1. 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.
- Author
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Møller, Peter, Azqueta, Amaya, Rodriguez-Garraus, Adriana, Bakuradze, Tamara, Richling, Elke, Bankoglu, Ezgi Eyluel, Stopper, Helga, Bastos, Victoria Claudino, Langie, Sabine A S, Jensen, Annie, Ristori, Sara, Scavone, Francesca, Giovannelli, Lisa, Wojewódzka, Maria, Kruszewski, Marcin, Valdiglesias, Vanessa, Laffon, Blanca, Costa, Carla, Costa, Solange, and Teixeira, João Paulo
- Subjects
COMETS ,DIMETHYL sulfoxide ,POTASSIUM ,DNA ,POTASSIUM channels ,DNA damage - Abstract
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 KBrO
3 . 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. [ABSTRACT FROM AUTHOR]- Published
- 2023
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