scholarly journals Cell survival after DNA damage in the comet assay

2021 ◽  
Author(s):  
Ezgi Eyluel Bankoglu ◽  
Carolin Schuele ◽  
Helga Stopper
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Comet Assay ◽  
Survival Data ◽  
Topoisomerase Ii ◽  
Basic Research ◽  
Human Biomonitoring ◽  
A Cell ◽  
Tk6 Cells ◽  
Genotoxicity Testing

AbstractThe comet assay is widely used in basic research, genotoxicity testing, and human biomonitoring. However, interpretation of the comet assay data might benefit from a better understanding of the future fate of a cell with DNA damage. DNA damage is in principle repairable, or if extensive, can lead to cell death. Here, we have correlated the maximally induced DNA damage with three test substances in TK6 cells with the survival of the cells. For this, we selected hydrogen peroxide (H2O2) as an oxidizing agent, methyl methanesulfonate (MMS) as an alkylating agent and etoposide as a topoisomerase II inhibitor. We measured cell viability, cell proliferation, apoptosis, and micronucleus frequency on the following day, in the same cell culture, which had been analyzed in the comet assay. After treatment, a concentration dependent increase in DNA damage and in the percentage of non-vital and apoptotic cells was found for each substance. Values greater than 20–30% DNA in tail caused the death of more than 50% of the cells, with etoposide causing slightly more cell death than H2O2 or MMS. Despite that, cells seemed to repair of at least some DNA damage within few hours after substance removal. Overall, the reduction of DNA damage over time is due to both DNA repair and death of heavily damaged cells. We recommend that in experiments with induction of DNA damage of more than 20% DNA in tail, survival data for the cells are provided.

scholarly journals DNA damage and cell death assessment in patients with severe multiple trauma using comet assay

Health ◽  
2010 ◽  
Vol 02 (05) ◽  
pp. 412-417 ◽  
Author(s):  
Aliy K. Zhanataev ◽  
Victor V. Moroz ◽  
Andrey D. Durnev ◽  
Maria Yu. Muravyeva ◽  
Vasiliy I. Reshetnyak
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Comet Assay ◽  
Multiple Trauma ◽  
Severe Multiple Trauma

scholarly journals EVALUATION OF CYTOTOXIC AND GENOTOXIC EFFECTS OF ZERUMBONE ON COLON ADENOCARCINOMA COLO205 CELLS AND HUMAN LYMPHOCYTES

2017 ◽  
Vol 9 (10) ◽  
pp. 92
Author(s):  
Rima Thiyam ◽  
Mangamoori Lakshmi Narasu
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Comet Assay ◽  
Apoptotic Cell ◽  
Morphological Changes ◽  
Human Lymphocytes ◽  
Genotoxic Effect ◽  
Colorectal Cancer Cell Line ◽  
Dependent Manner ◽  
Ic50 Values

Objective: The objective of the present study was to investigate the growth inhibitory effect, apoptosis initiation and genotoxic effect of zerumbone (ZER), a phytochemical and cisplatin, a chemotherapeutic drug on human colorectal cancer cell line COLO205 and normal human lymphocytes.Methods: The antiproliferative activity of ZER and cisplatin (positive control) on COLO205 cells and lymphocytes was analysed by 3( 4, 5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide (MTT) assay. Morphological analysis of the cells was studied by using inverted phase contrast microscope. Propidium iodide staining method was used to observe the apoptotic morphological changes in the treated cells. Finally comet assay was conducted to observe the extent of DNA damage induced by ZER and cisplatin on COLO205 and lymphocytes.Results: ZER and cisplatin exhibited growth inhibition in a dose and time dependent manner against COLO205 with no considerable effect on lymphocytes. The IC50 values of ZER on COLO205 for 24h, 48h and 72h were 19 µg/ml, 10 µg/ml and 5 µg/ml. Comparatively the IC50 values of cisplatin on COLO205 for 24h, 48h and 72h were 38 µg/ml, 24 µg/ml and 15 µg/ml.  Morphological changes such as cell shrinkage, membrane blebbing and nuclear condensation were observed in COLO205 while no significant change was observed in lymphocytes. Fluorescence imaging studies confirmed apoptotic cell death in treated COLO205 cells while no significant cell death was observed in treated lymphocytes. Comet assay revealed significant DNA damage in treated COLO205 cells.Conclusion: The present study demonstrated the cytotoxic and genotoxic effect of ZER and cisplatin on COLO205 cells. These drugs showed no significant effect on lymphocytes.

scholarly journals SIRT1 Promotes Cell Survival under Stress by Deacetylation-Dependent Deactivation of Poly(ADP-Ribose) Polymerase 1

2009 ◽  
Vol 29 (15) ◽  
pp. 4116-4129 ◽  
Author(s):  
Senthilkumar B. Rajamohan ◽  
Vinodkumar B. Pillai ◽  
Madhu Gupta ◽  
Nagalingam R. Sundaresan ◽  
Konstantin G. Birukov ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Cell Survival ◽  
Cross Talk ◽  
Gene Promoter ◽  
Stress Conditions ◽  
Transcriptional Level ◽  
A Cell

ABSTRACT Poly(ADP-ribose) polymerase 1 (PARP1) and SIRT1 deacetylase are two NAD-dependent enzymes which play major roles in the decision of a cell to live or to die in a stress situation. Because of the dependence of both enzymes on NAD, cross talk between them has been suggested. Here, we show that PARP1 is acetylated after stress of cardiomyocytes, resulting in the activation of PARP1, which is independent of DNA damage. SIRT1 physically binds to and deacetylates PARP1. Increased acetylation of PARP1 was also detected in hearts of SIRT1−/− mice, compared to that detected in the hearts of SIRT1+/+ mice, confirming a role of SIRT1 in regulating the PARP1 acetylation in vivo. SIRT1-dependent deacetylation blocks PARP1 activity, and it protects cells from PARP1-mediated cell death. We also show that SIRT1 negatively regulates the activity of the PARP1 gene promoter, thus suggesting that the deacetylase controls the PARP1 activity at the transcriptional level as well. These data demonstrate that the activity of PARP1 is under the control of SIRT1, which is necessary for survival of cells under stress conditions.

Comet assay analysis of DNA damage in T- and B-lymphocytes separated by MACS for human biomonitoring studies

2012 ◽  
Vol 26 (2) ◽  
pp. 369-372 ◽  
Author(s):  
So-Young Park ◽  
Eunkyung Cho ◽  
Eunha Oh ◽  
Donggeun Sul
Keyword(s):  
Dna Damage ◽  
Comet Assay ◽  
B Lymphocytes ◽  
Human Biomonitoring ◽  
T And B Lymphocytes

scholarly journals Effect of cryopreservation on DNA damage and DNA repair activity in human blood samples in the comet assay

2021 ◽  
Author(s):  
Ezgi Eyluel Bankoglu ◽  
Franzisca Stipp ◽  
Johanna Gerber ◽  
Florian Seyfried ◽  
August Heidland ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Dna Damage ◽  
Comet Assay ◽  
Human Blood ◽  
Ex Vivo ◽  
Human Biomonitoring ◽  
Dna Damage And Repair ◽  
Blood Samples ◽  
Repair Activity ◽  
Human Blood Samples

AbstractThe comet assay is a commonly used method to determine DNA damage and repair activity in many types of samples. In recent years, the use of the comet assay in human biomonitoring became highly attractive due to its various modified versions, which may be useful to determine individual susceptibility in blood samples. However, in human biomonitoring studies, working with large sample numbers that are acquired over an extended time period requires some additional considerations. One of the most important issues is the storage of samples and its effect on the outcome of the comet assay. Another important question is the suitability of different blood preparations. In this study, we analysed the effect of cryopreservation on DNA damage and repair activity in human blood samples. In addition, we investigated the suitability of different blood preparations. The alkaline and FPG as well as two different types of repair comet assay and an in vitro hydrogen peroxide challenge were applied. Our results confirmed that cryopreserved blood preparations are suitable for investigating DNA damage in the alkaline and FPG comet assay in whole blood, buffy coat and PBMCs. Ex vivo hydrogen peroxide challenge yielded its optimal effect in isolated PBMCs. The utilised repair comet assay with either UVC or hydrogen peroxide-induced lesions and an aphidicolin block worked well in fresh PBMCs. Cryopreserved PBMCs could not be used immediately after thawing. However, a 16-h recovery with or without mitotic stimulation enabled the application of the repair comet assay, albeit only in a surviving cell fraction.

scholarly journals New Application of the Comet Assay

2011 ◽  
Vol 59 (7) ◽  
pp. 655-660 ◽  
Author(s):  
Elva I. Cortés-Gutiérrez ◽  
Martha I. Dávila-Rodríguez ◽  
José Luís Fernández ◽  
Carmen López-Fernández ◽  
Altea Gosálbez ◽  
...  
Keyword(s):  
Dna Damage ◽  
Comet Assay ◽  
Single Cells ◽  
White Blood Cells ◽  
Human Biomonitoring ◽  
Cell Types ◽  
Dna Damage And Repair ◽  
Dna Migration ◽  
Sensitive Tool ◽  
Chromosome Isolation

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.

scholarly journals The Comet Assay to Determine the Mode of Cell Death for the Ultrasonic Delivery of Doxorubicin to Human Leukemia (HL-60 Cells) from Pluronic P105 Micelles

2005 ◽  
Vol 4 (6) ◽  
pp. 707-711 ◽  
Author(s):  
Ghaleb A. Husseini ◽  
Kim L. O'Neill ◽  
William G. Pitt
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Comet Assay ◽  
Propidium Iodide ◽  
Cell Killing ◽  
Human Leukemia ◽  
Dna Profile ◽  
Fluorescent Microscope

This notes examines the mode of cell death of HL-60 cells exposed to 70 kHz and 1.3 W/cm2 ultrasound in the presence of 1% Pluronic P105 and 1.67 μg/ml doxorubicin (Dox). The cells were ultrasonicated for 30, 60, and 120 minutes. They were then lysed, electrophorised, stained using propidium iodide, and their DNA profile captured using a fluorescent microscope. The gradual DNA damage observed and the comet tails captured after one and two hours of insonation suggest that the mode of cell killing is apoptosis.

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