The comet assay for human biomonitoring: Effect of cryopreservation on DNA damage in different blood cell preparations

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.

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Cell survival after DNA damage in the comet assay

Archives of Toxicology ◽

10.1007/s00204-021-03164-3 ◽

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.

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New Application of the Comet Assay

Journal of Histochemistry & Cytochemistry ◽

10.1369/0022155411410884 ◽

2011 ◽

Vol 59 (7) ◽

pp. 655-660 ◽

Cited By ~ 16

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.

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Assessment of reference values for DNA damage detected by the comet assay in human blood cell DNA

Mutation Research/Reviews in Mutation Research ◽

10.1016/j.mrrev.2005.10.001 ◽

2006 ◽

Vol 612 (2) ◽

pp. 84-104 ◽

Cited By ~ 105

Author(s):

P MOLLER

Keyword(s):

Dna Damage ◽

Comet Assay ◽

Blood Cell ◽

Reference Values ◽

Human Blood ◽

Human Blood Cell

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Leucocytes isolated from simply frozen whole blood can be used in human biomonitoring for DNA damage measurement with the comet assay

Cell Biochemistry and Function ◽

10.1002/cbf.3016 ◽

2013 ◽

Vol 32 (3) ◽

pp. 299-302 ◽

Cited By ~ 17

Author(s):

Maryam B. Akor-Dewu ◽

Naouale El Yamani ◽

Olena Bilyk ◽

Linda Holtung ◽

Torunn E. Tjelle ◽

...

Keyword(s):

Dna Damage ◽

Comet Assay ◽

Whole Blood ◽

Human Biomonitoring

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Application of the comet assay for the evaluation of DNA damage from frozen human whole blood samples: Implications for human biomonitoring

Toxicology Letters ◽

10.1016/j.toxlet.2019.11.010 ◽

2020 ◽

Vol 319 ◽

pp. 58-65 ◽

Cited By ~ 3

Author(s):

Goran Gajski ◽

Marko Gerić ◽

Tanja Živković Semren ◽

Blanka Tariba Lovaković ◽

Višnja Oreščanin ◽

...

Keyword(s):

Dna Damage ◽

Comet Assay ◽

Whole Blood ◽

Human Biomonitoring ◽

Blood Samples ◽

Human Whole Blood ◽

Whole Blood Samples

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B-Comet Assay (Comet Assay on Buccal Cells) for the Evaluation of Primary DNA Damage in Human Biomonitoring Studies

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph17249234 ◽

2020 ◽

Vol 17 (24) ◽

pp. 9234

Author(s):

Carla Russo ◽

Mattia Acito ◽

Cristina Fatigoni ◽

Milena Villarini ◽

Massimo Moretti

Keyword(s):

Dna Damage ◽

Comet Assay ◽

Gradient Centrifugation ◽

Venous Blood ◽

Smoking Habit ◽

Human Biomonitoring ◽

Peripheral Blood Lymphocytes ◽

Normal Weight ◽

Blood Collection ◽

Primary Dna Damage

Many subjects perceive venous blood collection as too invasive, and thus moving to better-accepted procedures for leukocytes collection might be crucial in human biomonitoring studies (e.g., biomonitoring of occupational or residential exposure to genotoxins) management. In this context, primary DNA damage was assessed in buccal lymphocytes (BLs), fresh whole venous, and capillary blood leukocytes, and compared with that in peripheral blood lymphocytes (PBLs)—the most frequently used cells—in 15 young subjects. Mouthwashes were collected after the volunteers rinsed their mouths with normal saline, and BLs were isolated by density gradient centrifugation. Blood samples were collected by venipuncture or by lancet. Anthropometric and lifestyle information was obtained by the administration of a structured questionnaire. As shown in the Bland-Altman plots, the level of agreement between BLs and PBLs lied within the accepted range, we thus enrolled a wider population (n = 54) to assess baseline DNA damage in BLs. In these cells, mean values of tail length (µm), tail intensity (%), and tail moment were 25.7 ± 0.9, 6.7 ± 0.4 and 1.0 ± 0.1, respectively. No significant association was observed between sex and smoking habit with any of the DNA damage parameters. Conversely, underweight subjects displayed significantly higher genomic instability compared with normal weight group (p < 0.05). In conclusion, we successfully managed to set up and update a non-invasive and well-accepted procedure for the isolation of BLs from saliva that could be useful in upcoming biomonitoring studies.

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