Naslov
Technical recommendations to perform the alkaline standard and enzymemodified comet assay in human biomonitoring studies
(Technical recommendations to perform the alkaline standard and enzyme-modified comet assay in human biomonitoring studies)

Autori
Azqueta, Amaya ; Muruzabal, Damian ; Boutet- Robinet, Elisa ; Milić, Mirta ; Dusinska, Maria ; Brunborg, Gunnar ; Møller, Peter ; Collins, Andrew R.

Izvornik
Mutation research. Genetic toxicology and environmental mutagenesis (1383-5718) 843 (2019); 24-32

Vrsta, podvrsta i kategorija rada
Radovi u časopisima, članak, znanstveni

Ključne riječi
standard comet assay ; Fpg-modified comet assay ; DNA damage ; protocol ; controls ; recommendations

Sažetak
The comet assay (single cell gel electrophoresis) is widely used as a biomonitoring tool to assess DNA damage – strand breaks, as well as oxidised bases ; it can also be adapted to measure DNA repair. It is based on the ability of breaks in the DNA to relax supercoiling, allowing DNA loops to extend from the nuclear core (nucleoid) under an electric field to form a comet-like tail. Most commonly, it is applied to white blood cells. The range of detection is between a few hundred breaks per cell and a few thousand, encompassing levels of damage that can be repaired and tolerated by human cells. Its applications include monitoring various diseases, studying the influence of nutrition on DNA stability, and investigating effects of environmental and occupational mutagens. Here we address the issue of inter-laboratory variation in comet assay results. This variation is largely due to differences in methods. Imposing a standard protocol is not practical, but users should be aware of the crucial parameters that affect performance of the assay. These include the concentration of agarose in which the cells are embedded ; the duration of cell lysis, and of enzyme incubation when oxidised bases are being measured ; the duration of alkaline unwinding ; the duration of electrophoresis and the voltage gradient applied ; and the method used to score the comets. Including reference standards in each experiment allows experimental variability to be monitored – and if variation is not extreme, results can be normalised using reference standard values. Reference standards are also essential for inter- laboratory comparison. Finally, we offer recommendations which, we believe, will limit variability and increase the usefulness of this assay in molecular epidemiology.

Izvorni jezik
Engleski

Znanstvena područja
Biologija, Interdisciplinarne prirodne znanosti, Javno zdravstvo i zdravstvena zaštita, Interdisciplinarne biotehničke znanosti, Biotehnologija u biomedicini (prirodno područje, biomedicina i zdravstvo, biotehničko područje)

Napomena
Acknowledgement: hCOMET CA12135 ; Ministry of Economy, Industry and Competitiveness (‘Ramón y Cajal’ programme, RYC-2013-14370) of the Spanish Government ; BIOGENSA project (AGL2015-70640-R) of the Ministry of Economy, Industry and Competitiveness of the Spanish Government and the European Regional Development Fund (ERDF) ; NILU internal project COST Comet assay 117029

POVEZANOST RADA