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

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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Using Comet Assay for Genotoxic Evaluation of Zataria multiflora Boiss., An Iranian Endemic Plant

Journal of Food Quality and Hazards Control ◽

10.18502/jfqhc.8.1.5463 ◽

2021 ◽

Author(s):

Z. Ayubi ◽

A.A. Jafari ◽

S.A. Ayatollahi-Mousavi ◽

H. Jafari ◽

M. Panddeh

Keyword(s):

Dna Damage ◽

Comet Assay ◽

B Lymphocytes ◽

Alcoholic Extract ◽

Endemic Plant ◽

Food Preservative ◽

Zataria Multiflora ◽

Significant Difference ◽

Spss Software ◽

Phosphate Buffered Saline

Background: Zataria multiflora Boiss. (Avishan-e Shirazi), as an Iranian endemic plant, belongs to the Lamiaceae family and may be used as a food preservative. This study aimed to detect potential genotoxic effects of Z. multiflora extract. Methods: Hydro-alcoholic extract of Z. multiflora was prepared. Human B lymphocytes were treated with 1% extract within 3 and 24 h. Sterile Phosphate Buffered Saline (PBS) and cisplatin were used as negative and positive controls, respectively. DNA damage profiles were examined using comet assay (Single Cell Gel Electrophoresis). Data were statistically analyzed by SPSS software v. 21. Results: No statistically significant (p=0.071) DNA damage was observed in B lymphocytes treated with either Z. multiflora extract or PBS after 3 and 24 h. However, there was a statistically significant difference (p=0.0001) between DNA damage in B lymphocytes that treated with cisplatin and Z. multiflora after 3 and 24 h. Conclusion: The comet assay used in the current study showed that Z. multiflora had no genotoxic effect.

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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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