lines), starting with cell pellets or with cell extracts provided by the coordinating...... laboratory. There was a large inter-laboratory variation, as evidenced by the range in the mean level of repair incisions between the laboratory with the lowest (0.002incisions/10(6)bp) and highest (0.988incisions/10(6)bp) incision activity. Nevertheless, six out of eight laboratories reported the same cell...

Evaluation of Antioxidant and DNA Damage Protection Activity of the Hydroalcoholic Extract of Desmostachya bipinnata L. Stapf

Directory of Open Access Journals (Sweden)

Upendarrao Golla

2014-01-01

Full Text Available Desmostachya bipinnata Stapf (Poaceae/Gramineae is an official drug of ayurvedic pharmacopoeia. Various parts of this plant were used extensively in traditional and folklore medicine to cure various human ailments. The present study was aimed to evaluate the antioxidant and DNA damage protection activity of hydroalcoholic extract of Desmostachya bipinnata both in vitro and in vivo, to provide scientific basis for traditional usage of this plant. The extract showed significant antioxidant activity in a dose-dependent manner with an IC50 value of 264.18±3.47 μg/mL in H2O2 scavenging assay and prevented the oxidative damage to DNA in presence of DNA damaging agent (Fenton’s reagent at a concentration of 50 μg/mL. Also, the presence of extract protected yeast cells in a dose-dependent manner against DNA damaging agent (Hydroxyurea in spot assay. Moreover, the presence of extract exhibited significant antioxidant activity in vivo by protecting yeast cells against oxidative stressing agent (H2O2. Altogether, the results of current study revealed that Desmostachya bipinnata is a potential source of antioxidants and lends pharmacological credence to the ethnomedical use of this plant in traditional system of medicine, justifying its therapeutic application for free-radical-induced diseases.

DNA damages induced in human lymphocytes by UV or X-rays and repair capacities of healthy donors and skin cancer patients

International Nuclear Information System (INIS)

Cebulska-Wasilewska, A.; Dyga, W.; Budzanowska, E.

1999-01-01

The aim of this study was to compare variation in the individual susceptibility of various donors to the induction of the DNA damage by genotoxic agents and their cellular capabilities to repair induced damage. DNA damages induced by UV or X-rays in lymphocytes and cellular repair capability of healthy donors and persons bearing various categories of skin cancer cells were investigated. Fresh blood was collected by venipuncture from 35 individuals (including nine prior to skin cancer treatment). All cancer patients were nonsmoking males, however 42.3 % of them were former smokers. All healthy donors were also males, an average age was 38.6 y and among them 68% were recent or former smokers. Immediately after collecting samples, lymphocytes were isolated and stored at -70 o C for further studies in vitro. Previously cryopreserved lymphocytes were defrosted and viability of the cells was investigated. The single cell gel electrophoresis assay (SCGE), known as a Comet assay, was performed in defrozen lymphocytes to evaluate individual DNA damage levels presented in lymphocytes at the time of sample's collection. To compare individual susceptibility to the induction of DNA damage by UV and ionizing radiation, lymphocytes were exposed to dose of 6 J/m 2 of UV or 2 Gy of X-rays and DNA damages were detected again with an application of the Comet assay. Additionally, to study variation in the individuals cellular capability to repair damages induced, prior to the DNA damage analysis an incubation of cells exposed was also done in presence or absence of phytohemagglutinin (cell divisions processes starting agent). Results showed in untreated lymphocytes of skin cancer patients significantly higher than in the reference group levels of the DNA damages. Significantly different responses to UV and significantly lower capabilities to repair UV induced damage in skin cancer patients were observed. On the average, no differences between reference group and skin cancer patients

Recent Advancements in DNA Damage-Transcription Crosstalk and High-Resolution Mapping of DNA Breaks.

Science.gov (United States)

Vitelli, Valerio; Galbiati, Alessandro; Iannelli, Fabio; Pessina, Fabio; Sharma, Sheetal; d'Adda di Fagagna, Fabrizio

2017-08-31

Until recently, DNA damage arising from physiological DNA metabolism was considered a detrimental by-product for cells. However, an increasing amount of evidence has shown that DNA damage could have a positive role in transcription activation. In particular, DNA damage has been detected in transcriptional elements following different stimuli. These physiological DNA breaks are thought to be instrumental for the correct expression of genomic loci through different mechanisms. In this regard, although a plethora of methods are available to precisely map transcribed regions and transcription start sites, commonly used techniques for mapping DNA breaks lack sufficient resolution and sensitivity to draw a robust correlation between DNA damage generation and transcription. Recently, however, several methods have been developed to map DNA damage at single-nucleotide resolution, thus providing a new set of tools to correlate DNA damage and transcription. Here, we review how DNA damage can positively regulate transcription initiation, the current techniques for mapping DNA breaks at high resolution, and how these techniques can benefit future studies of DNA damage and transcription.

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

Science.gov (United States)

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.

Lead induces DNA damage and alteration of ALAD and antioxidant genes mRNA expression in construction site workers.

Science.gov (United States)

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 gene.

LORD-Q: a long-run real-time PCR-based DNA-damage quantification method for nuclear and mitochondrial genome analysis

Science.gov (United States)

Lehle, Simon; Hildebrand, Dominic G.; Merz, Britta; Malak, Peter N.; Becker, Michael S.; Schmezer, Peter; Essmann, Frank; Schulze-Osthoff, Klaus; Rothfuss, Oliver

2014-01-01

DNA damage is tightly associated with various biological and pathological processes, such as aging and tumorigenesis. Although detection of DNA damage is attracting increasing attention, only a limited number of methods are available to quantify DNA lesions, and these techniques are tedious or only detect global DNA damage. In this study, we present a high-sensitivity long-run real-time PCR technique for DNA-damage quantification (LORD-Q) in both the mitochondrial and nuclear genome. While most conventional methods are of low-sensitivity or restricted to abundant mitochondrial DNA samples, we established a protocol that enables the accurate sequence-specific quantification of DNA damage in >3-kb probes for any mitochondrial or nuclear DNA sequence. In order to validate the sensitivity of this method, we compared LORD-Q with a previously published qPCR-based method and the standard single-cell gel electrophoresis assay, demonstrating a superior performance of LORD-Q. Exemplarily, we monitored induction of DNA damage and repair processes in human induced pluripotent stem cells and isogenic fibroblasts. Our results suggest that LORD-Q provides a sequence-specific and precise method to quantify DNA damage, thereby allowing the high-throughput assessment of DNA repair, genotoxicity screening and various other processes for a wide range of life science applications. PMID:24371283

DNA Damage Observed in Unaffected Individuals with Family History of T2DM

Science.gov (United States)

Ramesh, Nikhila; Abilash, V. G.

2017-11-01

Diabetes has been documented to cause high levels of DNA fragmentation in some cases. As diabetes is inheritable and influenced by both genetic and environmental factors, an investigation into the genomic stability of individuals who are strongly at risk of inheriting diabetes was conducted by inducing oxidative stress, as DNA damage in unaffected individuals could be a sign of onset of the disease or the presence of genetic alterations that reduce cellular defences against reactive oxygen species. In this study, alkaline comet assay was performed on isolated human leukocytes to determine whether individuals with a family history of Type 2 Diabetes Mellitus (T2DM) are more prone to DNA damage under oxidative stress. Visual scoring of comets showed that these individuals have higher degree of DNA damage compared to a control individual with no family history of Type 2 Diabetes Mellitus. Further studies with large sample could determine the presence of disabled cellular defences against oxidative stress in unaffected individuals and intervention with antioxidants could prevent or manage Type 2 Diabetes Mellitus and its complications.

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

Science.gov (United States)

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 (Padrenaline 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.

Modulation of X ray DNA damage by SR-2508 +/- buthionine sulfoximine

International Nuclear Information System (INIS)

Kinsella, T.J.; Dobson, P.P.; Russo, A.; Mitchell, J.B.; Fornace, A.J. Jr.

1986-01-01

It has been demonstrated that glutathione (GSH) depletion with buthionine sulfoximine (BSO) potentiates SR-2508 radiosensitization in hypoxic cells. We have measured the effect of SR-2508 alone, BSO alone, and combined treatment on radiation-induced DNA strand breaks in hypoxic V79 cells using alkaline and neutral elution. DNA base damage recognized by a damage specific endonuclease from M. luteus was also studied. Hypoxic irradiation markedly reduces the efficiency of single-strand (SSB) and double-strand breaks (DSB) to 10-20% compared to oxic irradiation. Hypoxia had less effect on the efficiency of base damage (ESS). BSO treatment alone, resulting in GSH depletion to less than 5% of controls, had little effect of hypoxic-SSB, DSB, and ESS. SR-2508 (5 mM) treatment alone in hypoxic cells increased the number of SSB, DSB and ESS to approximately half of that resulting from oxic irradiation. However, the combination of BSO and SR-2508 in hypoxic cells resulted in SSB and DSB comparable to oxic irradiation. This combined treatment resulted in less effect on ESS. We conclude that the observed hypoxic radiosensitization, using clonogenic survival assays with combined BSO-SR-2508, correlates with our results assessing DNA strand breaks and base damage

Influence of 1.8-GHz (GSM) radiofrequency radiation (RFR) on DNA damage and repair induced by X-rays in human leukocytes in vitro.

Science.gov (United States)

Zhijian, Chen; Xiaoxue, Li; Yezhen, Lu; Deqiang, Lu; Shijie, Chen; Lifen, Jin; Jianlin, Lou; Jiliang, He

2009-01-01

In the present study, the in vitro comet assay was used to determine whether 1.8-GHz radiofrequency radiation (RFR) can influence DNA repair in human leukocytes exposed to X-rays. The specific energy absorption rate (SAR) of 2 W/kg (the current European safety limit) was applied. The leukocytes from four young healthy donors were intermittently exposed to RFR for 24 h (fields on for 5 min, fields off for 10 min), and then irradiated with X-rays at doses of 0.25, 0.5, 1.0 and 2.0 Gy. DNA damage to human leukocytes was detected using the comet assay at 0, 15, 45, 90, 150 and 240 min after exposure to X-rays. Using the comet assay, the percent of DNA in the tail (% tail DNA) served as the indicator of DNA damage; the DNA repair percentage (DRP) served as the indicator of the DNA repair speed. The results demonstrated that (1) the DNA repair speeds of human leukocytes after X-ray exposure exhibited individual differences among the four donors; (2) the intermittent exposures of 1.8-GHz RFR at the SAR of 2 W/kg for 24 h did not directly induce DNA damage or exhibit synergistic effects with X-rays on human leukocytes.

Study of terahertz-radiation-induced DNA damage in human blood leukocytes

Energy Technology Data Exchange (ETDEWEB)

Angeluts, A A; Esaulkov, M N; Kosareva, O G; Solyankin, P M; Shkurinov, A P [International Laser Center, M. V. Lomonosov Moscow State University, Moscow (Russian Federation); Gapeyev, A B; Pashovkin, T N [Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region (Russian Federation); Matyunin, S N [Section of Applied Problems at the Presidium of the Russian Academy of Sciences, Moscow (Russian Federation); Nazarov, M M [Institute on Laser and Information Technologies, Russian Academy of Sciences, Shatura, Moscow Region (Russian Federation); Cherkasova, O P [Institute of Laser Physics, Siberian Branch, Russian Academy of Sciences, Novosibirsk (Russian Federation)

2014-03-28

We have carried out the studies aimed at assessing the effect of terahertz radiation on DNA molecules in human blood leukocytes. Genotoxic testing of terahertz radiation was performed in three different oscillation regimes, the blood leukocytes from healthy donors being irradiated for 20 minutes with the mean intensity of 8 – 200 μW cm{sup -2} within the frequency range of 0.1 – 6.5 THz. Using the comet assay it is shown that in the selected regimes such radiation does not induce a direct DNA damage in viable human blood leukocytes. (biophotonics)

DNA Damage Signaling Is Induced in the Absence of Epstein-Barr Virus (EBV) Lytic DNA Replication and in Response to Expression of ZEBRA.

Science.gov (United States)

Wang'ondu, Ruth; Teal, Stuart; Park, Richard; Heston, Lee; Delecluse, Henri; Miller, George

2015-01-01

Epstein Barr virus (EBV), like other oncogenic viruses, modulates the activity of cellular DNA damage responses (DDR) during its life cycle. Our aim was to characterize the role of early lytic proteins and viral lytic DNA replication in activation of DNA damage signaling during the EBV lytic cycle. Our data challenge the prevalent hypothesis that activation of DDR pathways during the EBV lytic cycle occurs solely in response to large amounts of exogenous double stranded DNA products generated during lytic viral DNA replication. In immunofluorescence or immunoblot assays, DDR activation markers, specifically phosphorylated ATM (pATM), H2AX (γH2AX), or 53BP1 (p53BP1), were induced in the presence or absence of viral DNA amplification or replication compartments during the EBV lytic cycle. In assays with an ATM inhibitor and DNA damaging reagents in Burkitt lymphoma cell lines, γH2AX induction was necessary for optimal expression of early EBV genes, but not sufficient for lytic reactivation. Studies in lytically reactivated EBV-positive cells in which early EBV proteins, BGLF4, BGLF5, or BALF2, were not expressed showed that these proteins were not necessary for DDR activation during the EBV lytic cycle. Expression of ZEBRA, a viral protein that is necessary for EBV entry into the lytic phase, induced pATM foci and γH2AX independent of other EBV gene products. ZEBRA mutants deficient in DNA binding, Z(R183E) and Z(S186E), did not induce foci of pATM. ZEBRA co-localized with HP1β, a heterochromatin associated protein involved in DNA damage signaling. We propose a model of DDR activation during the EBV lytic cycle in which ZEBRA induces ATM kinase phosphorylation, in a DNA binding dependent manner, to modulate gene expression. ATM and H2AX phosphorylation induced prior to EBV replication may be critical for creating a microenvironment of viral and cellular gene expression that enables lytic cycle progression.

Ultrastructural and DNA damaging effects of lead nitrate in the liver.

Science.gov (United States)

Narayana, K; Al-Bader, Maie

2011-01-01

A ubiquitous environmental toxicant - lead is known to affect several organ systems. This study was designed to investigate the effects of lead nitrate exposure on liver structure and DNA fragmentation. Adult male Wistar rats were treated orally with lead nitrate at the dose levels of 0%, 0.5% and 1% for 60 days and sacrificed on the next day. The liver was processed for thick sections and evaluated after toludine blue staining and by electron microscopy after staining with uranyl acetate and lead citrate. The DNA damage was assessed by DNA fragmentation assay. The liver weight was not significantly affected in the experimental groups. Hepatocyte nuclei were not shrunk, instead lead was mitogenic to hepatocytes as indicated by an increase in the number of binucleated hepatocytes (Plead-treated groups, these changes were not significantly different from that in control as evaluated by optical density. In conclusion, lead induces necrotic changes with simultaneous mitogenic activity; however, it does not induce significant DNA damage in the liver. Copyright © 2009 Elsevier GmbH. All rights reserved.

Analysis of DNA damage in sea lamprey (Petromyzon marinus) spermatozoa by UV, hydrogen peroxide, and the toxicant bisazir

International Nuclear Information System (INIS)

Ciereszko, Andrzej; Wolfe, Tobie D.; Dabrowski, Konrad

2005-01-01

In this study we sought to demonstrate that Comet assay can be applied to sea lamprey sperm DNA fragmentation and used to describe the relationship between sperm DNA damage and sperm fertilizing ability. We show that the assay can be used reliably and accurately, and unlike in the case of mammals, there is no need for additional steps related to improvement of efficacy of lysis and DNA decondensation. This agrees with the presence of histone proteins in lamprey sperm. An increase in DNA fragmentation was noted during short-term storage of milt on ice (0-4 days). We demonstrated genotoxic effects of UV radiation and oxidative stress (exposure to hydrogen peroxide) and found that oxidative damage to sperm DNA was likely repaired after fertilization in the embryo. Repairing capacity of the oocyte toward sperm DNA lesions caused by UV was restricted. Toxic effect of p,p-bis-(1-aziridinyl)-N-methylphosphinothioic acid (p,p-bis(1-aziridinyl)-N-methylphosphinothioic amide), a sea lamprey chemosterilant, could not be linked to DNA fragmentation in the in vitro tests. Its genotoxicity in vivo may possibly be associated with other mechanisms of DNA degradation (oxidation or DNA-protein and DNA-DNA cross-linking). In conclusion, this study demonstrates that Comet assay can be successfully applied to monitor effects of environmental disturbances and imposed injuries in sea lamprey spermatozoa and possibly other species of ancient fish with acrosomal sperm

Analysis of DNA damage in sea lamprey (Petromyzon marinus) spermatozoa by UV, hydrogen peroxide, and the toxicant bisazir

Energy Technology Data Exchange (ETDEWEB)

Ciereszko, Andrzej [School of Natural Resources, Ohio State University, 210 Kottman Hall, 2021 Coffey Rd., Columbus, OH 434210 (United States); Semen Biology Group, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, 10-747 Olsztyn (Poland); Wolfe, Tobie D. [School of Natural Resources, Ohio State University, 210 Kottman Hall, 2021 Coffey Rd., Columbus, OH 434210 (United States); Dabrowski, Konrad [School of Natural Resources, Ohio State University, 210 Kottman Hall, 2021 Coffey Rd., Columbus, OH 434210 (United States)]. E-mail: dabrowski.1@osu.edu

2005-06-15

In this study we sought to demonstrate that Comet assay can be applied to sea lamprey sperm DNA fragmentation and used to describe the relationship between sperm DNA damage and sperm fertilizing ability. We show that the assay can be used reliably and accurately, and unlike in the case of mammals, there is no need for additional steps related to improvement of efficacy of lysis and DNA decondensation. This agrees with the presence of histone proteins in lamprey sperm. An increase in DNA fragmentation was noted during short-term storage of milt on ice (0-4 days). We demonstrated genotoxic effects of UV radiation and oxidative stress (exposure to hydrogen peroxide) and found that oxidative damage to sperm DNA was likely repaired after fertilization in the embryo. Repairing capacity of the oocyte toward sperm DNA lesions caused by UV was restricted. Toxic effect of p,p-bis-(1-aziridinyl)-N-methylphosphinothioic acid (p,p-bis(1-aziridinyl)-N-methylphosphinothioic amide), a sea lamprey chemosterilant, could not be linked to DNA fragmentation in the in vitro tests. Its genotoxicity in vivo may possibly be associated with other mechanisms of DNA degradation (oxidation or DNA-protein and DNA-DNA cross-linking). In conclusion, this study demonstrates that Comet assay can be successfully applied to monitor effects of environmental disturbances and imposed injuries in sea lamprey spermatozoa and possibly other species of ancient fish with acrosomal sperm.

DNA damage in grasshopper Chorthippus brunneus (Orthoptera) hatchlings following paraquat exposure.

Science.gov (United States)

Augustyniak, M; Nocoń, Ł; Kędziorski, A; Łaszczyca, P; Sawczyn, T; Tarnawska, M; Zawisza-Raszka, A

2015-04-01

Comet assay was applied to study genotoxic damage induced by paraquat (PQ) in brain cells of Chorthippus brunneus (Insecta: Orthoptera) hatchlings. Percentage of the comet fluorescence in the tail (TDNA), length of the comet tail (TL) and Olive tail moment (OTM) were used for quantitative assessment of the DNA damage. Multiple regression analysis supplemented standard statistical elaboration of the results. Increasing PQ concentrations applied either directly to the brain cells suspension (10, 50, and 250 μM PQ final concentration--in vitro protocol) or indirectly (50, 250, and 1250 μM PQ final concentration--in vivo protocol) provoked significant increase of oxidative damage to DNA (higher median TDNA and OTM values). The damage increased with time of exposure (0, 5, 15, and 30 min) following in vitro application, but decreased in longer interval (3 vs 24 h) after in vivo administration of paraquat. On contrary, median TL values did not correlate with paraquat concentration irrespectively of the exposure protocol. Possible reason of this discrepancy in light of paraquat toxicity is discussed. Copyright © 2015 Elsevier Ltd. All rights reserved.

Assessment of DNA damage using comet assay in middle-aged overweight/obese subjects after following a hypocaloric diet supplemented with cocoa extract.

Science.gov (United States)

Ibero-Baraibar, Idoia; Azqueta, Amaya; Lopez de Cerain, Adela; Martinez, J Alfredo; Zulet, M Angeles

2015-01-01

Nutrient excess and unbalanced diets can result in overproduction of reactive oxygen species (ROS), which are associated with oxidative stress. Cocoa extract contains antioxidants that inhibit the harmful effects of ROS. This trial analysed the effect of cocoa extract consumption integrated as a bioactive compound into ready-to-eat meals, on oxidative stress at the level of DNA in overweight/obese subjects. Fifty volunteers [57.26(5.24) years, 30.59(2.33)kg/m(2)] participated in a 4-week double-blind, randomised, placebo-controlled parallel nutritional intervention. Half of the volunteers received meals supplemented with 1.4 g/day cocoa extract, while the other half received control meals, both within a 15% energy restriction diet. Lymphocytes were isolated and endogenous strand breaks, oxidised bases and resistance to H2O2-induced damage were measured by the comet assay. The intake of ready-to-eat meals supplemented with cocoa extract did not show relevant changes in the oxidative status of DNA. However, in the cocoa group, oxidised bases negatively correlated with methyl epicatechin-O-sulphate (r = -0.76; P = -0.007) and epicatechin sulphate (r = -0.61; P = -0.046). When volunteers of both groups were analysed together, a marginal decrease (P = 0.072) in oxidised bases was observed, which attributed to weight loss. Subjects who started the intervention with higher levels of damage showed a greater reduction in oxidised bases after 4 weeks (P = 0.040) compared to those who had lower baseline levels. In conclusion, even if 1.4 g of cocoa supplementation for 4 weeks did not show notable changes in terms of antioxidant status of DNA, the energy restriction showed a slightly decrease in oxidised bases and this was seen to a greater extent in subjects who started the intervention with higher levels of damage. On the other hand, the inverse associations found between oxidised bases and some cocoa-derived metabolites suggest that a protective effect might be seen in a

Carcinogen-induced damage to DNA

International Nuclear Information System (INIS)

Strauss, B.; Altamirano, M.; Bose, K.; Sklar, R.; Tatsumi, K.

1979-01-01

Human cells respond to carcinogen-induced damage in their DNA in at least two ways. The first response, excision repair, proceeds by at least three variations, depending on the nature of the damage. Nucleotide excision results in relatively large repair patches but few free DNA breaks, since the endonuclease step is limiting. Apurinic repair is characterized by the appearance of numerous breaks in the DNA and by short repair patches. The pathways behave as though they function independently. Lymphoic cells derived from a xeroderma pigmentosum complementation group C patient are deficient in their ability to perform nucleotide excision and also to excise 6 methoxyguanine adducts, but they are apurinic repair competent. Organisms may bypass damage in their DNA. Lymphoblastoid cells, including those derived from xeroderma pigmentosum treated with 3 H-anti-BPDE, can replicate their DNA at low doses of carcinogen. Unexcised 3 H is found in the light or parental strand of the resulting hybrid DNA when replication occurs in medium with BrdUrd. This observation indicates a bypass reaction occurring by a mechanism involving branch migration at DNA growing points. Branch migration in DNA preparations have been observed, but the evidence is that most occurs in BrdUrd-containing DNA during cell lysis. The measurement of the bifilarly substituted DNA resulting from branch migration is a convenient method of estimating the proportion of new synthesis remaining in the vicinity of the DNA growing point. Treatment with carcinogens or caffeine results in accumulation of DNA growing points accompanied by the synthesis of shortened pieces of daughter DNA

Sea urchin coelomocytes are resistant to a variety of DNA damaging agents

Energy Technology Data Exchange (ETDEWEB)

Loram, Jeannette; Raudonis, Renee; Chapman, Jecar; Lortie, Mae [Bermuda Institute of Ocean Sciences, St. George' s, Bermuda, GE 01 (Bermuda); Bodnar, Andrea, E-mail: andrea.bodnar@bios.edu [Bermuda Institute of Ocean Sciences, St. George' s, Bermuda, GE 01 (Bermuda)

2012-11-15

Increasing anthropogenic activities are creating environmental pressures that threaten marine ecosystems. Effective environmental health assessment requires the development of rapid, sensitive, and cost-effective tools to predict negative impacts at the individual and ecosystem levels. To this end, a number of biological assays using a variety of cells and organisms measuring different end points have been developed for biomonitoring programs. The sea urchin fertilization/development test has been useful for evaluating environmental toxicology and it has been proposed that sea urchin coelomocytes represent a novel cellular biosensor of environmental stress. In this study we investigated the sensitivity of coelomocytes from the sea urchin Lytechinus variegatus to a variety of DNA-damaging agents including ultraviolet (UV) radiation, hydrogen peroxide (H{sub 2}O{sub 2}), methylmethane sulfonate (MMS) and benzo[a]pyrene (BaP). LD{sub 50} values determined for coelomocytes after 24 h of exposure to these DNA damaging agents indicated a high level of resistance to all treatments. Significant increases in the formation of apurinic/apyrimidinic (AP or abasic) sites in DNA were only detected using high doses of H{sub 2}O{sub 2}, MMS and UV radiation. Comparison of sea urchin coelomocytes with hemocytes from the gastropod mollusk Aplysia dactylomela and the decapod crustacean Panulirus argus indicated that sensitivity to different DNA damaging agents varies between species. The high level of resistance to genotoxic agents suggests that DNA damage may not be an informative end point for environmental health assessment using sea urchin coelomocytes however, natural resistance to DNA damaging agents may have implications for the occurrence of neoplastic disease in these animals.

Repair of DNA damage in Deinococcus radiodurans

International Nuclear Information System (INIS)

Evans, D.M.

1984-01-01

The repair of DNA lesions in Deinococcus radiodurans was examined with particular reference to DNA excision repair of ultraviolet light (UV) induced pyrimidine dimers. The characteristics of excision repair via UV endonucleases α and β in vivo varied with respect to (a) the substrate range of the enzymes, (b) the rate of repair of DNA damage (c) the requirement for a protein synthesised in response to DNA damage to attenuate exonuclease action at repairing regions. UV endonuclease α is postulated to incise DNA in a different manner from UV endonuclease β thus defining the method of subsequent repair. Several DNA damage specific endonuclease activities independent of α and β are described. Mutations of the uvsA, uvsF and uvsG genes resulted in an increase in single-strand breaks in response to DNA damage producing uncontrolled DNA degradation. Evidence is presented that these genes have a role in limiting the access of UV endonuclease β to DNA lesions. uvsF and uvsG are also shown to be linked to the mtoA gene. Mutation of uvsH and reo-1 produces further distinct phenotypes which are discussed. An overall model of excision repair of DNA damage in Deinococcus radiodurans is presented. (author)

DNA damage mediated transcription arrest: Step back to go forward.

Science.gov (United States)

Mullenders, Leon

2015-12-01

The disturbance of DNA helix conformation by bulky DNA damage poses hindrance to transcription elongating due to stalling of RNA polymerase at transcription blocking lesions. Stalling of RNA polymerase provokes the formation of R-loops, i.e. the formation of a DNA-RNA hybrid and a displaced single stranded DNA strand as well as displacement of spliceosomes. R-loops are processed into DNA single and double strand breaks by NER factors depending on TC-NER factors leading to genome instability. Moreover, stalling of RNA polymerase induces a strong signal for cell cycle arrest and apoptosis. These toxic and mutagenic effects are counteracted by a rapid recruitment of DNA repair proteins to perform transcription coupled nucleotide excision repair (TC-NER) to remove the blocking DNA lesions and to restore transcription. Recent studies have highlighted the role of backtracking of RNA polymerase to facilitate TC-NER and identified novel factors that play key roles in TC-NER and in restoration of transcription. On the molecular level these factors facilitate stability of the repair complex by promotion and regulation of various post-translational modifications of NER factors and chromatin substrate. In addition, the continuous flow of new factors that emerge from screening assays hints to several regulatory levels to safeguard the integrity of transcription elongation after disturbance by DNA damage that have yet to be explored. Copyright © 2015 Elsevier B.V. All rights reserved.

Molecular mechanisms in radiation damage to DNA

International Nuclear Information System (INIS)

Osman, R.

1991-01-01

The objectives of this work are to elucidate the molecular mechanisms that are responsible for radiation-induced DNA damage. The overall goal is to understand the relationship between the chemical and structural changes produced by ionizing radiation in DNA and the resulting impairment of biological function expressed as carcinogenesis or cell death. The studies are based on theoretical explorations of possible mechanisms that link initial radiation damage in the form of base and sugar damage to conformational changes in DNA. These mechanistic explorations should lead to the formulation of testable hypothesis regarding the processes of impairment of regulation of gene expression, alternation in DNA repair, and damage to DNA structure involved in cell death or cancer

A fluorescence sedimentation assay for dsDNA antibodies

DEFF Research Database (Denmark)

Duus, K; Draborg, A H; Güven, E

2017-01-01

The Farr assay is a radioimmunoassay (RIA) for dsDNA antibodies, based on antibody precipitation using ammonium sulphate and quantification using radio-labelled dsDNA. The RIA-Farr assay offers outstanding clinical specificity and sensitivity for systemic lupus erythematosus (SLE) compared to other...... on precipitation with polyethylene glycol (PEG) and fluorescence of EvaGreen intercalated in dsDNA as detection principle. As dsDNA antibodies are quantified using fluorescence, the disadvantages of working with radioactivity are eliminated. The Fluoro-Farr assay was developed and validated, and the diagnostic...

Preferential binding of yeast Rad4-Rad23 complex to damaged DNA

International Nuclear Information System (INIS)

Jansen, L.E.T.; Verhage, R.A.; Brouwer, J.

1998-01-01

The yeast Rad4 and Rad23 proteins form a complex that is involved in nucleotide excision repair (NER). Their function in this process is not known yet, but genetic data suggest that they act in an early step in NER. We have purified an epitope-tagged Rad4.Rad23 (tRad4. Rad23) complex from yeast cells, using a clone overproducing Rad4 with a hemagglutinin-tag at its C terminus. tRad4.Rad23 complex purified by both conventional and immuno-affinity chromatography complements the in vitro repair defect of rad4 and rad23 mutant extracts, demonstrating that these proteins are functional in NER. Using electrophoretic mobility shift assays, we show preferential binding of the tRad4.Rad23 complex to damaged DNA in vitro. UV-irradiated, as well as N-acetoxy-2-(acetylamino)fluorene-treated DNA, is efficiently bound by the protein complex. These data suggest that Rad4.Rad23 interacts with DNA damage during NER and may play a role in recognition of the damage

Autophagy in DNA Damage Response

Directory of Open Access Journals (Sweden)

Piotr Czarny

2015-01-01

Full Text Available DNA damage response (DDR involves DNA repair, cell cycle regulation and apoptosis, but autophagy is also suggested to play a role in DDR. Autophagy can be activated in response to DNA-damaging agents, but the exact mechanism underlying this activation is not fully understood, although it is suggested that it involves the inhibition of mammalian target of rapamycin complex 1 (mTORC1. mTORC1 represses autophagy via phosphorylation of the ULK1/2–Atg13–FIP200 complex thus preventing maturation of pre-autophagosomal structures. When DNA damage occurs, it is recognized by some proteins or their complexes, such as poly(ADPribose polymerase 1 (PARP-1, Mre11–Rad50–Nbs1 (MRN complex or FOXO3, which activate repressors of mTORC1. SQSTM1/p62 is one of the proteins whose levels are regulated via autophagic degradation. Inhibition of autophagy by knockout of FIP200 results in upregulation of SQSTM1/p62, enhanced DNA damage and less efficient damage repair. Mitophagy, one form of autophagy involved in the selective degradation of mitochondria, may also play role in DDR. It degrades abnormal mitochondria and can either repress or activate apoptosis, but the exact mechanism remains unknown. There is a need to clarify the role of autophagy in DDR, as this process may possess several important biomedical applications, involving also cancer therapy.

Gymnemagenin-a triterpene saponin prevents γ-radiation induced cellular DNA damage

International Nuclear Information System (INIS)

Arunachalam, Kantha Deivi; Arun, Lilly Baptista; Annamalai, Sathesh Kumar; Hari, Shanmugasundaram

2014-01-01

Gymnema sylvestre an ethno-medicinally important plant was investigated for its protecting activity against radiation induced DNA damage. The major bioactive component present in Gymnema sylvestre such as gymnemic acid and gymnemagenin a triterpene saponin, were tested for its radioprotective effects against 60 Co irradiation induced DNA damage in fish model using fresh water fish Pangasius sutchi. Fishes subjected to a dose of 133 Gy of gamma radiation and observed for eight days. The genotoxic assessment by micronucleus assay showed us that that the plant extract helped in reducing the frequency of micronucleated and binucleated erythrocytes compared to the irradiated control group. The genotoxic assessment by alkaline comet assay by single gel electrophoresis shows that pretreatment with the plant extract appreciably decreased the percentage of tail DNA towards the levels close to those of normal control group. The gradual increase in the level of the antioxidant enzymes: superoxide dismutase (SOD) and catalase (CAT) during the course of the experiment indicates that the antioxidant enzyme activities play an important role in protecting organisms against gamma radiation-induced cellular oxidative stress. In conclusion the leaf extracts of Gymnema sylvstre exerts its radio protective potential by suppressing the toxic assault of ROS generated by the ionizing radiation through its ability to boost the levels of antioxidant enzymes (CAT and SOD) due to the presence of its phytochemicals like gymnemgenenin- a Triterpene Saponin. (author)

Mechanisms for radiation damage in DNA

International Nuclear Information System (INIS)

Sevilla, M.D.

1993-12-01

In this project the author has proposed several mechanisms for radiation damage to DNA and its constituents, and has detailed a series of experiments utilizing electron spin resonance spectroscopy, HPLC, GC-mass spectroscopy and ab initio molecular orbital calculations to test the proposed mechanisms. In this years work he has completed several experiments on the role of hydration water on DNA radiation damage, continued the investigation of the localization of the initial charges and their reactions on DNA, investigated protonation reactions in DNA base anions, and employed ab initio molecular orbital theory to gain insight into the initial events of radiation damage to DNA. Ab initio calculations have provided an understanding of the energetics evolved in anion and cation formation, ion radical transfer in DNA as well as proton transfer with DNA base pair radical ions. This has been extended in this years work to a consideration of ionization energies of various components of the DNA deoxyribose backbone and resulting neutral sugar radicals. This information has aided the formation of new radiation models for the effect of radiation on DNA. During this fiscal year four articles have been published, four are in press, one is submitted and several more are in preparation. Four papers have been presented at scientific meetings. This years effort will include another review article on the open-quotes Electron Spin Resonance of Radiation Damage to DNAclose quotes

Influence of the complexity of radiation-induced DNA damage on enzyme recognition

International Nuclear Information System (INIS)

Palmer, Philip

2002-01-01

Ionising radiation is unique in inducing DNA clustered damage together with the simple isolated lesions. Understanding how these complex lesions are recognised and repaired by the cell is key to understanding the health risks associated with radiation exposure. This study focuses on whether ionising radiation-induced complex single-strand breaks (SSB) are recognised by DNA-PK and PARP, and whether the complexity of DSB influence their ligation by either DNA ligase lV/XRCC4 (LX) complex or T4 DNA ligase. Plasmid DNA, irradiated in aqueous solution using sparsely ionising γ-rays and densely ionising α-particles produce different yields of complex DNA damages, used as substrates for in vitro DNA-PK and PARP activity assays. The activity of DNA-PK to phosphorylate a peptide was determined using HF19 cell nuclear extracts as a source of DNA-PK. PARP ADP-ribosylation activity was determined using purified PARP enzyme. The activation of DNA-PK and PARP by irradiated DNA is due to SSB and not the low yield of DSB (linear plasmid DNA <10%). A ∼2 fold increase in DNA-PK activation and a ∼3-fold reduction in PARP activity seen on increasing the ionising density of the radiation (proportion of complex damage) are proposed to reflect changes in the complexity of SSB and may relate to damage signalling. Complex DSB synthesised as double-stranded oligonucleotides, with a 2 bp 5'-overhang, and containing modified lesions, 8-oxoguanine and abasic sites, at known positions relative to the termini were used as substrates for in vitro ligation by DNA ligase IV/XRCC4 or T4 ligase. The presence of a modified lesion 2 or 3 bp but not 4 bp from the 3'-termini and 2 or 6 bp from the 5'-termini caused a drastic reduction in the extent of ligation. Therefore, the presence of modified lesions near to the termini of a DSB may compromise their rejoining by non-homologous end-joining (NHEJ) involving the LX complex. (author)

Characterization of antibodies specific for UV-damaged DNA by ELISA

Energy Technology Data Exchange (ETDEWEB)

Eggset, G; Volden, G; Krokan, H

1987-04-01

The specificity of affinity purified antibodies raised against UV-irradiated DNA was examined using an enzyme-linked immunosorbent assay. DNA irradiated with UV doses higher than needed for saturation with pyrimidine dimers bound increasing amounts of antibody. Photosensitized DNA, containing high amounts of pyrimidine dimers, showed very poor binding of antibody. When UV-irradiated DNA was given a second dose of 340-nm UV light, the binding of antibodies was inhibited. Taken together, this indicates a major specificity for (6-4)-photoproducts, which are photochemically reversed by UV light in the 340-nm region. The antibodies also showed little but detectable binding to pyrimidine glycols produced in DNA by oxidation with OsO/sub 4/. Previously, we have used these antibodies for the detection of UV-induced DNA damage and its repair in human skin in vivo. These findings indicate that (6-4)-photoproducts, considered highly mutagenic, are repaired in human skin.

Characterization of antibodies specific for UV-damaged DNA by ELISA

International Nuclear Information System (INIS)

Eggset, G.; Volden, G.; Krokan, H.; Norsk Hydro Research Centre, Porsgrunn

1987-01-01

The specificity of affinity purified antibodies raised against UV-irradiated DNA was examined using an enzyme-linked immunosorbent assay. DNA irradiated with UV doses higher than needed for saturation with pyrimidine dimers bound increasing amounts of antibody. Photosensitized DNA, containing high amounts of pyrimidine dimers, showed very poor binding of antibody. When UV-irradiated DNA was given a second dose of 340-nm UV light, the binding of antibodies was inhibited. Taken together, this indicates a major specificity for (6-4)-photoproducts, which are photochemically reversed by UV light in the 340-nm region. The antibodies also showed little but detectable binding to pyrimidine glycols produced in DNA by oxidation with OsO 4 . Previously, we have used these antibodies for the detection of UV-induced DNA damage and its repair in human skin in vivo. These findings indicate that (6-4)-photoproducts, considered highly mutagenic, are repaired in human skin. (author)

SIRT participates at DNA damage response

Energy Technology Data Exchange (ETDEWEB)

Yun, Mi Yong; Joeng, Jae Min; Lee, Kee Ho [Korea Cancer Center Hospital, Seoul (Korea, Republic of); Park, Gil Hong [College of Medicine, Korea University, Seoul (Korea, Republic of)

2009-05-15

Sir2 maintains genomic stability in multiple ways in yeast. As a NAD{sup +}-dependent histone deacetylase, Sir2 has been reported to control chromatin silencing. In both budding yeast and Drosophila, overexpression of Sir2 extends life span. Previous reports have also demonstrated that Sir2 participate at DNA damage repair. A protein complex containing Sir2 has been reported to translocate to DNA double-strand breaks. Following DNA damage response, SIRT1 deacetylates p53 protein and attenuates its ability as a transcription factor. Consequently, SIRT1 over-expression increases cell survival under DNA damage inducing conditions. These previous observations mean a possibility that signals generated during the process of DNA repair are delivered through SIRT1 to acetylated p53. We present herein functional evidence for the involvement of SIRT1 in DNA repair response to radiation. In addition, this modulation of DNA repair activity may be connected to deacetylation of MRN proteins.

The Intertwined Roles of DNA Damage and Transcription

OpenAIRE

Di Palo, Giacomo

2016-01-01

DNA damage and transcription are two interconnected events. Transcription can induce damage and scheduled DNA damage can be required for transcription. Here, we analyzed genome-wide distribution of 8oxodG-marked oxidative DNA damage obtained by OxiDIP-Seq, and we found a correlation with transcription of protein coding genes.

DNA damage-induced inflammation and nuclear architecture.

Science.gov (United States)

Stratigi, Kalliopi; Chatzidoukaki, Ourania; Garinis, George A

2017-07-01

Nuclear architecture and the chromatin state affect most-if not all- DNA-dependent transactions, including the ability of cells to sense DNA lesions and restore damaged DNA back to its native form. Recent evidence points to functional links between DNA damage sensors, DNA repair mechanisms and the innate immune responses. The latter raises the question of how such seemingly disparate processes operate within the intrinsically complex nuclear landscape and the chromatin environment. Here, we discuss how DNA damage-induced immune responses operate within chromatin and the distinct sub-nuclear compartments highlighting their relevance to chronic inflammation. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Increased DNA damage in progression of COPD: a response by poly(ADP-ribose polymerase-1.

Directory of Open Access Journals (Sweden)

Ingrid Oit-Wiscombe

Full Text Available Chronic oxidative stress (OS, a major mechanism of chronic obstructive pulmonary disease (COPD, may cause significant damage to DNA. Poly(ADP-ribose polymerase (PARP-1 is rapidly activated by OS-induced DNA lesions. However, the degree of DNA damage along with the evolution of COPD is unclear. In peripheral blood mononuclear cells of non-smoking individuals, non-obstructive smokers, patients with COPD of all stages and those with COPD exacerbation, we evaluated DNA damage, PARP activity and PARP-1 mRNA expression using Comet Assay IV, biotinylated-NAD incorporation assay and qRT-PCR, respectively and subjected results to ordinal logistic regression modelling. Adjusted for demographics, smoking-related parameters and lung function, novel comet parameters, tail length/cell length ratio and tail migration/cell length ratio, showed the greatest increase along the study groups corresponding to the evolution of COPD [odds ratio (OR 7.88, 95% CI 4.26-14.57; p<0.001 and OR 3.91, 95% CI 2.69-5.66; p<0.001, respectively]. Analogously, PARP activity increased significantly over the groups (OR = 1.01; 95%; p<0.001. An antioxidant tetrapeptide UPF17 significantly reduced the PARP-1 mRNA expression in COPD, compared to that in non-obstructive individuals (p = 0.040. Tail length/cell length and tail migration/cell length ratios provide novel progression-sensitive tools for assessment of DNA damage. However, it remains to be elucidated whether inhibition of an elevated PARP-1 activity has a safe enough potential to break the vicious cycle of the development and progression of COPD.

DNA damage in lens epithelium of cataract patients in vivo and ex vivo.

Science.gov (United States)

Øsnes-Ringen, Oyvind; Azqueta, Amaia O; Moe, Morten C; Zetterström, Charlotta; Røger, Magnus; Nicolaissen, Bjørn; Collins, Andrew R

2013-11-01

DNA damage has been described in the human cataractous lens epithelium, and oxidative stress generated by UV radiation and endogenous metabolic processes has been suggested to play a significant role in the pathogenesis of cataract. In this study, the aim was to explore the quality and relative quantity of DNA damage in lens epithelium of cataract patients in vivo and after incubation in a cell culture system. Capsulotomy specimens were analysed, before and after 1 week of ex vivo cultivation, using the comet assay to measure DNA strand breaks, oxidized purine and pyrimidine bases and UV-induced cyclobutane pyrimidine dimers. DNA strand breaks were barely detectable, oxidized pyrimidines and pyrimidine dimers were present at low levels, whereas there was a relatively high level of oxidized purines, which further increased after cultivation. The observed levels of oxidized purines in cataractous lens epithelium may support a theory consistent with light damage and oxidative stress as mediators of molecular damage to the human lens epithelium. Damage commonly associated with UV-B irradiation was relatively low. The levels of oxidized purines increased further in a commonly used culture system. This is of interest considering the importance and versatility of ex vivo systems in studies exploring the pathogenesis of cataract. © 2012 The Authors. Acta Ophthalmologica © 2012 Acta Ophthalmologica Scandinavica Foundation.

Establishing a model for assessing DNA damage in murine brain cells as a molecular marker of chemotherapy-associated cognitive impairment.

Science.gov (United States)

Krynetskiy, Evgeny; Krynetskaia, Natalia; Rihawi, Diana; Wieczerzak, Katarzyna; Ciummo, Victoria; Walker, Ellen

2013-10-17

Chemotherapy-associated cognitive impairment often follows cancer chemotherapy. We explored chemotherapy-induced DNA damage in the brain cells of mice treated with 5-fluorouracil (5FU), an antineoplastic agent, to correlate the extent of DNA damage to behavioral functioning in an autoshaping-operant mouse model of chemotherapy-induced learning and memory deficits (Foley et al., 2008). Male, Swiss-Webster mice were injected once with saline or 75 mg/kg 5FU at 0, 12, and 24h and weighed every 24h. Twenty-four h after the last injection, the mice were tested in a two-day acquisition and the retention of a novel response task for food reinforcement. Murine brain cells were analyzed for the presence of single- and double-strand DNA breaks by the single cell gel electrophoresis assay (the Comet assay). We detected significant differences (p<0.0001) for all DNA damage characteristics (DNA "comet" tail shape, migration pattern, tail moment and olive moments) between control mice cohort and 5FU-treated mice cohort: tail length - 119 vs. 153; tail moment - 101 vs. 136; olive moment - 60 vs. 82, correspondingly. We found a positive correlation between increased response rates (r=0.52, p<0.05) and increased rate of errors (r=0.51, p<0.05), and DNA damage on day 1. For all 15 mice (saline-treated and 5FU-treated mice), we found negative correlations between DNA damage and weight (r=-0.75, p<0.02). Our results indicate that chemotherapy-induced DNA damage changes the physiological status of the brain cells and may provide insights to the mechanisms for cognitive impairment after cancer chemotherapy. Copyright © 2013 Elsevier Inc. All rights reserved.

Radiosensitivity evaluation of human tumor cell lines by detecting 4977 bp deletion in mitochondrial DNA and comet assay

International Nuclear Information System (INIS)

Chu Liping; Liu Qiang; Wang Qin; Li Jin; Yue Jingyin; Mu Chuanjie; Fan Feiyue

2008-01-01

Objective: To explore the feasibility of determining radiosensitivity of human tumor cell lines in vitro using the assay of mtDNA 4977 bp deletion and comet assay. Methods: Three human tumor cell lines were selected in this study, HepG 2 , EC-9706 and MCF-7. The surviving fraction(SF), the ratio of mtDNA 4977 bp deletion and DNA damage were detected by MTY assay, nested PCR technique and comet assay, respectively. Results: The results of MTT assay showed that the radiosensitivity of HepG 2 and EC-9706 was higher than that of MCF-7. The ratio of mtDNA 4977 bp deletion of HepG 2 and EC-9706 was higher significantly than that of MCF-7 (P 2 and EC-9706 was higher than that of MCF-7. The difference of radiosensitivity among these three tumor cell lines was significant after 8 Gy γ-ray irradiation. Conclusions: Combination of many biological parameter is helpful to evaluate the radiosensitivity of tumor cells more accurately. (authors)

Neocarzinostatin-mediated DNA damage and repair in wild-type and repair-deficient Chinese hamster ovary cells

International Nuclear Information System (INIS)

Kuo, W.L.; Meyn, R.E.; Haidle, C.W.

1984-01-01

The formation and repair of neocarzinostatin (NCS)-mediated DNA damage were examined in two strains of Chinese hamster ovary cells. The response in strain EM9, a mutant line selected for its sensitivity to ethyl methanesulfonate and shown to have a defect in the repair of X-ray-induced DNA breaks, was compared with that observed in the parental strain (AA8). The DNA strand breaks and their subsequent rejoining were measured using the method of elution of DNA from filters under either alkaline (for single-strand breaks), or nondenaturing conditions (for double-strand breaks). Colony survival assays showed that the mutant was more sensitive to the action of NCS than was the parental strain by a factor of approximately 1.5. Elution analyses showed that the DNA from both strains was damaged by NCS; the mutant displayed more damage than the parent under the same treatment conditions. Single-strand breaks were produced with a frequency of about 10 to 15 times the frequency of double-strand breaks. Both strains were able to rejoin both single-strand breaks and double-strand breaks induced by NCS treatment. The strand break data suggest that the difference in NCS-mediated cytotoxicity between EM9 and AA8 cells may be directly related to the enhanced production of DNA strand breaks in EM9. However, the fact that much higher doses of NCS were required in the DNA studies compared to the colony survival assays implies that either a small number of DNA breaks occur in a critical region of the genome, or that lesions other than DNA strand breaks are partly responsible for the observed cytotoxicity

Human embryonic stem cells have enhanced repair of multiple forms of DNA damage

DEFF Research Database (Denmark)

Maynard, Scott; Swistowska, Anna Maria; Lee, Jae Wan

2008-01-01

cells compared with various differentiated murine cells. Using single-cell gel electrophoresis (comet assay) we found that human embryonic stem cells (BG01, I6) have more efficient repair of different types of DNA damage (generated from H2O2, UV-C, ionizing radiation, or psoralen) than human primary...

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

Science.gov (United States)

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...

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

Science.gov (United States)

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

X-Ray induced DNA damage – why use plants?

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John William Einset

2015-06-01

Full Text Available The comet assay was used to monitor DNA repair after X-ray exposures caused by 0.2-15 Gy. A clear distinction in the time course of DNA repair after 2 Gy was observed with an early ‘rapid phase’, lasting 20-40 minutes, being followed by a ‘slow phase’ which actually consists of a period of negligible repair and then rapid repair during 140-160 minutes. The fact that homozygous mutants for both ATM and BRCA1 fail to repair DNA completely during 3 hours after 2 Gy exposures indicates that repair processes occurring during the ‘slow phase’ involve ds breaks in DNA. Both BRCA1 and Rad51 expression are strongly upregulated by X-rays in Arabidopsis. Rye grass, Norway spruce and Sawara cypress also have ‘slow phase’ repair similar to Arabidopsis, suggesting that the requisite enzymes have to be induced in these plants as well. To look at the effect of genome size in relation to sensitivity to DNA damage, we exposed isolated nuclei from Norway spruce (19.2 Gbp genome, celery (14.1 Gbp, spinach (12.6 Gbp Sawara cypress (8.9 Gbp, lettuce (2.6 Gbp and Arabidopsis (0.135 Gbp to X-rays. After a 1 Gy exposure, a linear relationship was seen between % tails and genome size, confirming the idea that larger genomes are more sensitive to X-ray damage.

DNA damage repair and radiosensitivity

International Nuclear Information System (INIS)

Suzuki, Norio

2003-01-01

Tailored treatment is not new in radiotherapy; it has been the major subject for the last 20-30 years. Radiation responses and RBE (relative biological effectiveness) depend on assay systems, endpoints, type of tissues and tumors, radiation quality, dose rate, dose fractionation, physiological and environmental factors etc, Latent times to develop damages also differ among tissues and endpoints depending on doses and radiation quality. Recent progress in clarification of radiation induced cell death, especially of apoptotic cell death, is quite important for understanding radiosensitivity of tumor cure process as well as of tumorigenesis. Apoptotic cell death as well as dormant cells had been unaccounted and missed into a part of reproductive cell death. Another area of major progress has been made in clarifying repair mechanisms of radiation damage, i.e., non-homologous end joining (NHEJ) and homologous recombinational repair (HRR). New approaches and developments such as cDNA or protein micro arrays and so called informatics in addition to basic molecular biological analysis are expected to aid identifying molecules and their roles in signal transduction pathways, which are multi-factorial and interactive each other being involved in radiation responses. (authors)

Influence of Heavy Metal Stress on Antioxidant Status and DNA Damage in Urtica dioica

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Darinka Gjorgieva

2013-01-01

Full Text Available Heavy metals have the potential to interact and induce several stress responses in the plants; thus, effects of heavy metal stress on DNA damages and total antioxidants level in Urtica dioica leaves and stems were investigated. The samples are sampled from areas with different metal exposition. Metal content was analyzed by Inductively Coupled Plasma-Atomic Emission Spectrometer (ICP-AES, for total antioxidants level assessment the Ferric-Reducing Antioxidant Power (FRAP assay was used, and genomic DNA isolation from frozen plant samples was performed to obtain DNA fingerprints of investigated plant. It was found that heavy metal contents in stems generally changed synchronously with those in leaves of the plant, and extraneous metals led to imbalance of mineral nutrient elements. DNA damages were investigated by Random Amplified Polymorphic DNA (RAPD technique, and the results demonstrated that the samples exposed to metals yielded a large number of new fragments (total 12 in comparison with the control sample. This study showed that DNA stability is highly affected by metal pollution which was identified by RAPD markers. Results suggested that heavy metal stress influences antioxidant status and also induces DNA damages in U. dioica which may help to understand the mechanisms of metals genotoxicity.

Influence of heavy metal stress on antioxidant status and DNA damage in Urtica dioica.

Science.gov (United States)

Gjorgieva, Darinka; Kadifkova Panovska, Tatjana; Ruskovska, Tatjana; Bačeva, Katerina; Stafilov, Trajče

2013-01-01

Heavy metals have the potential to interact and induce several stress responses in the plants; thus, effects of heavy metal stress on DNA damages and total antioxidants level in Urtica dioica leaves and stems were investigated. The samples are sampled from areas with different metal exposition. Metal content was analyzed by Inductively Coupled Plasma-Atomic Emission Spectrometer (ICP-AES), for total antioxidants level assessment the Ferric-Reducing Antioxidant Power (FRAP) assay was used, and genomic DNA isolation from frozen plant samples was performed to obtain DNA fingerprints of investigated plant. It was found that heavy metal contents in stems generally changed synchronously with those in leaves of the plant, and extraneous metals led to imbalance of mineral nutrient elements. DNA damages were investigated by Random Amplified Polymorphic DNA (RAPD) technique, and the results demonstrated that the samples exposed to metals yielded a large number of new fragments (total 12) in comparison with the control sample. This study showed that DNA stability is highly affected by metal pollution which was identified by RAPD markers. Results suggested that heavy metal stress influences antioxidant status and also induces DNA damages in U. dioica which may help to understand the mechanisms of metals genotoxicity.

Pretreatment with mixed-function oxidase inducers increases the sensitivity of the hepatocyte/DNA repair assay

International Nuclear Information System (INIS)

Shaddock, J.G.; Heflich, R.H.; McMillan, D.C.; Hinson, J.A.; Casciano, D.A.

1989-01-01

A recent National Toxicology Program evaluation indicates that the rat hepatocyte/DNA repair assay has a high false-negative rate and that it is insensitive to some genotoxic hepatocarcinogens as well as other species and organ-specific carcinogens. In this study, the authors examined whether the sensitivity of the hepatocyte/DNA repair assay might be increased through animal pretreatment with various hepatic mixed-function oxidase inducers, i.e., Aroclor 1254, phenobarbital, and 3,3',4,4'-tetrachloroazobenzene (TCAB). The effects on unscheduled DNA synthesis (UDS), a measured of DNA damage and repair, were studied in cultures exposed to known and/or potential carcinogens that had been evaluated as negative or questionable or that produced conflicting results with hepatocytes isolated from uninduced animals. 4,4'-Oxydianiline, 1-nitropy-rene, and TCAB produced concentration-dependent increases in UDS in hepatocytes from rats pretreated with Aroclor 1254. 4,4'-Oxydianiline and TCAB also induced a dose-dependent increase in DNA repair in hepatocytes from rats pretreated with phenobarbital, whereas 1-nitropyrene was negative. These data indicate that the limited sensitivity to chemical carcinogens displayed by the hepatocyte/DNA repair assay may be increased by using hepatocytes isolated from animals exposed to hepatic mixed-function oxidase inducers

Pretreatment with mixed-function oxidase inducers increases the sensitivity of the hepatocyte/DNA repair assay

Energy Technology Data Exchange (ETDEWEB)

Shaddock, J.G.; Heflich, R.H.; McMillan, D.C.; Hinson, J.A.; Casciano, D.A. (National Center for Toxicological Research, Jefferson, AK (USA) Univ. of Arkansas for Medical Sciences, Little Rock (USA))

1989-01-01

A recent National Toxicology Program evaluation indicates that the rat hepatocyte/DNA repair assay has a high false-negative rate and that it is insensitive to some genotoxic hepatocarcinogens as well as other species and organ-specific carcinogens. In this study, the authors examined whether the sensitivity of the hepatocyte/DNA repair assay might be increased through animal pretreatment with various hepatic mixed-function oxidase inducers, i.e., Aroclor 1254, phenobarbital, and 3,3{prime},4,4{prime}-tetrachloroazobenzene (TCAB). The effects on unscheduled DNA synthesis (UDS), a measured of DNA damage and repair, were studied in cultures exposed to known and/or potential carcinogens that had been evaluated as negative or questionable or that produced conflicting results with hepatocytes isolated from uninduced animals. 4,4{prime}-Oxydianiline, 1-nitropy-rene, and TCAB produced concentration-dependent increases in UDS in hepatocytes from rats pretreated with Aroclor 1254. 4,4{prime}-Oxydianiline and TCAB also induced a dose-dependent increase in DNA repair in hepatocytes from rats pretreated with phenobarbital, whereas 1-nitropyrene was negative. These data indicate that the limited sensitivity to chemical carcinogens displayed by the hepatocyte/DNA repair assay may be increased by using hepatocytes isolated from animals exposed to hepatic mixed-function oxidase inducers.

Direct Binding to Replication Protein A (RPA)-coated Single-stranded DNA Allows Recruitment of the ATR Activator TopBP1 to Sites of DNA Damage*

Science.gov (United States)

Acevedo, Julyana; Yan, Shan; Michael, W. Matthew

2016-01-01

A critical event for the ability of cells to tolerate DNA damage and replication stress is activation of the ATR kinase. ATR activation is dependent on the BRCT (BRCA1 C terminus) repeat-containing protein TopBP1. Previous work has shown that recruitment of TopBP1 to sites of DNA damage and stalled replication forks is necessary for downstream events in ATR activation; however, the mechanism for this recruitment was not known. Here, we use protein binding assays and functional studies in Xenopus egg extracts to show that TopBP1 makes a direct interaction, via its BRCT2 domain, with RPA-coated single-stranded DNA. We identify a point mutant that abrogates this interaction and show that this mutant fails to accumulate at sites of DNA damage and that the mutant cannot activate ATR. These data thus supply a mechanism for how the critical ATR activator, TopBP1, senses DNA damage and stalled replication forks to initiate assembly of checkpoint signaling complexes. PMID:27129245

Lead-induced DNA damage in Vicia faba root cells: Potential involvement of oxidative stress

OpenAIRE

Pourrut, Bertrand; Jean, Séverine; Silvestre, Jérôme; Pinelli, Eric

2011-01-01

Genotoxic effects of lead (0–20 µM) were investigated in whole-plant roots of Vicia faba L., grown hydroponically under controlled conditions. Lead-induced DNA damage in V. faba roots was evaluated by use of the comet assay, which allowed the detection of DNA strand-breakage and with the V. faba micronucleus test, which revealed chromosome aberrations. The results clearly indicate that lead induced DNA fragmentation in a dose-dependant manner with a maximum effect at 10 µM. In addition, at th...

Cellular Responses to Cisplatin-Induced DNA Damage

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Alakananda Basu

2010-01-01

Full Text Available Cisplatin is one of the most effective anticancer agents widely used in the treatment of solid tumors. It is generally considered as a cytotoxic drug which kills cancer cells by damaging DNA and inhibiting DNA synthesis. How cells respond to cisplatin-induced DNA damage plays a critical role in deciding cisplatin sensitivity. Cisplatin-induced DNA damage activates various signaling pathways to prevent or promote cell death. This paper summarizes our current understandings regarding the mechanisms by which cisplatin induces cell death and the bases of cisplatin resistance. We have discussed various steps, including the entry of cisplatin inside cells, DNA repair, drug detoxification, DNA damage response, and regulation of cisplatin-induced apoptosis by protein kinases. An understanding of how various signaling pathways regulate cisplatin-induced cell death should aid in the development of more effective therapeutic strategies for the treatment of cancer.

Role of DNA repair in repair of cytogenetic damages. Slowly repaired DNA injuries involved in cytogenetic damages repair

International Nuclear Information System (INIS)

Zaichkina, S.I.; Rozanova, O.M.; Aptikaev, G.F.; Ganassi, E.Eh.

1989-01-01

Caffeine was used to study the kinetics of cytogenetic damages repair in Chinese hamster fibroblasts. Its half-time (90 min) was shown to correlate with that of repair of slowly repaired DNA damages. The caffeine-induced increase in the number of irreparable DNA damages, attributed to inhibition of double-strand break repair, is in a quantitative correlation with the effect of the cytogenetic damage modification

Age and metabolic risk factors associated with oxidatively damaged DNA in human peripheral blood mononuclear cells

DEFF Research Database (Denmark)

Løhr, Mille; Jensen, Annie; Eriksen, Louise

2015-01-01

Aging is associated with oxidative stress-generated damage to DNA and this could be related to metabolic disturbances. This study investigated the association between levels of oxidatively damaged DNA in peripheral blood mononuclear cells (PBMCs) and metabolic risk factors in 1,019 subjects, aged...... 18-93 years. DNA damage was analyzed as strand breaks by the comet assay and levels of formamidopyrimidine (FPG-) and human 8-oxoguanine DNA glycosylase 1 (hOGG1)-sensitive sites There was an association between age and levels of FPG-sensitive sites for women, but not for men. The same tendency......, cholesterol and glycosylated hemoglobin (HbA1c). In the group of men, there were significant positive associations between alcohol intake, HbA1c and FPG-sensitive sites in multivariate analysis. The levels of metabolic risk factors were positively associated with age, yet only few subjects fulfilled all...

Impact of air pollution and genotype variability on DNA damage in Prague policemen

Czech Academy of Sciences Publication Activity Database

Novotná, Božena; Topinka, Jan; Solanský, I.; Chvátalová, Irena; Lněničková, Zdena; Šrám, Radim

2007-01-01

Roč. 172, - (2007), s. 37-47 ISSN 0378-4274 R&D Projects: GA MŽP SL/5/160/05 Institutional research plan: CEZ:AV0Z50390512 Keywords : oxidative DNA damage * comet assay * carcinogenic polycyclic aromatic hydrocarbons Subject RIV: DN - Health Impact of the Environment Quality Impact factor: 2.826, year: 2007

Role of DNA repair in repair of cytogenetic damages. Contribution of repair of single-strand DNA breaks to cytogenetic damages repair

International Nuclear Information System (INIS)

Rozanova, O.M.; Zaichkina, S.I.; Aptikaev, G.F.; Ganassi, E.Eh.

1989-01-01

The comparison was made between the results of the effect of poly(ADP-ribosylation) ingibitors (e.g. nicotinamide and 3-aminobenzamide) and a chromatin proteinase ingibitor, phenylmethylsulfonylfluoride, on the cytogenetic damages repair, by a micronuclear test, and DNA repair in Chinese hamster fibroblasts. The values of the repair half-periods (5-7 min for the cytogenetic damages and 5 min for the rapidly repaired DNA damages) and a similar modyfying effect with regard to radiation cytogenetic damages and kynetics of DNA damages repair were found to be close. This confirms the contribution of repair of DNA single-strand breaks in the initiation of structural damages to chromosomes

Optimization of a DNA Nicking Assay to Evaluate Oenocarpus bataua and Camellia sinensis Antioxidant Capacity

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Louis-Jérôme Leba

2014-10-01

Full Text Available This study was aimed at assessing the DNA damage protective activity of different types of extracts (aqueous, methanolic and acetonic using an in vitro DNA nicking assay. Several parameters were optimized using the pUC18 plasmid, especially FeSO4, EDTA, solvent concentrations and incubation time. Special attention has been paid to removing the protective and damaging effect of the solvent and FeSO4 respectively, as well as to identifying the relevant positive and negative controls. For each solvent, the optimal conditions were determined: (i for aqueous extracts, 0.33 mM of FeSO4 and 0.62 mM of EDTA were incubated for 20 min at 37 °C; (ii for acetone extracts, 1.16% solvent were incubated for 15 min at 37 °C with 1.3 mM of FeSO4 and 2.5 mM of EDTA and (iii for methanol extracts, 0.16% solvent, were incubated for 1.5 h at 37 °C with 0.33 mM of FeSO4 and 0.62 mM of EDTA. Using the optimized conditions, the DNA damage protective activity of aqueous, methanolic and acetonic extracts of an Amazonian palm berry (Oenocarpus bataua and green tea (Camellia sinensis was assessed. Aqueous and acetonic Oenocarpus bataua extracts were protective against DNA damage, whereas aqueous, methanolic and acetonic extracts of Camellia sinensis extracts induced DNA damage.

Transcription and DNA Damage: Holding Hands or Crossing Swords?

Science.gov (United States)

D'Alessandro, Giuseppina; d'Adda di Fagagna, Fabrizio

2017-10-27

Transcription has classically been considered a potential threat to genome integrity. Collision between transcription and DNA replication machinery, and retention of DNA:RNA hybrids, may result in genome instability. On the other hand, it has been proposed that active genes repair faster and preferentially via homologous recombination. Moreover, while canonical transcription is inhibited in the proximity of DNA double-strand breaks, a growing body of evidence supports active non-canonical transcription at DNA damage sites. Small non-coding RNAs accumulate at DNA double-strand break sites in mammals and other organisms, and are involved in DNA damage signaling and repair. Furthermore, RNA binding proteins are recruited to DNA damage sites and participate in the DNA damage response. Here, we discuss the impact of transcription on genome stability, the role of RNA binding proteins at DNA damage sites, and the function of small non-coding RNAs generated upon damage in the signaling and repair of DNA lesions. Copyright © 2016 Elsevier Ltd. All rights reserved.

Grape (Vitis vinifera) extracts protect against radiation-induced oxidative stress and DNA damage

International Nuclear Information System (INIS)

Singha, Indrani; Das, Subir Kumar; Saxena, S.; Gautam, S.

2016-01-01

Ionizing radiation (IR) causes oxidative stress through the overwhelming generation of reactive oxygen species (ROS) in the living cells leading further to the oxidative damage to biomolecules. Grapes (Vitis vinifera) contain several bioactive phytochemicals and are the richest source of antioxidant. In this study, we investigated and compared in vitro antioxidant activity and DNA damage protective property of the grape extracts of four different cultivars, including the Thompson seedless, Flame seedless, Kishmish chorni and Red globe. The activities of ascorbic acid oxidase and catalase significantly (p<0.01) differed among extracts within the same cultivar, while that of peroxidase and polyphenol oxidase did not differ significantly among extracts of any cultivar. In vitro antioxidant activities were assessed by ferric-reducing antioxidant power (FRAP) assay and ABTS. The superoxide radical-scavenging activity was higher in the seed as compared to the skin or pulp of the same cultivar. DNA damage was evaluated in acellular system using pBR322 plasmid relaxation. Grape extract was able to effectively scavenge free radicals in vitro. It could significantly prevent radiation-induced DNA damage. Furthermore, the protective action of grape depends on the source of extract and type of the cultivars. (author)

Synthesis of water soluble CdS nanoparticles and study of their DNA damage activity

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Kumar Suranjit Prasad

2017-05-01

Full Text Available This study reports a novel method for preparation of water soluble CdS nanoparticles using leaf extract of a plant, Asparagus racemosus. The extract of the leaf tissue which worked as a stabilizing and capping agent, assisted the formation of nanoparticles. Nanoparticles were characterized using a UV–vis spectrophotometer, Photoluminescence, TEM, EDAX, XRD and FT-IR. Transmission electron microscopy followed by selected area electron diffraction pattern analysis indicated the formation of spherical, polydispersed, crystalline, CdS of diameter ranging from 2 to 8 nm. X-ray diffraction studies showed the formation of 111, 220 and 311 planes of face-centered cubic (fcc CdS. EDAX analysis confirmed the presence of Cd and S in nanosphere. The cytotoxicity test using MTT assay as well as DNA damage analysis using comet assay revealed that synthesized nano CdS quantum dots (QDs caused less DNA damage and cell death of lymphocytes than pure CdS nanoparticles.

Mechanisms for radiation damage in DNA

International Nuclear Information System (INIS)

Sevilla, M.D.

1985-07-01

Radiation damage to DNA results from the direct interaction of radiation with DNA where positive ions, electrons and excited states are formed in the DNA, and the indirect effect where radical species formed in the surrounding medium by the radiation attack the DNA. The primary mechanism proposed for radiation damage, by the direct effect, is that positive and negative ions formed within the DNA strand migrate through the stacked DNA bases. The ions can then recombine, react with the DNA bases most likely to react by protonation of the anion and deprotonation or hydroxylation of the cation or transfer out of the DNA chain to the surrounding histone protein. This work as aimed at understanding the possible reactions of the DNA base ion radicals, as well as their initial distribution in the DNA strand. 31 refs

Modulation of radiation induced DNA damage by natural products in hemopoietic tissue of mice

International Nuclear Information System (INIS)

Jayakumar, S.; Bhilwade, H.N.; Chaubey, R.C.

2014-01-01

Ionizing radiation is known to induce oxidative stress through generation of ROS leading to a variety of DNA lesions. However, the most dangerous DNA lesions which are responsible for the origin of lethal effects, mutagenesis, genomic instability and carcinogenesis are the DSBs. During recent years efforts are being made to identify phytochemicals, antioxidants or neutraxeuticals which can reduce harmful effect of radiation during accidental exposure or prevent normal tissue injury during radiotherapy. In the present study, we have investigated the radioprotective role of curcumin, a dietary antioxidant, taurine, malabaricone-C, and umbelliferone, for their radioprotective properties in hemopoietic cells of mice. Groups of mice-were fed 1% of curcumin in diet for three weeks. Similarly other groups of mice were injected i.p. with 50 mg/kg body weight of taurine for five consecutive days. After the completion of the treatment mice pre-treated with curcumin and taurine were exposed to 3 Gy of gamma rays. Malabaricone-C was tested for its radiomodulation potential in vitro, in spleenocytes of mouse. Spleenocytes were isolated and treated with different concentrations (0.5-25 ìM) of malabaricone-C. Immediately after irradiation, alkaline comet assay were performed using standard procedures. Twenty four post radiation exposure mice were sacrificed for micronucleus test. Results of these studies showed significant reduction in DNA damage by curcumin. The micronucleus data showed marginal increase in the frequency of micronucleated erythrocytes in curcumin fed group as compared to the controls. Mice receiving curcumin for 3 weeks in diet followed by gamma radiation (3 Gy), showed approximately 50% reduction in the frequency of micro nucleated polychromatic erythrocytes. Pre-treatment of mice with taurine significantly (p < 0.01) reduced the frequency of gamma rays induced mn-PCEs in bone marrow tissue. Malabaricone-C at 1.5 ìM concentration showed very good protection

Seasonal variation of DNA damage and repair in patients with non-melanoma skin cancer and referents with and without psoriasis

DEFF Research Database (Denmark)

Møller, P; Knudsen, Lisbeth E.; Frentz, G

1998-01-01

Quadruples of skin cancer patients with and without psoriasis and referents with and without psoriasis (4 x 20 study persons) were identified and examined for DNA damage by single cell gel electrophoresis (comet-assay) and DNA-repair by UV-induced unscheduled DNA synthesis (UDS) in mononuclear...... to solar radiation. When the comet tail moment data were stratified by sampling period, an interaction between psoriasis and skin cancer was detected, with patients with psoriasis and skin cancer exhibiting more DNA damage. Patients with psoriasis and skin cancer also had lower UDS compared to healthy...

Is the Oxidative DNA Damage Level of Human Lymphocyte Correlated with the Antioxidant Capacity of Serum or the Base Excision Repair Activity of Lymphocyte?

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Yi-Chih Tsai

2013-01-01

Full Text Available A random screening of human blood samples from 24 individuals of nonsmoker was conducted to examine the correlation between the oxidative DNA damage level of lymphocytes and the antioxidant capacity of serum or the base excision repair (BER activity of lymphocytes. The oxidative DNA damage level was measured with comet assay containing Fpg/Endo III cleavage, and the BER activity was estimated with a modified comet assay including nuclear extract of lymphocytes for enzymatic cleavage. Antioxidant capacity was determined with trolox equivalent antioxidant capacity assay. We found that though the endogenous DNA oxidation levels varied among the individuals, each individual level appeared to be steady for at least 1 month. Our results indicate that the oxidative DNA damage level is insignificantly or weakly correlated with antioxidant capacity or BER activity, respectively. However, lymphocytes from carriers of Helicobacter pylori (HP or Hepatitis B virus (HBV tend to give higher levels of oxidative DNA damage (P<0.05. Though sera of this group of individuals show no particular tendency with reduced antioxidant capacity, the respective BER activities of lymphocytes are lower in average (P<0.05. Thus, reduction of repair activity may be associated with the genotoxic effect of HP or HBV infection.