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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
    Publication . Møller, Peter; Azqueta, Amaya; Rodriguez-Garraus, Adriana; Bakuradze, Tamara; Richling, Elke; Bankoglu, Ezgi Eyluel; Stopper, Helga; Claudino Bastos, Victoria; Langie, Sabine A.S.; Jensen, Annie; Ristori, Sara; Scavone, Francesca; Giovannelli, Lisa; Wojewódzka, Maria; Kruszewski, Marcin; Valdiglesias, Vanessa; Laffon, Blanca; Costa, Carla; Costa, Solange; Paulo Teixeira, João; Marino, Mirko; Del Bo’, Cristian; Riso, Patrizia; Zheng, Congying; Shaposhnikov, Sergey; Collins, Andrew
    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.
    2023-10-14Journal article Restricted access Show more
  • DNA strand break levels in cryopreserved mononuclear blood cell lines measured by the alkaline comet assay: results from the hCOMET ring trial
    Publication . Møller, Peter; Azqueta, Amaya; Rodriguez-Garraus, Adriana; Bakuradze, Tamara; Richling, Elke; Bankoglu, Ezgi Eyluel; Stopper, Helga; Claudino Bastos, Victoria; Langie, Sabine A.S.; Jensen, Annie; Ristori, Sara; Scavone, Francesca; Giovannelli, Lisa; Wojewódzka, Maria; Kruszewski, Marcin; Valdiglesias, Vanessa; Laffon, Blanca; Costa, Carla; Costa, Solange; Paulo Teixeira, João; Marino, Mirko; Del Bo, Cristian; Riso, Patrizia; Zheng, Congying; Shaposhnikov, Sergey; Collins, Andrew
    The comet assay is widely used in biomonitoring studies for the analysis of DNA damage in leukocytes and peripheral blood mononuclear cells. Rather than processing blood samples directly, it can be desirable to cryopreserve whole blood or isolated cells for later analysis by the comet assay. However, this creates concern about artificial accumulation of DNA damage during cryopreservation. In this study, 10 laboratories used standardized cryopreservation and thawing procedures of monocytic (THP-1) or lymphocytic (TK6) cells. Samples were cryopreserved in small aliquots in 50% foetal bovine serum, 40% cell culture medium, and 10% dimethyl sulphoxide. Subsequently, cryopreserved samples were analysed by the standard comet assay on three occasions over a 3-year period. Levels of DNA strand breaks in THP-1 cells were increased (four laboratories), unaltered (four laboratories), or decreased (two laboratories) by long-term storage. Pooled analysis indicates only a modest positive association between storage time and levels of DNA strand breaks in THP-1 cells (0.37% Tail DNA per year, 95% confidence interval: -0.05, 0.78). In contrast, DNA strand break levels were not increased by cryopreservation in TK6 cells. There was inter-laboratory variation in levels of DNA strand breaks in THP-1 cells (SD = 3.7% Tail DNA) and TK6 reference sample cells (SD = 9.4% Tail DNA), whereas the intra-laboratory residual variation was substantially smaller (i.e. SD = 0.4%-2.2% Tail DNA in laboratories with the smallest and largest variation). In conclusion, the study shows that accumulation of DNA strand breaks in cryopreserved mononuclear blood cell lines is not a matter of concern.
    2023-10-14Journal article Restricted access Show more