Peter Møller, Amaya Azqueta, Adriana Rodriguez-Garraus, Tamara Bakuradze, Elke Richling, Ezgi Eyluel Bankoglu, Helga Stopper, Victoria Claudino Bastos, Sabine A S Langie, Annie Jensen, 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 Zheng, Sergey Shaposhnikov, Andrew Collins
{"title":"溴酸钾阳性测定对照的长期冷冻保存,通过Fpg修饰的彗星测定法测量氧化损伤的DNA:来自hCOMET环试验的结果。","authors":"Peter Møller, Amaya Azqueta, Adriana Rodriguez-Garraus, Tamara Bakuradze, Elke Richling, Ezgi Eyluel Bankoglu, Helga Stopper, Victoria Claudino Bastos, Sabine A S Langie, Annie Jensen, 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 Zheng, Sergey Shaposhnikov, Andrew Collins","doi":"10.1093/mutage/gead020","DOIUrl":null,"url":null,"abstract":"<p><p>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.</p>","PeriodicalId":18889,"journal":{"name":"Mutagenesis","volume":" ","pages":"264-272"},"PeriodicalIF":2.5000,"publicationDate":"2023-10-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"2","resultStr":"{\"title\":\"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.\",\"authors\":\"Peter Møller, Amaya Azqueta, Adriana Rodriguez-Garraus, Tamara Bakuradze, Elke Richling, Ezgi Eyluel Bankoglu, Helga Stopper, Victoria Claudino Bastos, Sabine A S Langie, Annie Jensen, 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 Zheng, Sergey Shaposhnikov, Andrew Collins\",\"doi\":\"10.1093/mutage/gead020\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>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.</p>\",\"PeriodicalId\":18889,\"journal\":{\"name\":\"Mutagenesis\",\"volume\":\" \",\"pages\":\"264-272\"},\"PeriodicalIF\":2.5000,\"publicationDate\":\"2023-10-14\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"2\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Mutagenesis\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.1093/mutage/gead020\",\"RegionNum\":4,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"GENETICS & HEREDITY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Mutagenesis","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1093/mutage/gead020","RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"GENETICS & HEREDITY","Score":null,"Total":0}
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.
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.
期刊介绍:
Mutagenesis is an international multi-disciplinary journal designed to bring together research aimed at the identification, characterization and elucidation of the mechanisms of action of physical, chemical and biological agents capable of producing genetic change in living organisms and the study of the consequences of such changes.