The enzyme-modified comet assay: Past, present and future

Muruzabal, Damián; Collins, Andrew; Azqueta, Amaya

doi:10.1016/j.fct.2020.111865

Cited by 51 publications

(40 citation statements)

References 158 publications

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“…Addition of such enzymes induces additional breaks in the DNA in locations of DNA lesions, which causes further migration of genetic material during the electrophoresis step. Several different enzymes have been used with comet assay for detection of specific DNA lesions: Endonuclease III (oxidized pyrimidines), Formamidopyrimidine DNA glycosylase (oxidized purines, formamidopyrimidines), 8-hydroxyguanine DNA-glycosylase (oxidized purines, formamidopyrimidines), T4 endonuclease V (cyclobutane pyrimidine dimers), 3-methyladenine DNA glycosylase II (alkylated bases, hypoxanthine), uracil-DNA glycosylase (uracil residue, deamination products) [42]. Detection of these types of damage finds use predominantly in measurement of genotoxic effects of chemicals, however, DNA lesions such as complex oxidative damage have been measured using comet assay with endonucleolytic enzymes in cells exposed to low doses of gamma rays [43].…”

Section: Alkaline Comet Assaymentioning

confidence: 99%

Use of Biological Dosimetry for Monitoring Medical Workers Occupationally Exposed to Ionizing Radiation

Piotrowski

Dawid²,

Kulcenty

et al. 2021

Radiation

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Medical workers are the largest group exposed to man-made sources of ionizing radiation. The annual doses received by medical workers have decreased over the last several decades, however for some applications, like fluoroscopically guided procedures, the occupational doses still remain relatively high. Studies show that for some procedures the operator and staff still use insufficient protective and dosimetric equipment, which might cause an underestimation of medical exposures. Physical dosimetry methods are a staple for estimating occupational exposures, although due to the inconsistent use of protection measures, an alternative method such as biological dosimetry might complement the physical methods to achieve a more complete picture. Such methods were used to detect exposures to doses as low as 0.1 mSv/year, and could be useful for a more accurate assessment of genotoxic effects of ionizing radiation in medical workers. Biological dosimetry is usually based on the measurement of the effects present in peripheral blood lymphocytes. Although some methods, such as chromosome aberration scoring or micronucleus assay, show promising results, currently there is no one method recognized as most suitable for dosimetric application in the case of chronic, low-dose exposures. In this review we decided to evaluate different methods used for biological dosimetry in assessment of occupational exposures of medical workers.

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Section: Alkaline Comet Assaymentioning

confidence: 99%

Use of Biological Dosimetry for Monitoring Medical Workers Occupationally Exposed to Ionizing Radiation

Piotrowski

Dawid²,

Kulcenty

et al. 2021

Radiation

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“…This method has been optimized on mammalian cell lines, but it has the potential for adaptation and use with other mammalian cell types, particularly those frequently employed in conventional comet assays (e.g., in peripheral blood mononuclear cells). The use of cells from other animal and non-animal species, including plants that require specific cell lysis conditions to isolate the nuclei, within enzyme modified comet assays has also been reported (see more comprehensive reviews on this topic [17,18]). However, we would recommend that the assay is thoroughly validated and optimized using the specific cell type of interest.…”

Section: Introductionmentioning

confidence: 99%

The Enzyme-Modified Neutral Comet (EMNC) Assay for Complex DNA Damage Detection

Fabbrizi

Hughes

Parsons

2021

MPs

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The comet assay is a versatile, simple, and sensitive gel electrophoresis–based method that can be used to measure and accurately quantify DNA damage, particularly single and double DNA strand breaks, in single cells. While generally this is used to measure variation in DNA strand break levels and repair capacity within a population of cells, the technique has more recently been adapted and evolved into more complex analysis and detection of specific DNA lesions, such as oxidized purines and pyrimidines, achieved through the utilization of damage-specific DNA repair enzymes following cell lysis. Here, we detail a version of the enzyme-modified neutral comet (EMNC) assay for the specific detection of complex DNA damage (CDD), defined as two or more DNA damage lesions within 1–2 helical turns of the DNA. CDD induction is specifically relevant to ionizing radiation (IR), particularly of increasing linear energy transfer (LET), and is known to contribute to the cell-killing effects of IR due to the difficult nature of its repair. Consequently, the EMNC assay reveals important details regarding the extent and complexity of DNA damage induced by IR, but also has potential for the study of other genotoxic agents that may induce CDD.

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“…Comet assay may be used to assess oxidative stress-induced genotoxicity by introducing an additional step of incubation with some lesion-specific endonucleases. The human 8-oxoguanine DNA glycosylase (hOGG1) is the most effective enzyme in specifically detecting 8-oxo-7,8-dihydroguanine (8-oxoGua), the most frequent DNA oxidative lesion, since unlike formamidopyrimidine glycosylase (fpg) or endonuclease III (endoIII) it does not recognize alkylated bases (Smith et al 2006); therefore, it appears to give more reliable estimates of DNA oxidation damage (Muruzabal et al 2021). hOGG1-modified comet assay has been widely used in human biomonitoring studies to detect oxidative DNA damage (Azqueta et al 2020).…”

Section: Discussionmentioning

confidence: 99%

Salivary leucocytes as suitable biomatrix for the comet assay in human biomonitoring studies

et al. 2021

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Peripheral blood leucocytes (PBL) have been traditionally used to investigate DNA damage by the comet assay in population studies, but validating alternative non-invasive samples would expand the application of this assay in human biomonitoring. The objectives of this study were (i) to test the validity of salivary leucocytes as a proper biomatrix for the comet assay, (ii) to evaluate the ability of this approach to detect different types of primary and oxidative DNA damage, and (iii) to determine whether frozen salivary leucocytes are still suitable for displaying those types of DNA damage. Fresh and frozen leucocytes isolated from saliva samples (six healthy non-smoking volunteers), were exposed to four genotoxic agents inducing different types of DNA damage, both primary (methyl methanesulfonate, actinomycin-D, ultraviolet radiation) and oxidative (potassium bromate), and standard or enzyme-modified comet assay was conducted. Results were compared with those obtained from PBL. Cells exposed to the four genotoxic agents showed dose-dependent increases of primary and oxidative DNA damage, demonstrating the suitability of all these samples to detect genetic damage from different origin. When comparing baseline levels of DNA damage, just a slight significant increase in primary DNA damage was observed in frozen salivary leucocytes regarding the other biomatrices, but similar results were obtained regarding sensitivity to DNA damage induction by all agents tested. This study demonstrates that salivary leucocytes can be employed in comet assay as an alternative or complement to blood samples. Frozen salivary leucocytes were proved to be a very convenient sample in large biomonitoring studies.

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The enzyme-modified comet assay: Past, present and future

Cited by 51 publications

References 158 publications

Use of Biological Dosimetry for Monitoring Medical Workers Occupationally Exposed to Ionizing Radiation

Use of Biological Dosimetry for Monitoring Medical Workers Occupationally Exposed to Ionizing Radiation

The Enzyme-Modified Neutral Comet (EMNC) Assay for Complex DNA Damage Detection

Salivary leucocytes as suitable biomatrix for the comet assay in human biomonitoring studies

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