scholarly journals Comet assay: a versatile but complex tool in genotoxicity testing

2021 ◽  
Author(s):  
Eugenia Cordelli ◽  
Margherita Bignami ◽  
Francesca Pacchierotti
Keyword(s):  
Dna Damage ◽  
Comet Assay ◽  
Full Potential ◽  
Food Contaminants ◽  
Exposure Limits ◽  
Mechanisms Of Toxicity ◽  
Interstrand Crosslinks ◽  
Dna Strand ◽  
Protocol Standardization

Abstract The comet assay is a versatile method for measuring DNA strand breaks in individual cells. It can also be applied to cells isolated from treated animals. In this review, we highlight advantages and limitations of this in vivo comet assay in a regulatory context. Modified versions of the standard protocol detect oxidized DNA bases and may be used to reveal sites of DNA base loss, DNA interstrand crosslinks, and the extent of DNA damage induced indirectly by reactive oxygen species elicited by chemical-induced oxidative stress. The assay is, however, at best semi-quantitative, and we discuss possible approaches to improving DNA damage quantitation and highlight the necessity of optimizing protocol standardization to enhance the comparability of results between laboratories. As a genotoxicity test in vivo, the in vivo comet assay has the advantage over the better established micronucleus erythrocyte test that it can be applied to any organ, including those that are specific targets of chemical carcinogens or those that are the first sites of contact of ingested or inhaled mutagens. We illustrate this by examples of its use in risk assessment for the food contaminants ochratoxin and furan. We suggest that improved quantitation is required to reveal the full potential of the comet assay and enhance its role in the battery of in vivo approaches to characterize the mechanisms of toxicity and carcinogenicity of chemicals and to aid the determination of safe human exposure limits.

Chromium(VI) Toxicity: Uptake, Reduction, and DNA Damage

1989 ◽  
Vol 8 (7) ◽  
pp. 1275-1283 ◽  
Author(s):  
Andrew M. Standeven ◽  
Karen E. Wetterhahn
Keyword(s):  
Dna Damage ◽  
Anion Channel ◽  
Functional Changes ◽  
Interstrand Crosslinks ◽  
Chromium Vi ◽  
Chain Complexes ◽  
Dna Strand ◽  
Dt Diaphorase

Much recent data supports the “uptake-reduction” model explaining the carcinogenicity of chromium(VI) compounds and the lack of carcinogenicity of chromium(III) com pounds. Cr(VI) readily enters cells by diffusion through a nonspecific anion channel, whereas cells are relatively impermeable to Cr(III). Glutathione appears to facilitate Cr(VI) uptake by reducing Cr(VI) to Cr(III) after it enters the cell, presumably keeping intracellular Cr(VI) concentration low and allowing for further Cr(VI) uptake. Some other nonenzymatic factors, for example, ascorbate and riboflavin, as well as enzymes, such as cytochrome P-450, DT-diaphorase, and the mitochondrial electron transport chain complexes, are capable of reducing Cr(VI) in vitro, but their contribution in vivo is not clear. Cr(VI), once reduced intracellularly, produces various forms of DNA damage including DNA interstrand crosslinks, DNA-protein crosslinks, DNA strand breaks, and Cr-DNA adducts. The pathway of Cr(VI) metabolism in different tissues appears to influence the type of “reactive intermediates” produced, for example, Cr(V) and radical species, and thus the nature and extent of DNA damage. This DNA damage presumably accounts for observed functional changes in DNA replication and transcription which may be crucial to the carcinogenicity of chromium(VI) compounds.

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

2010 ◽  
Vol 29 (9) ◽  
pp. 721-729 ◽  
Author(s):  
B. Marczynski ◽  
M. Raulf-Heimsoth ◽  
B. Pesch ◽  
B. Kendzia ◽  
HU Käfferlein ◽  
...  
Keyword(s):  
Dna Damage ◽  
Comet Assay ◽  
Rank Correlation ◽  
Dna Strand Breaks ◽  
Strand Breaks ◽  
Blood Lymphocytes ◽  
Exposed Workers ◽  
Before And After ◽  
Dna Strand ◽  
Post Shift

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

In vivo exposure to microcystins induces DNA damage in the haemocytes of the zebra mussel,Dreissena polymorpha, as measured with the comet assay

2007 ◽  
Vol 48 (1) ◽  
pp. 22-29 ◽  
Author(s):  
Guillaume Juhel ◽  
John O'Halloran ◽  
Sarah C. Culloty ◽  
Ruth M. O'Riordan ◽  
John Davenport ◽  
...  
Keyword(s):  
Dna Damage ◽  
Comet Assay ◽  
Zebra Mussel ◽  
Dreissena Polymorpha

scholarly journals Comet assay measures of DNA damage are predictive of bladder cancer cell treatment sensitivity in vitro and outcome in vivo

2013 ◽  
Vol 134 (5) ◽  
pp. 1102-1111 ◽  
Author(s):  
Karen J. Bowman ◽  
Manar M. Al‐Moneef ◽  
Benedict T. Sherwood ◽  
Alexandra J. Colquhoun ◽  
Jonathan C. Goddard ◽  
...  
Keyword(s):  
Dna Damage ◽  
Bladder Cancer ◽  
Comet Assay ◽  
Cancer Cell ◽  
Bladder Cancer Cell ◽  
Treatment Sensitivity

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

2013 ◽  
Vol 33 (5) ◽  
pp. 488-495
Author(s):  
K Narumi ◽  
H Takasawa ◽  
W Ohyama ◽  
K Kaneko
Keyword(s):  
Dna Damage ◽  
Comet Assay ◽  
Peripheral Blood ◽  
Clinical Signs ◽  
Enzyme Reaction ◽  
Glandular Stomach ◽  
Alternative Methods ◽  
Sprague Dawley ◽  
Genotoxic Carcinogens

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

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

2014 ◽  
Vol 155 (47) ◽  
pp. 1872-1875 ◽  
Author(s):  
János Megyesi ◽  
Anna Biró ◽  
László Wigmond ◽  
Jenő Major ◽  
Anna Tompa
Keyword(s):  
Dna Damage ◽  
Occupational Exposure ◽  
Comet Assay ◽  
Oxidative Dna Damage ◽  
Microscopic Method ◽  
Monitoring Method ◽  
Strand Breaks ◽  
Cell Counts ◽  
Polycyclic Aromatic ◽  
Dna Strand

Introduction: The comet assay is a fluorescent microscopic method that is able to detect DNA strand-breaks even in non-proliferative cells in samples with low cell counts. Aim: The aim of the authors was to measure genotoxic DNA damage and assess oxidative DNA damage caused by occupational exposure in groups exposed to benzene, polycyclic aromatic carbohydrates and styrene at the workplace in order to clarify whether the comet assay can be used as an effect marker tool in genotoxicology monitoring. Method: In addition to the basic steps of the comet assay, one sample was treated with formamido-pirimidine-DNA-glycolase restriction-enzyme that measures oxidative DNA damage. Results: An increase was observed in tail moments in each group of untreated and Fpg-treated samples compared to the control. Conclusions: It can be concluded that occupational exposure can be detected with the method. The comet assay may prove to be an excellent effect marker and a supplementary technique for monitoring the presence or absence of genotoxic effects. Orv. Hetil., 2014, 155(47), 1872–1875.

scholarly journals Effects of photopolymerisation on genotoxicity of composite adhesives in the comet assay

Genetika ◽  
2016 ◽  
Vol 48 (2) ◽  
pp. 617-627
Author(s):  
Stefan Dacic ◽  
Ninoslav Djelic ◽  
Milena Radakovic ◽  
Nada Lakic ◽  
Aleksandar Veselinovic ◽  
...  
Keyword(s):  
Dna Damage ◽  
Comet Assay ◽  
Human Lymphocytes ◽  
Negative Control ◽  
In Vivo Studies ◽  
Halogen Lamp ◽  
Genotoxic Effects ◽  
Significant Difference ◽  
Isolated Human Lymphocytes

Certain in vivo studies have shown that the application of adhesives directly onto the open pulp or on a thin layer of dentin causes inflammation and pulpal abscesses. This reaction is related to toxic effects of monomers from adhesives. It has been confirmed that after proper illumination the adhesives become less toxic. The aim of the study was to examine genotoxicity of non-polymerised, partly polymerised and polymerised adhesives on isolated human lymphocytes using the alkaline Comet assay. Adper Single bond2 and Adper Easy One/3M ESPE adhesive photopolymerisation was performed by Elipar Highlight 3M ESPE halogen lamp for 0, 10 and 40 sec, at final concentrations of 100, 200, 500 and 1000 ?g/mL. With both adhesives, photopolymerisation at 0 and 10 seconds showed statistically significant increase in DNA damage in comparision to the negative control (solvent). On the other hand, after 40 seconds of photopolymerisation of both adhesives in all tested concentrations, the degree of DNA damage in Comet assay had no significant difference (P>0.05, ?2 test) compared to the negative control. Therefore, only the 40 seconds of photopolymerisation prevented genotoxic effects of both adhesives in the Comet assay.

scholarly journals DNA damage in human whole blood caused by radiopharmaceuticals evaluated by the comet assay

Mutagenesis ◽  
2019 ◽  
Vol 34 (3) ◽  
pp. 239-244 ◽  
Author(s):  
Heinz H Schmeiser ◽  
Karl-Rudolf Muehlbauer ◽  
Walter Mier ◽  
Ann-Christin Baranski ◽  
Oliver Neels ◽  
...  
Keyword(s):  
Dna Damage ◽  
Comet Assay ◽  
Dna Strand Breaks ◽  
Strand Breaks ◽  
Human Whole Blood ◽  
Time Points ◽  
Dna Migration ◽  
Activity Concentrations ◽  
Positron Emitters ◽  
Dna Strand

Abstract Radiopharmaceuticals used for diagnosis or therapy induce DNA strand breaks, which may be detectable by single-cell gel electrophoresis (called comet assay). Blood was taken from patients before and at different time points after treatment with radiopharmaceuticals; blood cells were investigated by the comet assay using the percentage of DNA in the tail as the critical parameter. Whereas [225Ac]Ac-prostate-specific membrane antigen (PSMA)-617 alpha therapy showed no difference relative to the blood sample taken before treatment, beta therapy with [177Lu]Lu-PSMA-617 3 h post-injection revealed a small but significant increase in DNA strand breaks. In blood of patients who underwent positron emission tomography (PET) with either [18F]2-fluor-2-deoxy-D-glucose (FDG) or [68Ga]Ga-PSMA-11, an increase of DNA migration determined by the comet assay was not found when analysed at different time points (2–70 min) after intravenous tracer injection. Human whole blood was incubated with the targeted clinically relevant therapeutic radiopharmaceuticals [225Ac]Ac-PSMA-617, [177Lu]Lu-PSMA-617 and [90Y]Y-DOTA(0)-Phe(1)-Tyr(3)-octreotide (DOTA-TOC) at different activity concentrations (kBq/ml) for 5 days and then analysed by the comet assay. DNA damage increased with higher concentrations of all radiolabeled compounds tested. [177Lu]Lu-PSMA-617 caused higher blood cell radiotoxicity than equal activity concentrations of [90Y]Y-DOTA-TOC. Likewise, whole human blood was exposed to the positron emitters [18F]FDG and [68Ga]Ga-PSMA-11 in vitro for 24 h with activity concentrations ranging between 5 and 40 MBq/ml. The same activity concentration dependent elevated DNA migration was observed for both compounds although decay energies are different. This study demonstrated that the amount of DNA damage detected by the comet assay in whole human blood is similar among different positron emitters and divergent by a factor of 200 between alpha particles and beta radiation.

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