An optimized comet-based in vitro DNA repair assay to assess base and nucleotide excision repair activity

Vodenková, Soňa; Azqueta, Amaya; Collins, Andrew; Dušinská, Maria; Gaivão, Isabel; Möller, Peter; Opattová, Alena; Vodička, Pavel; Godschalk, Roger; Langie, Sabine A. S.

doi:10.1038/s41596-020-0401-x

Cited by 37 publications

(28 citation statements)

References 95 publications

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“…It should be noted that the DNA damaging reagents used in this study can be removed from the cells, but the NPs cannot be easily removed. Because of its simplicity and sensitivity, the comet assay is widely used in many frontier fields, including biological monitoring, [142,143] DNA repair, [144,145] genotoxicity evaluation, [146,147] and extensive studies on the cytotoxicity of metal-based NPs (Figure 5d). [138] The micronucleus (MN) assay is another genotoxicity evaluation method used to detect chromosomal or mitotic organ damage (Figure 5e).…”

Section: Evaluation Methods For Dna Damagementioning

confidence: 99%

Cytotoxicity of Metal‐Based Nanoparticles: From Mechanisms and Methods of Evaluation to Pathological Manifestations

Xiong

Xiao-hong

et al. 2022

Advanced Science

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Metal-based nanoparticles (NPs) are particularly important tools in tissue engineering-, drug carrier-, interventional therapy-, and biobased technologies. However, their complex and varied migration and transformation pathways, as well as their continuous accumulation in closed biological systems, cause various unpredictable toxic effects that threaten human and ecosystem health. Considerable experimental and theoretical efforts have been made toward understanding these cytotoxic effects, though more research on metal-based NPs integrated with clinical medicine is required. This review summarizes the mechanisms and evaluation methods of cytotoxicity and provides an in-depth analysis of the typical effects generated in the nervous, immune, reproductive, and genetic systems. In addition, the challenges and opportunities are discussed to enhance future investigations on safer metal-based NPs for practical commercial adoption.

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Section: Evaluation Methods For Dna Damagementioning

confidence: 99%

Cytotoxicity of Metal‐Based Nanoparticles: From Mechanisms and Methods of Evaluation to Pathological Manifestations

Xiong

Xiao-hong

et al. 2022

Advanced Science

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“…We are also integrating this device with a ubiquitous smartphone for image capturing and data processing, which is well suited for ready-to-go testing in low-resource settings. Compared with the commonly used alkaline comet assay and TUNEL assay, 9,18 this method provides rapid, high throughput, and absolute quantitative measurements of multiple DNA damages induced by exogenous chemical agents. We envision that the method described here will find useful applications in drug development, genotoxicity testing, and environmental toxicology.…”

Section: Discussionmentioning

confidence: 99%

Quantifying DNA damage on paper sensors via controlled template-independent DNA polymerization

et al. 2022

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“…The extract is then incubated with substrate cells harboring specifically induced lesions (Vodenkova et al 2020). In this variation, any excision of lesions must be due to DNA repair.…”

Section: Discussionmentioning

confidence: 99%

“…The third popular version of measuring DNA repair activity using comet assay is to use a cellular extract containing repair enzymes. The extract is then incubated with substrate cells harboring specifically induced lesions (Vodenkova et al 2020 ). In this variation, any excision of lesions must be due to DNA repair.…”

Section: Discussionmentioning

confidence: 99%

Cell survival after DNA damage in the comet assay

2021

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The 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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An optimized comet-based in vitro DNA repair assay to assess base and nucleotide excision repair activity

Abstract: The comet-based in vitro DNA repair assay: a standardized method to assess an individual's DNA repair activity.

Cited by 37 publications

References 95 publications

Cytotoxicity of Metal‐Based Nanoparticles: From Mechanisms and Methods of Evaluation to Pathological Manifestations

Cytotoxicity of Metal‐Based Nanoparticles: From Mechanisms and Methods of Evaluation to Pathological Manifestations

Quantifying DNA damage on paper sensors via controlled template-independent DNA polymerization

Cell survival after DNA damage in the comet assay

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