title: γH2AX assay as DNA damage biomarker for human population studies: defining experimental conditions.
abstract: H2AX histone phosphorylation represents an early event in the cellular response against DNA double-strand breaks (DSBs), and plays a central role in sensing and repairing DNA damage. Therefore, the analysis of H2AX phosphorylated (γH2AX) may be possibly used as biomarker of genotoxicity and genomic instability with a number of applications in human epidemiology. However, the lack of an experimental standard leads to a wide heterogeneity in the results obtained and their interpretation, affecting the reliability of the assay. To address the most critical issues limiting the use of the γH2AX assay in human population studies, a flow cytometry analysis was performed to establish differences in γH2AX levels between fresh or cryopreserved peripheral blood lymphocytes, and to assess the influence of phytohemagglutinin (PHA) stimulation. To this purpose, cells were treated with 4 known genotoxic chemicals with different mechanisms of DSB induction, ie, bleomycin, methyl methanesulfonate, camptothecin, and actinomycin. According to our results, both unstimulated and stimulated fresh lymphocytes can be efficiently employed to evaluate γH2AX levels, but the sensitivity of the assay is depending upon the kind of damage observed. On the other hand, cryopreserved lymphocytes require PHA stimulation since unstimulated cells showed too high basal damage. Consequently, the protocol conditions will depend on the expected mechanism of production of DSB and the characteristics of the study design (sample collection and storage conditions, type of epidemiological study). Further studies are required to standardize the protocol of γH2AX assay to be employed as biomarker of genotoxicity or genomic instability in human population studies.
authors.affiliation: *DICOMOSA Group, Department of Psychology, Area of Psychobiology, Universidade da Coruña, Edificio de Servicios Centrales de Investigación, Campus Elviña s/n, 15071 Coruña, Spain and Unit of Clinical and Molecular Epidemiology, IRCCS San Raffaele Pisana, Via di Val Cannuta, 247, 00166 Rome, Italy.
authors.forename: María
authors.initials: M
authors.lastname: Sánchez-Flores
authors.affiliation: *DICOMOSA Group, Department of Psychology, Area of Psychobiology, Universidade da Coruña, Edificio de Servicios Centrales de Investigación, Campus Elviña s/n, 15071 Coruña, Spain and Unit of Clinical and Molecular Epidemiology, IRCCS San Raffaele Pisana, Via di Val Cannuta, 247, 00166 Rome, Italy.
authors.forename: Eduardo
authors.initials: E
authors.lastname: Pásaro
authors.affiliation: *DICOMOSA Group, Department of Psychology, Area of Psychobiology, Universidade da Coruña, Edificio de Servicios Centrales de Investigación, Campus Elviña s/n, 15071 Coruña, Spain and Unit of Clinical and Molecular Epidemiology, IRCCS San Raffaele Pisana, Via di Val Cannuta, 247, 00166 Rome, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Bonassi
authors.affiliation: *DICOMOSA Group, Department of Psychology, Area of Psychobiology, Universidade da Coruña, Edificio de Servicios Centrales de Investigación, Campus Elviña s/n, 15071 Coruña, Spain and Unit of Clinical and Molecular Epidemiology, IRCCS San Raffaele Pisana, Via di Val Cannuta, 247, 00166 Rome, Italy.
authors.forename: Blanca
authors.initials: B
authors.lastname: Laffon
authors.affiliation: *DICOMOSA Group, Department of Psychology, Area of Psychobiology, Universidade da Coruña, Edificio de Servicios Centrales de Investigación, Campus Elviña s/n, 15071 Coruña, Spain and Unit of Clinical and Molecular Epidemiology, IRCCS San Raffaele Pisana, Via di Val Cannuta, 247, 00166 Rome, Italy vvaldiglesias@udc.es.
authors.forename: Vanessa
authors.initials: V
authors.lastname: Valdiglesias
mesh_terms: D000328:Adult; D015415:Biomarkers / Q000378:metabolism*; D002478:Cells, Cultured; D004249:DNA Damage; D004781:Environmental Exposure; D004785:Environmental Pollutants / Q000633:toxicity*; D005260:Female; D005434:Flow Cytometry; D006657:Histones / Q000378:metabolism*; D006801:Humans; D008214:Lymphocytes / Q000187:drug effects / Q000378:metabolism
chemical_list: D015415:Biomarkers; D004785:Environmental Pollutants; C468783:H2AX protein, human; D006657:Histones
keywords: DNA damage response; genomic instability; genotoxicity; human population studies; γH2AX assay