A newly adapted pulsed-field gel electrophoresis technique allows to detect distinct types of DNA damage at low frequencies in human dermal fibroblasts upon exposure to non-toxic H2O2 concentrations.
Reactive oxygen species (ROS) comprise several oxygen containing compounds, among them hydrogen peroxide (H2O2), which are generated by internal and external sources and play pleiotropic roles in physiological and pathological states. Skin cells as well as cells from other tissues have developed antioxidant defense mechanisms to protect themselves from high concentrations of ROS. Although biological and pathological roles of ROS have previously been elucidated, so far only limited knowledge exists regarding ROS-mediated generation of DNA breaks and base lesions occurring at low frequency in intact skin cells. This study was therefore designed to probe a newly adapted pulsed-field gel electrophoresis technique for the adequate measurement of high molecular weight DNA fragments as well as to investigate the protective role of the antioxidant enzyme catalase against H2O2-mediated damage in human dermal fibroblasts. We stably transfected and overexpressed the full-length catalase cDNA in the human dermal fibroblast cell line 1306 in culture and found that these cells are significantly more protected from cytotoxicity, overall DNA strand breaks, and 8-oxodeoxyguanine base lesions resulting from H2O2-triggered oxidative stress compared to vector-transfected 1306 cells or secondary dermal fibroblasts. This work has outlined the importance of catalase in the protection from H2O2-mediated cytotoxicity and DNA damage which--if unbalanced--even when occurring at low frequency are known to lead to genomic instability, a hallmark in carcinogenesis and premature aging.
Brenneisen
P
P
Department of Dermatology, University of Cologne, Germany.
Wenk
J
J
Wlaschek
M
M
Blaudschun
R
R
Scharffetter-Kochanek
K
K
D000080242:8-Hydroxy-2'-Deoxyguanosine; D016590:Aphidicolin / Q000494:pharmacology; D002374:Catalase / Q000235:genetics / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects; D002478:Cells, Cultured; D004249:DNA Damage; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000032:analysis; D004586:Electrophoresis / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000737:chemistry* / Q000187:drug effects* / Q000201:enzymology; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D006861:Hydrogen Peroxide / Q000008:administration & dosage / Q000494:pharmacology*; D012503:Saponins / Q000494:pharmacology; D012867:Skin / Q000166:cytology; D014162:Transfection
D004791:Enzyme Inhibitors; D012503:Saponins; D016590:Aphidicolin; D000080242:8-Hydroxy-2'-Deoxyguanosine; D006861:Hydrogen Peroxide; D002374:Catalase; D003849:Deoxyguanosine