title: Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation.
abstract: Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high radiation doses. Addressing questions related to environmental, occupational and public health safety in a proper and scientifically justified manner heavily relies on the ability to accurately measure the biological effects of low dose pollutants, such as ionizing radiation and chemical substances. DNA damage and repair are the most important early indicators of health risks due to their potential long term consequences, such as cancer. Here we describe a protocol to study the effect of chronic in vivo exposure to low doses of γ- and β-radiation on DNA damage and repair in mouse spleen cells. Using a commonly accepted marker of DNA double-strand breaks, phosphorylated histone H2AX called γH2AX, we demonstrate how it can be used to evaluate not only the levels of DNA damage, but also changes in the DNA repair capacity potentially produced by low dose in vivo exposures. Flow cytometry allows fast, accurate and reliable measurement of immunofluorescently labeled γH2AX in a large number of samples. DNA double-strand break repair can be evaluated by exposing extracted splenocytes to a challenging dose of 2 Gy to produce a sufficient number of DNA breaks to trigger repair and by measuring the induced (1 hr post-irradiation) and residual DNA damage (24 hrs post-irradiation). Residual DNA damage would be indicative of incomplete repair and the risk of long-term genomic instability and cancer. Combined with other assays and end-points that can easily be measured in such in vivo studies (e.g., chromosomal aberrations, micronuclei frequencies in bone marrow reticulocytes, gene expression, etc.), this approach allows an accurate and contextual evaluation of the biological effects of low level stressors.
authors.affiliation: Radiological Protection Research and Instrumentation, Canadian Nuclear Laboratories.
authors.forename: Matthew
authors.initials: M
authors.lastname: Flegal
authors.affiliation: Radiological Protection Research and Instrumentation, Canadian Nuclear Laboratories.
authors.forename: Melinda S
authors.initials: MS
authors.lastname: Blimkie
authors.affiliation: Radiological Protection Research and Instrumentation, Canadian Nuclear Laboratories.
authors.forename: Heather
authors.initials: H
authors.lastname: Wyatt
authors.affiliation: Radiological Protection Research and Instrumentation, Canadian Nuclear Laboratories.
authors.forename: Michelle
authors.initials: M
authors.lastname: Bugden
authors.affiliation: Radiological Protection Research and Instrumentation, Canadian Nuclear Laboratories.
authors.forename: Joel
authors.initials: J
authors.lastname: Surette
authors.affiliation: Radiological Protection Research and Instrumentation, Canadian Nuclear Laboratories; Dmitry.Klokov@cnl.ca.
authors.forename: Dmitry
authors.initials: D
authors.lastname: Klokov
mesh_terms: D000818:Animals; D001610:Beta Particles; D001853:Bone Marrow; D004247:DNA / Q000528:radiation effects*; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair / Q000528:radiation effects*; D005434:Flow Cytometry / Q000379:methods*; D005720:Gamma Rays; D006657:Histones / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D010766:Phosphorylation / Q000528:radiation effects; D011897:Random Allocation
chemical_list: C468783:H2AX protein, human; D006657:Histones; D004247:DNA