title: Oxygen-induced DNA damage in freshly isolated brain cells compared with cultured astrocytes in the Comet assay.
abstract: Brain cells are continuously exposed to reactive oxygen species generated by oxidative metabolism, and in certain pathological conditions defence mechanisms against oxygen radicals may be weakened and/or overwhelmed. DNA is a potential target for oxidative damage, and genomic damage can contribute to neuropathogenesis. It is important, therefore, to identify tools for the quantitative analysis of DNA damage in models of neurological disorders. The aim of this study was to compare the susceptibility of DNA to oxidative stress in cells freshly dissociated from the mouse brain, to that in cultured brain cells. Both primary cultures and a continuous cell line of astrocytes were considered. All cells were treated by xanthine/xanthine oxidase, a superoxide generator or hydrogen peroxide, applied alone or in the presence of the oxygen radical scavengers, superoxide dismutase, catalase, or ascorbic acid. DNA damage, quantified with the Comet assay, was consistent in all the different cell preparations exposed to oxidative stress, and was attenuated in similar ways by superoxide dismutase and catalase, scavengers of superoxide anion and hydrogen peroxide, respectively. The results with ascorbic acid were more variable, presumably because this compound may switch from anti- to pro-oxidant status depending on its concentration and other experimental conditions. Overall, similar responses were found in freshly dissociated and cultured brain cells. These results suggest that the Comet assay can be directly applied to cells freshly dissociated from the brain of rodents, including models of neurological disorders, such as stroke models and animals with targeted mutations that mimic human diseases.
authors.affiliation: Department of Biomedical Sciences, University of Bradford, Bradford, United Kingdom.
authors.forename: Eduardo
authors.initials: E
authors.lastname: Cemeli
authors.forename: Ian F
authors.initials: IF
authors.lastname: Smith
authors.forename: Chris
authors.initials: C
authors.lastname: Peers
authors.forename: Jutta
authors.initials: J
authors.lastname: Urenjak
authors.forename: Oleg V
authors.initials: OV
authors.lastname: Godukhin
authors.forename: Tihomir P
authors.initials: TP
authors.lastname: Obrenovitch
authors.forename: Diana
authors.initials: D
authors.lastname: Anderson
mesh_terms: D000818:Animals; D001205:Ascorbic Acid / Q000378:metabolism; D001253:Astrocytes / Q000187:drug effects*; D001921:Brain / Q000187:drug effects* / Q000378:metabolism; D002374:Catalase / Q000378:metabolism; D002478:Cells, Cultured; D020552:Comet Assay; D004247:DNA / Q000187:drug effects*; D004249:DNA Damage; D016166:Free Radical Scavengers / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018384:Oxidative Stress; D010100:Oxygen / Q000633:toxicity*; D051381:Rats; D017208:Rats, Wistar; D013482:Superoxide Dismutase / Q000378:metabolism; D013481:Superoxides / Q000494:pharmacology; D014969:Xanthine Oxidase / Q000378:metabolism; D014970:Xanthines / Q000378:metabolism
chemical_list: D016166:Free Radical Scavengers; D014970:Xanthines; D013481:Superoxides; D004247:DNA; D006861:Hydrogen Peroxide; D002374:Catalase; D013482:Superoxide Dismutase; D014969:Xanthine Oxidase; D001205:Ascorbic Acid; D010100:Oxygen