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

Research output: Contribution to journalJournal articleResearchpeer-review

  • Amaya Azqueta
  • Adriana Rodriguez-Garraus
  • Tamara Bakuradze
  • Elke Richling
  • Ezgi Eyluel Bankoglu
  • Helga Stopper
  • Victoria Claudino Bastos
  • Sabine A S Langie
  • Sara Ristori
  • Francesca Scavone
  • Lisa Giovannelli
  • Maria Wojewódzka
  • Marcin Kruszewski
  • Vanessa Valdiglesias
  • Blanca Laffon
  • Carla Costa
  • Solange Costa
  • João Paulo Teixeira
  • Mirko Marino
  • Cristian Del Bo'
  • Patrizia Riso
  • Congying Zhang
  • Sergey Shaposhnikov
  • Andrew Collins

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.

Original languageEnglish
Issue number5
Pages (from-to)264–272
Number of pages9
Publication statusPublished - 2023

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© 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.

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