High‐Throughput Assays to Quantify the Formation of DNA Strand Breaks

Moreno‐Villanueva, María; Bürkle, Alexander

doi:10.1002/9781118538203.ch14

Cited by 4 publications

(3 citation statements)

References 52 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Even though these techniques provide useful tools for measuring DNA strand breaks in a high-throughput fashion, they have some disadvantages. A comprehensive comparison between these assays has been published before [ 11 ]. Briefly, ?H2AX assay is considered a very sensitive method that specifically detects double DNA strand breaks however in some cases the presence of ?H2AX foci in the absence of DNA damage has been demonstrated.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Mathematical modelling of the automated FADU assay for the quantification of DNA strand breaks and their repair in human peripheral mononuclear blood cells

et al. 2014

View full text Add to dashboard Cite

BackgroundCells continuously undergo DNA damage from exogenous agents like irradiation or genotoxic chemicals or from endogenous radicals produced by normal cellular metabolic activities. DNA strand breaks are one of the most common genotoxic lesions and they can also arise as intermediates of DNA repair activity. Unrepaired DNA damage can lead to genomic instability, which can massively compromise the health status of organisms. Therefore it is important to measure and quantify DNA damage and its repair.ResultsWe have previously published an automated method for measuring DNA strand breaks based on fluorimetric detection of alkaline DNA unwinding [1], and here we present a mathematical model of the FADU assay, which enables to an analytic expression for the relation between measured fluorescence and the number of strand breaks.ConclusionsAssessment of the formation and also the repair of DNA strand breaks is a crucial functional parameter to investigate genotoxicity in living cells. A reliable and convenient method to quantify DNA strand breakage is therefore of significant importance for a wide variety of scientific fields, e.g. toxicology, pharmacology, epidemiology and medical sciences.

show abstract

Section: Discussionmentioning

confidence: 99%

“…We have previously published a modified and automated version of the FADU assay [ 1 ],[ 11 ],[ 12 ]. Compared with the original FADU assay [ 2 ], the automated FADU assay shows comparable sensitivity, yet increased robustness and throughput, and decreased operator time.…”

Section: Discussionmentioning

confidence: 99%

Mathematical modelling of the automated FADU assay for the quantification of DNA strand breaks and their repair in human peripheral mononuclear blood cells

et al. 2014

View full text Add to dashboard Cite

show abstract

“…This control value is called the ‘T-value’ and set as 100% of DNA amount. Endogenous DNA damage can be assessed in samples, which are not exposed to external DNA damaging agents, resulting in the so called ‘P0 value’ (Figure 4) (113). All steps, beginning from the addition of suspension buffer until SybrGreen staining, are fully automated using a modified commercially available pipetting robot.…”

Section: Introductionmentioning

confidence: 99%

Emerging metrology for high-throughput nanomaterial genotoxicology

Nelson

Wright

Ibuki

et al. 2016

MUTAGE

View full text Add to dashboard Cite

The rapid development of the engineered nanomaterial (ENM) manufacturing industry has accelerated the incorporation of ENMs into a wide variety of consumer products across the globe. Unintentionally or not, some of these ENMs may be introduced into the environment or come into contact with humans or other organisms resulting in unexpected biological effects. It is thus prudent to have rapid and robust analytical metrology in place that can be used to critically assess and/or predict the cytotoxicity, as well as the potential genotoxicity of these ENMs. Many of the traditional genotoxicity test methods [e.g. unscheduled DNA synthesis assay, bacterial reverse mutation (Ames) test, etc.,] for determining the DNA damaging potential of chemical and biological compounds are not suitable for the evaluation of ENMs, due to a variety of methodological issues ranging from potential assay interferences to problems centered on low sample throughput. Recently, a number of sensitive, high-throughput genotoxicity assays/platforms (CometChip assay, flow cytometry/micronucleus assay, flow cytometry/γ-H2AX assay, automated ‘Fluorimetric Detection of Alkaline DNA Unwinding’ (FADU) assay, ToxTracker reporter assay) have been developed, based on substantial modifications and enhancements of traditional genotoxicity assays. These new assays have been used for the rapid measurement of DNA damage (strand breaks), chromosomal damage (micronuclei) and for detecting upregulated DNA damage signalling pathways resulting from ENM exposures. In this critical review, we describe and discuss the fundamental measurement principles and measurement endpoints of these new assays, as well as the modes of operation, analytical metrics and potential interferences, as applicable to ENM exposures. An unbiased discussion of the major technical advantages and limitations of each assay for evaluating and predicting the genotoxic potential of ENMs is also provided.

show abstract