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
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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. / 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; Teixeira, João Paulo; Marino, Mirko; Del Bo', Cristian; Riso, Patrizia; Zhang, Congying; Shaposhnikov, Sergey; Collins, Andrew.
I: Mutagenesis, Bind 38, Nr. 5, 2023, s. 264–272.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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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 -
ID: 360132881