title: Development of a DNA-binding TEMPO derivative for evaluation of nuclear oxidative stress and its application in living cells.
abstract: Oxidative stress in nuclei is known to induce either oxidative modification of DNA bases or single/double-strand breaks, which may lead to carcinogenesis. To evaluate the redox status in nuclei in living cells, we designed a novel nucleus-localizing redox spin probe, F-DisT, which contains a fluorescein fluorophore linked to a DNA minor-groove-binding moiety. Nuclear distribution of the probe was easily confirmed by colocalization with a nuclear stain, Hoechst 33342, in confocal microscopy. Measurement of oxidative stress with F-DisT in a murine macrophage cell line exposed to endotoxin (lipopolysaccharide) showed a remarkable increase in the ESR signal decay rate. This increase was significantly inhibited by N(ω)-nitro-l-arginine (nitric oxide synthase inhibitor) and diphenyleneiodonium chloride (NADPH oxidase inhibitor). These results indicate that nitric oxide and superoxide contribute to oxidative stress in nuclei. Similar studies in membrane or mitochondria using respective organelle-specific spin probes indicated that the redox microenvironments in these organelles are markedly different from that in nuclei. Thus, subcellular redox microenvironments show marked variability in endotoxin-stimulated living cells.
authors.affiliation: Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Aichi, Japan.
authors.forename: Mamiko
authors.initials: M
authors.lastname: Ikeda
authors.forename: Hidehiko
authors.initials: H
authors.lastname: Nakagawa
authors.forename: Shizuka
authors.initials: S
authors.lastname: Ban
authors.forename: Hiroki
authors.initials: H
authors.lastname: Tsumoto
authors.forename: Takayoshi
authors.initials: T
authors.lastname: Suzuki
authors.forename: Naoki
authors.initials: N
authors.lastname: Miyata
mesh_terms: D000818:Animals; D001562:Benzimidazoles / Q000493:pharmacokinetics; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002477:Cells / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D003497:Cyclic N-Oxides / Q000737:chemistry / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D051379:Mice; D008954:Models, Biological; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology; D013113:Spin Labels / Q000138:chemical synthesis; D014018:Tissue Distribution
chemical_list: D001562:Benzimidazoles; D003497:Cyclic N-Oxides; D005456:Fluorescent Dyes; D013113:Spin Labels; D004247:DNA; C017807:bisbenzimide ethoxide trihydrochloride; C003959:TEMPO