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:

Møller Peter1ORCID,Azqueta Amaya2,Rodriguez-Garraus Adriana2,Bakuradze Tamara3,Richling Elke3,Bankoglu Ezgi Eyluel4,Stopper Helga4,Claudino Bastos Victoria5,Langie Sabine A S5,Jensen Annie1,Ristori Sara6,Scavone Francesca6,Giovannelli Lisa6,Wojewódzka Maria7,Kruszewski Marcin78,Valdiglesias Vanessa910ORCID,Laffon Blanca1011,Costa Carla121314,Costa Solange121314,Paulo Teixeira João121314ORCID,Marino Mirko15ORCID,Del Bo’ Cristian15,Riso Patrizia15,Zheng Congying516,Shaposhnikov Sergey17,Collins Andrew1617

Affiliation:

1. Department of Public Health, Section of Environmental Health, University of Copenhagen , Øster Farimagsgade 5A, DK-1014 Copenhagen K , Denmark

2. Department of Pharmacology and Toxicology, School of Pharmacy and Nutrition, University of Navarra , C/Irunlarrea 1, 31009 Pamplona , Spain

3. Food Chemistry and Toxicology, Department of Chemistry, Rhineland-Palatinate Technical University Kaiserslautern-Landau , Erwin-Schroedinger-Str. 52, D-67663 Kaiserslautern , Germany

4. Institute of Pharmacology and Toxicology, University of Wuerzburg , Versbacher Str. 9, 97078 Wuerzburg , Germany

5. Department of Pharmacology and Toxicology, School for Nutrition and Translational Research in Metabolism (NUTRIM), Maastricht University , Maastricht , The Netherlands

6. Department of Neuroscience, Psychology, Pharmacology and Child Health (NEUROFARBA), Section Pharmacology and Toxicology, University of Florence , Florence , Italy

7. Centre for Radiobiology and Biological Dosimetry, Institute of Nuclear Chemistry and Technology , Dorodna 16, 01-310 Warsaw , Poland

8. Department of Molecular Biology and Translational Research, Institute of Rural Health , Jaczewskiego 2, 20-090 Lublin , Poland

9. Universidade da Coruña, Grupo NanoToxGen, Centro Interdisciplinar de Química e Bioloxía – CICA, Departamento de Biología , A Coruña , Spain

10. Instituto de Investigación Biomédica de A Coruña (INIBIC) , A Coruña , Spain

11. Universidade da Coruña, Grupo DICOMOSA, Centro Interdisciplinar de Química e Bioloxía – CICA, Departamento de Psicología , A Coruña , Spain

12. Environmental Health Department, National Institute of Health , Porto , Portugal

13. EPIUnit—Instituto de Saúde Pública, Universidade do Porto , Porto , Portugal

14. Laboratory for Integrative and Translational Research in Population Health (ITR) , Porto , Portugal

15. Department of Food, Environmental and Nutritional Sciences (DeFENS), Università degli Studi di Milano , 20133 Milan , Italy

16. Department of Nutrition, University of Oslo , Norway

17. NorGenotech AS , Oslo , Norway

Abstract

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 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.

Funder

Spanish Ministry of Science and Innovation

Università degli Studi di Firenze

Publisher

Oxford University Press (OUP)

Subject

Health, Toxicology and Mutagenesis,Genetics (clinical),Toxicology,Genetics

Reference45 articles.

1. DNA repair as a human biomonitoring tool: comet assay approaches;Azqueta;Mutat Res,2019

2. The comet assay in animal models: from bugs to whales—(Part 1 invertebrates);Gajski;Mutat Res,2019

3. The comet assay in animal models: from bugs to whales—(Part 2 vertebrates);Gajski;Mutat Res,2019

4. Application of the comet assay in human biomonitoring: an hCOMET perspective;Azqueta;Mutat Res,2020

5. Measurement of DNA damage with the comet assay in high-prevalence diseases: current status and future directions;Møller;Mutagenesis,2020

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