title: Studies on the photosensitized reduction of resorufin and implications for the detection of oxidative stress with Amplex Red.
abstract: The photosensitized reduction of resorufin (RSF), the fluorescent product of Amplex Red, was investigated using electron spin resonance (ESR), optical absorption/fluorescence, and oxygen consumption measurements. Anaerobic reaction of RSF in the presence of the electron donor reduced nicotinamide adenine dinucleotide (NADH) demonstrated that during visible light irradiation (λ > 300 nm), RSF underwent one-electron reduction to produce a semiquinoneimine-type anion radical (RSF(•) ‾) as demonstrated by direct ESR. Spin-trapping studies of incubations containing RSF, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and NADH demonstrated, under irradiation with visible light, the production of the superoxide dismutase (SOD)-sensitive DMPO/(•)OOH adduct. Both absorption and fluorescence spectra of RSF in the presence of NADH demonstrated that the RSF(•) ‾ was further reduced during irradiation with formation of its colorless dihydroquinoneimine form, dihydroresorufin (RSFH₂). Both RSF(•) ‾ and RSFH₂, when formed in an aerobic system, were immediately oxidized by oxygen, which regenerated the dye and formed superoxide. Oxygen consumption measurements with a Clark-type oxygen electrode showed that molecular oxygen was consumed in a light-dependent process. The suppression of oxygen consumption by addition of SOD or catalase further confirmed the production of superoxide and hydrogen peroxide.
authors.affiliation: Laboratory of Toxicology and Pharmacology, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC 27709, USA. zhaob2@niehs.nih.gov
authors.forename: Baozhong
authors.initials: B
authors.lastname: Zhao
authors.forename: Kalina
authors.initials: K
authors.lastname: Ranguelova
authors.forename: Jinjie
authors.initials: J
authors.lastname: Jiang
authors.forename: Ronald P
authors.initials: RP
authors.lastname: Mason
mesh_terms: D016227:Benzoquinones / Q000138:chemical synthesis; D003497:Cyclic N-Oxides / Q000378:metabolism; D005456:Fluorescent Dyes; D006861:Hydrogen Peroxide / Q000138:chemical synthesis; D008027:Light; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D010078:Oxazines / Q000138:chemical synthesis / Q000737:chemistry*; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D017319:Photosensitizing Agents / Q000737:chemistry*; D017382:Reactive Oxygen Species / Q000138:chemical synthesis*; D013194:Staining and Labeling; D013482:Superoxide Dismutase / Q000378:metabolism
chemical_list: D016227:Benzoquinones; D003497:Cyclic N-Oxides; D005456:Fluorescent Dyes; D010078:Oxazines; D017319:Photosensitizing Agents; D017382:Reactive Oxygen Species; C568681:dihydroresorufin; D009243:NAD; C470430:Amplex Red; C025232:semiquinone radicals; C014180:resorufin; C017245:5,5-dimethyl-1-pyrroline-1-oxide; D006861:Hydrogen Peroxide; D013482:Superoxide Dismutase