Browsing by Author "Koppen, Gudrun"
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- Application of the comet assay in human biomonitoring: an hCOMET perspectivePublication . Azqueta, Amaya; Ladeira, Carina; Giovannelli, Lisa; Boutet-Robinet, Elisa; Bonassi, Stefano; Neri, Monica; Gajski, Goran; Duthie, Susan; Del Bo’, Cristian; Riso, Patrizia; Koppen, Gudrun; Basaran, Nursen; Collins, Andrew; Møller, PeterThe comet assay is a well-accepted biomonitoring tool to examine the effect of dietary, lifestyle, environmental and occupational exposure on levels of DNA damage in human cells. With such a wide range of determinants for DNA damage levels, it becomes challenging to deal with confounding and certain factors are interrelated (e.g. poor nutritional intake may correlate with smoking status). This review describes the effect of intrinsic (i.e. sex, age, tobacco smoking, occupational exposure, and obesity) and extrinsic (season, environmental exposures, diet, physical activity, and alcohol consumption) factors on the level of DNA damage measured by the standard or enzyme-modified comet assay. Although each factor influences at least one comet assay endpoint, the collective evidence does not indicate single factors have a large impact. Thus, controlling for confounding may be necessary for a biomonitoring study, but none of the factors is strong enough to be regarded as a priori as a confounder. Controlling for confounding in the comet assay requires a case-by-case approach. Inter-laboratory variation in levels of DNA damage and to some extent also reproducibility in biomonitoring studies are issues that have haunted the users of the comet assay for years. Procedures to collect specimens, and their storage, are not standardized. Likewise, statistical issues related to both sample-size calculation (before sampling of specimens) and statistical analysis of the results vary between studies. This review gives guidance to statistical analysis of the typically complex exposure, co-variate, and effect relationships in human biomonitoring studies.
- Collection and storage of human white blood cells for analysis of DNA damage and repair activity using the comet assay in molecular epidemiology studiesPublication . Møller, Peter; Bankoglu, Ezgi Eyluel; Stopper, Helga; Giovannelli, Lisa; Ladeira, Carina; Koppen, Gudrun; Gajski, Goran; Collins, Andrew; Valdiglesias, Vanessa; Laffon, Blanca; Boutet-Robinet, Elisa; Perdry, Hervé; Del Bo’, Cristian; Langie, Sabine A S; Dusinska, Maria; Azqueta, AmayaDNA damage and repair activity are often assessed in blood samples from humans in different types of molecular epidemiology studies. However, it is not always feasible to analyze the samples on the day of collection without any type of storage. For instance, certain studies use repeated sampling of cells from the same subject or samples from different subjects collected at different time points, and it is desirable to analyze all these samples in the same comet assay experiment. In addition, flawless comet assay analyses on frozen samples open up the possibility of using this technique on biobank material. In this article, we discuss the use of cryopreserved peripheral blood mononuclear cells (PBMCs), buffy coat (BC), and whole blood (WB) for analysis of DNA damage and repair using the comet assay. The published literature and the authors' experiences indicate that various types of blood samples can be cryopreserved with only a minor effect on the basal level of DNA damage. There is evidence to suggest that WB and PBMCs can be cryopreserved for several years without much effect on the level of DNA damage. However, care should be taken when cryopreserving WB and BCs. It is possible to use either fresh or frozen samples of blood cells, but results from fresh and frozen cells should not be used in the same dataset. The article outlines detailed protocols for the cryopreservation of PBMCs, BCs, and WB samples.
- DNA damage in circulating leukocytes measured with the comet assay may predict the risk of deathPublication . Bonassi, Stefano; Ceppi, Marcello; Møller, Peter; Azqueta, Amaya; Milić, Mirta; Monica, Neri; Brunborg, Gunnar; Godschalk, Roger; Koppen, Gudrun; Langie, Sabine A. S.; Teixeira, João Paulo; Bruzzone, Marco; Da Silva, Juliana; Benedetti, Danieli; Cavallo, Delia; Ursini, Cinzia Lucia; Giovannelli, Lisa; Moretti, Silvia; Riso, Patrizia; Del Bo’, Cristian; Russo, Patrizia; Dobrzyńska, Malgorzata; Goroshinskaya, Irina A.; Surikova, Ekaterina I.; Staruchova, Marta; Barančokova, Magdalena; Volkovova, Katarina; Kažimirova, Alena; Smolkova, Bozena; Laffon, Blanca; Valdiglesias, Vanessa; Pastor, Susana; Marcos, Ricard; Hernández, Alba; Gajski, Goran; Spremo-Potparević, Biljana; Živković, Lada; Boutet-Robinet, Elisa; Perdry, Hervé; Lebailly, Pierre; Perez, Carlos L.; Basaran, Nursen; Nemeth, Zsuzsanna; Safar, Anna; Dusinska, Maria; Collins, Andrew; Anderson, Diana; Andrade, Vanessa; Pereira, Cristiana Costa; Costa, Solange; Gutzkow, Kristine B.; Ladeira, Carina; Moretti, Massimo; Costa, Carla; Orlow, Irene; Rojas, Emilio; Pourrut, Bertrand; Kruszewski, Marcin; Knasmueller, Siegfried; Shaposhnikov, Sergey; Žegura, Bojana; Stopper, HelgaThe comet assay or single cell gel electrophoresis is the most common method used to measure strand breaks and a variety of other DNA lesions in human populations. To estimate the risk of overall mortality, mortality by cause, and cancer incidence associated with DNA damage, a cohort of 2,403 healthy individuals (25,978 person-years) screened in 16 laboratories using the comet assay between 1996 and 2016 was followed-up. Kaplan–Meier analysis indicated a worse overall survival in the medium and high tertile of DNA damage (p < 0.001). The effect of DNA damage on survival was modeled according to Cox proportional hazard regression model. The adjusted hazard ratio (HR) was 1.42 (1.06–1.90) for overall mortality and 1.94 (1.04–3.59) for diseases of the circulatory system in subjects with the highest tertile of DNA damage. The findings of this study provide epidemiological evidence encouraging the implementation of the comet assay in preventive strategies for non-communicable diseases.
- Measuring DNA modifications with the comet assay: a compendium of protocolsPublication . Collins, Andrew; Møller, Peter; Gajski, Goran; Vodenková, Soňa; Abdulwahed, Abdulhadi; Anderson, Diana; Bankoglu, Ezgi Eyluel; Bonassi, Stefano; Boutet-Robinet, Elisa; Brunborg, Gunnar; Chao, Christy; Cooke, Marcus S.; Costa, Carla; Costa, Solange; Dhawan, Alok; de Lapuente, Joaquin; Bo’, Cristian Del; Dubus, Julien; Dusinska, Maria; Duthie, Susan J.; Yamani, Naouale El; Engelward, Bevin; Gaivão, Isabel; Giovannelli, Lisa; Godschalk, Roger; Guilherme, Sofia; Gutzkow, Kristine B.; Habas, Khaled; Hernández, Alba; Herrero, Oscar; Isidori, Marina; Jha, Awadhesh N.; Knasmüller, Siegfried; Kooter, Ingeborg M.; Koppen, Gudrun; Kruszewski, Marcin; Ladeira, Carina; Laffon, Blanca; Larramendy, Marcelo; Hégarat, Ludovic Le; Lewies, Angélique; Lewinska, Anna; Liwszyc, Guillermo E.; de Cerain, Adela López; Manjanatha, Mugimane; Marcos, Ricard; Milić, Mirta; de Andrade, Vanessa Moraes; Moretti, Massimo; Muruzabal, Damian; Novak, Matjaž; Oliveira, Rui; Olsen, Ann-Karin; Owiti, Norah; Pacheco, Mário; Pandey, Alok K.; Pfuhler, Stefan; Pourrut, Bertrand; Reisinger, Kerstin; Rojas, Emilio; Rundén-Pran, Elise; Sanz-Serrano, Julen; Shaposhnikov, Sergey; Sipinen, Ville; Smeets, Karen; Stopper, Helga; Teixeira, João Paulo; Valdiglesias, Vanessa; Valverde, Mahara; van Acker, Frederique; van Schooten, Frederik-Jan; Vasquez, Marie; Wentzel, Johannes F.; Wnuk, Maciej; Wouters, Annelies; Žegura, Bojana; Zikmund, Tomas; Langie, Sabine A. S.; Azqueta, AmayaThe comet assay is a versatile method to detect nuclear DNA damage in individual eukaryotic cells, from yeast to humans. The types of damage detected encompass DNA strand breaks and alkali-labile sites (e.g., apurinic/apyrimidinic sites), alkylated and oxidized nucleobases, DNA-DNA crosslinks, UV-induced cyclobutane pyrimidine dimers, some chemically induced DNA adducts. Depending on the specimen type, there are important modifications to the comet assay protocol to avoid the formation of additional DNA damage during the processing of samples and to ensure sufficient sensitivity to detect differences in damage levels between sample groups. Various applications of the comet assay have been validated by research groups in academia, industry, and regulatory agencies, and its strengths are highlighted by the adoption of the comet assay as an in vivo test for genotoxicity in animal organs by the Organisation for Economic Co-operation and Development. The present document includes a series of consensus protocols that describe the application of the comet assay to a wide variety of cell types, species, and types of DNA damage, thereby demonstrating its versatility.
- The comet assay for human biomonitoring: effect of cryopreservation on DNA damage in different blood cell preparationsPublication . Ladeira, Carina; Koppen, Gudrun; Scavone, Francesca; Giovannelli, LisaThis study was designed within the frame of the COST Action hCOMET 15132 (Working Group 6), with the aim of comparing different peripheral blood cell preparations for their feasibility in human biomonitoring studies, using the comet assay for the evaluation of DNA damage. Basal levels of strand breaks/ALS and formamidopyrimidine DNA glycosylase (Fpg) - sites, and H2O2 (500 μM)-induced strand breaks, were measured in whole blood, peripheral blood mononuclear cells - lymphocytes and monocytes - and buffy coat; in fresh and 1, 4 and 12 weeks-frozen samples. The comparison among the fresh preparations showed that the basal levels of DNA damage were all very low and similar in the three samples. Frozen whole blood samples stored in cryostraws without cryoprotection showed similar basal levels of DNA damage as fresh samples, indicating that this preparation, often chosen for biobanks, resists efficiently freezing/thawing artifacts. However, long-term storage of frozen buffy coat samples in cryostraws and with no cryopreservative did not appear feasible. Storage up to 3 months of frozen cryoprotected peripheral blood mononuclear cells induced small increases in basal strand breaks and no other statistically significant modification. Altogether, this study suggests that whole blood could be the most suitable sample to be used to perform comet assay in human epidemiological biomonitoring for genotoxicity assessment in frozen samples, such as those stored in biobanks.
- The hCOMET project: international database comparison of results with the comet assay in human biomonitoring (baseline frequency of DNA damage and effect of main confounders)Publication . Milić, Mirta; Ceppi, Marcello; Bruzzone, Marco; Azqueta, Amaya; Brunborg, Gunnar; Godschalk, Roger; Koppen, Gudrun; Langie, Sabine; Møller, Peter; Teixeira, João Paulo; Alija, Avdulla; Anderson, Diana; Andrade, Vanessa; Andreoli, Cristina; Asllani, Fisnik; Bangkoglu, Ezgi Eyluel; Barančoková, Magdalena; Basaran, Nursen; Boutet-Robinet, Elisa; Buschini, Annamaria; Cavallo, Delia; Costa Pereira, Cristiana; Costa, Carla; Costa, Solange; Da Silva, Juliana; Del Boˊ, Cristian; Dimitrijević Srećković, Vesna; Djelić, Ninoslav; Dobrzyńska, Malgorzata; Duračková, Zdenka; Dvořáková, Monika; Gajski, Goran; Galati, Serena; García Lima, Omar; Giovannelli, Lisa; Goroshinskaya, Irina A.; Grindel, Annemarie; Gutzkow, Kristine B.; Hernández, Alba; Hernández, Carlos; Holven, Kirsten B.; Ibero-Baraibar, Idoia; Ottestad, Inger; Kadioglu, Ela; Kažimirová, Alena; Kuznetsova, Elena; Ladeira, Carina; Laffon, Blanca; Lamonaca, Palma; Lebailly, Pierre; Louro, Henriqueta; Mandina Cardoso, Tania; Marcon, Francesca; Marcos, Ricard; Moretti, Massimo; Moretti, Silvia; Najafzadeh, Mojgan; Nemeth, Zsuzsanna; Neri, Monica; Novotna, Bozena; Orlow, Irene; Paduchova, Zuzana; Pastor, Susana; Perdry, Hervé; Spremo-Potparević, Biljana; Ramadhani, Dwi; Riso, Patrizia; Rohr, Paula; Rojas, Emilio; Rossner, Pavel; Safar, Anna; Sardas, Semra; Silva, Maria João; Sirota, Nikolay; Smolkova, Bozena; Staruchova, Marta; Stetina, Rudolf; Stopper, Helga; Surikova, Ekaterina I.; Ulven, Stine M.; Ursini, Cinzia Lucia; Valdiglesias, Vanessa; Valverde, Mahara; Vodicka, Pavel; Volkovova, Katarina; Wagner, Karl-Heinz; Živković, Lada; Dušinská, Maria; Collins, Andrew R.; Bonassi, StefanoThe alkaline comet assay, or single cell gel electrophoresis, is one of the most popular methods for assessing DNA damage in the human population. One of the open issues concerning this assay is the identification of those factors that can explain the large inter-individual and inter-laboratory variation. International collaborative initiatives such as the hCOMET project - a COST Action launched in 2016 - represent a valuable tool to meet this challenge. The aims of hCOMET were to establish reference values for the level of DNA damage in humans, to investigate the effect of host factors, lifestyle, and exposure to genotoxic agents, and to compare different sources of assay variability. A database of 19,320 subjects was generated, pooling data from 105 studies run by 44 laboratories in 26 countries between 1999 and 2019. A mixed random effect log-linear model, in parallel with a classic meta-analysis, was applied to take into account the extensive heterogeneity of data, due to descriptor, specimen, and protocol variability. As a result of this analysis interquartile intervals of DNA strand breaks (which includes alkali-labile sites) were reported for tail intensity, tail length, and tail moment (comet assay descriptors). A small variation by age was reported in some datasets, suggesting higher DNA damage in oldest age-classes, while no effect could be shown for sex or smoking habit, although the lack of data on heavy smokers has still to be considered. Finally, highly significant differences in DNA damage were found for most exposures investigated in specific studies. In conclusion, these data, which confirm that DNA damage measured by the comet assay is an excellent biomarker of exposure in several conditions, may contribute to improving the quality of study design and to the standardization of results of the comet assay in humans populations.