scholarly journals Measurement of oxidatively damaged DNA in mammalian cells using the comet assay: reflections on validity, reliability and variability

2021 ◽  
pp. 503423
Author(s):  
Peter Møller
Keyword(s):  
Comet Assay ◽  
Mammalian Cells ◽  
Damaged Dna

Calibration of the comet assay for the measurement of DNA damage in mammalian cells

2006 ◽  
Vol 40 (11) ◽  
pp. 1149-1154 ◽  
Author(s):  
Vanessa Pitozzi ◽  
Stefania Pallotta ◽  
Manuela Balzi ◽  
Marta Bucciolini ◽  
Aldo Becciolini ◽  
...  
Keyword(s):  
Dna Damage ◽  
Comet Assay ◽  
Mammalian Cells

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

2016 ◽  
Vol 57 (3) ◽  
pp. 312-317 ◽  
Author(s):  
Tomomi Kurashige ◽  
Mika Shimamura ◽  
Yuji Nagayama
Keyword(s):  
Comet Assay ◽  
Biological Effect ◽  
Genomic Dna ◽  
Mammalian Cells ◽  
Micronucleus Assay ◽  
Strand Break ◽  
Dna Double Strand Breaks ◽  
Ros Scavenging ◽  
Focus Formation ◽  
Multiple Parameters

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

scholarly journals Using a Human Papillomavirus Model to Study DNA Replication and Repair of Wild Type and Damaged DNA Templates in Mammalian Cells

2020 ◽  
Vol 21 (20) ◽  
pp. 7564
Author(s):  
Dipon Das ◽  
Molly L. Bristol ◽  
Pietro Pichierri ◽  
Iain M. Morgan
Keyword(s):  
Dna Replication ◽  
Viral Genome ◽  
Mammalian Cells ◽  
Human Papillomaviruses ◽  
Lacz Gene ◽  
Dna Templates ◽  
Protein Protein Interaction ◽  
Dna Replication And Repair ◽  
Cellular Components ◽  
Damaged Dna

Human papillomaviruses have 8kbp DNA episomal genomes that replicate autonomously from host DNA. During initial infection, the virus increases its copy number to 20–50 copies per cell, causing torsional stress on the replicating DNA. This activates the DNA damage response (DDR) and HPV replicates its genome, at least in part, using homologous recombination. An active DDR is on throughout the HPV life cycle. Two viral proteins are required for replication of the viral genome; E2 binds to 12bp palindromic sequences around the A/T rich origin of replication and recruits the viral helicase E1 via a protein–protein interaction. E1 forms a di-hexameric complex that replicates the viral genome in association with host factors. Transient replication assays following transfection with E1–E2 expression plasmids, along with an origin containing plasmid, allow monitoring of E1-E2 replication activity. Incorporating a bacterial lacZ gene into the origin plasmid allows for the determination of replication fidelity. Here we describe how we exploited this system to investigate replication and repair in mammalian cells, including using damaged DNA templates. We propose that this system has the potential to enhance the understanding of cellular components involved in DNA replication and repair.

scholarly journals Is the Comet Assay a Sensitive Procedure for Detecting Genotoxicity?

2010 ◽  
Vol 2010 ◽  
pp. 1-8 ◽  
Author(s):  
Satomi Kawaguchi ◽  
Takanori Nakamura ◽  
Ayumi Yamamoto ◽  
Gisho Honda ◽  
Yu F. Sasaki
Keyword(s):  
Comet Assay ◽  
Mammalian Cells ◽  
Positive Response ◽  
Micronucleus Test ◽  
Strand Breaks ◽  
Low Level ◽  
Genotoxic Potential ◽  
Dna Migration ◽  
Single Strand Breaks ◽  
Methyl Nitrosourea

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

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

2006 ◽  
Vol 17 (3) ◽  
pp. 228-232 ◽  
Author(s):  
Daniel Araki Ribeiro ◽  
Mariângela Esther Alencar Marques ◽  
Daisy Maria Fávero Salvador
Keyword(s):  
Comet Assay ◽  
Single Cell ◽  
Mammalian Cells ◽  
Lymphoma Cells ◽  
Trypan Blue Exclusion ◽  
Trypan Blue Exclusion Test ◽  
Gutta Percha ◽  
Mouse Lymphoma Cells ◽  
Mouse Lymphoma

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

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