TY - JOUR

T1 - Long-term cryopreservation of potassium bromate positive assay controls for measurement of oxidatively damaged DNA by the Fpg-modified comet assay

T2 - results from the hCOMET ring trial

AU - Møller, Peter

AU - Azqueta, Amaya

AU - Rodriguez-Garraus, Adriana

AU - Bakuradze, Tamara

AU - Richling, Elke

AU - Bankoglu, Ezgi Eyluel

AU - Stopper, Helga

AU - Bastos, Victoria Claudino

AU - Langie, Sabine A S

AU - Jensen, Annie

AU - Ristori, Sara

AU - Scavone, Francesca

AU - Giovannelli, Lisa

AU - Wojewódzka, Maria

AU - Kruszewski, Marcin

AU - Valdiglesias, Vanessa

AU - Laffon, Blanca

AU - Costa, Carla

AU - Costa, Solange

AU - Teixeira, João Paulo

AU - Marino, Mirko

AU - Del Bo', Cristian

AU - Riso, Patrizia

AU - Zhang, Congying

AU - Shaposhnikov, Sergey

AU - Collins, Andrew

N1 - © The Author(s) 2023. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

PY - 2023

Y1 - 2023

N2 - 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 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 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 three-year period. All laboratories obtained 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 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.

AB - 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 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 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 three-year period. All laboratories obtained 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 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.

U2 - 10.1093/mutage/gead020

DO - 10.1093/mutage/gead020

M3 - Journal article

C2 - 37357815

VL - 38

SP - 264

EP - 272

JO - Mutagenesis

JF - Mutagenesis

SN - 0267-8357

IS - 5

ER -