Establishment of a non-radioactive cleavage assay to assess the DNA repair capacity towards oxidatively damaged DNA in subcellular and cellular systems and the impact of copper.
Oxidative stress is involved in many diseases, and the search for appropriate biomarkers is one major focus in molecular epidemiology. 8-Oxoguanine (8-oxoG), a potentially mutagenic DNA lesion, is considered to be a sensitive biomarker for oxidative stress. Another approach consists in assessing the repair capacity towards 8-oxoG, mediated predominantly by the human 8-oxoguanine DNA glycosylase 1 (hOGG1). With respect to the latter, during the last few years so-called cleavage assays have been described, investigating the incision of (32)P-labelled and 8-oxoG damaged oligonucleotides by cell extracts. Within the present study, a sensitive non-radioactive test system based on a Cy5-labelled oligonucleotide has been established. Sources of incision activity are isolated proteins or extracts prepared from cultured cells and peripheral blood mononuclear cells (PBMC). After comparing different oligonucleotide structures, a hairpin-like structure was selected which was not degraded by cell extracts. Applying this test system the impact of copper on the activity of isolated hOGG1 and on hOGG activity in A549 cells was examined, showing a distinct inhibition of the isolated protein at low copper concentration as compared to a modest inhibition of hOGG activity in cells at beginning cytotoxic concentrations. For investigating PBMC, all reaction conditions, including the amounts of oligonucleotide and cell extract as well as the reaction time have been optimized. The incision activities of PBMC protein extracts obtained from different donors have been investigated, and inter-individual differences have been observed. In summary, the established method is as sensitive and even faster than the radioactive technique, and additionally, offers the advantage of reduced costs and low health risk.
Hamann
Ingrit
I
Fachgebiet Lebensmittelchemie und Toxikologie, Institut für Lebensmitteltechnologie und Lebensmittelchemie, Technische Universität Berlin, Berlin, Germany.
Schwerdtle
Tanja
T
Hartwig
Andrea
A
D000080242:8-Hydroxy-2'-Deoxyguanosine; D000818:Animals; D003114:Colony-Forming Units Assay; D019327:Copper Sulfate / Q000494:pharmacology*; D004249:DNA Damage; D045647:DNA Glycosylases / Q000378:metabolism* / Q000502:physiology*; D004260:DNA Repair; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000378:metabolism; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism; D051379:Mice; D013347:Subcellular Fractions; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism
D000080242:8-Hydroxy-2'-Deoxyguanosine; D045647:DNA Glycosylases; C485990:Ogg1 protein, mouse; C479058:oxoguanine glycosylase 1, human; D003849:Deoxyguanosine; D019327:Copper Sulfate