Detalle Publicación

Long-term cryopreservation of potassium bromate positive assay controls for measurement of oxidatively damaged DNA by the Fpg-modified comet assay: results from the hCOMET ring trial

Autores: Moller, P. (Autor de correspondencia); Azqueta Oscoz, Amaya; Rodriguez Garraus, Adriana; Bakuradze, T.; Richling, E.; Bankoglu, E. E.; Stopper, H.; Bastos, V. C.; Langie, S. A. S.; Jensen, A.; Ristori, S.; Svocane, F.; Giovannelli, L.; Wojewodzka, M.; Kruszewski, M.; Valdiglesias, V.; Laffon, B.; Costa, C.; Costa, S.; Teixeira, J. P.; Marino, M.; Del Bo', C.; Riso, P.; Zheng, C. Y.; Shaposhnikov, S.; Collins, A.
Título de la revista: MUTAGENESIS
ISSN: 0267-8357
Volumen: 38
Número: 5
Páginas: 264 - 272
Fecha de publicación: 2023
Resumen:
The formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay is widely used for the measurement of oxidatively generated damage to DNA. However, there has not been a recommended long-term positive control for this version of the comet assay. We have investigated potassium bromate as a positive control for the Fpg-modified comet assay because it generates many Fpg-sensitive sites with a little concurrent generation of DNA strand breaks. Eight laboratories used the same procedure for the treatment of monocytic THP-1 cells with potassium bromate (0, 0.5, 1.5, and 4.5 mM) and subsequent cryopreservation in a freezing medium consisting of 50% foetal bovine serum, 40% RPMI-1640 medium, and 10% dimethyl sulphoxide. The samples were analysed by the Fpg-modified comet assay three times over a 3-year period. All laboratories obtained a positive concentration-response relationship in cryopreserved samples (linear regression coefficients ranging from 0.79 to 0.99). However, there was a wide difference in the levels of Fpg-sensitive sites between the laboratory with the lowest (4.2% Tail DNA) and highest (74% Tail DNA) values in THP-1 cells after exposure to 4.5 mM KBrO3. In an attempt to assess sources of inter-laboratory variation in Fpg-sensitive sites, comet images from one experiment in each laboratory were forwarded to a central laboratory for visual scoring. There was high consistency between measurements of %Tail DNA values in each laboratory and the visual score of the same comets done in the central laboratory (r = 0.98, P < 0.001, linear regression). In conclusion, the results show that potassium bromate is a suitable positive comet assay control.