title: Photoaging of retinal pigment epithelial melanosomes: The effect of photobleaching on morphology and reactivity of the pigment granules.
abstract: To elucidate the mechanism of age-related changes in antioxidant and photoprotective properties of human retinal pigment epithelium (RPE) melanosomes, the effect of in vitro photoaging of bovine RPE melanosomes was examined employing an array of complementary spectroscopic and analytical methods. Electron paramagnetic resonance (EPR) spectroscopy, saturation recovery EPR, atomic force microscopy (AFM) and dynamic light scattering (DLS) were used to determine melanin content of control and photobleached melanosomes, and to monitor changes in their morphology. Methylene blue (MB), TEMPO choline, dysprosium(III) ions and singlet oxygen were employed as molecular probes to characterize the efficiency of control and photobleached melanosomes to interact with different reagents. EPR oximetry, UV-vis absorption spectroscopy, iodometric assay of lipid hydroperoxides and time-resolved singlet oxygen phosphorescence were used to analyze the efficiency of photobleached and untreated melanosomes to inhibit MB-photosensitized oxidation of liposomal lipids. The obtained results revealed that, compared to untreated melanosomes, moderately photobleached melanosomes protected unsaturated lipids less efficiently against photosensitized peroxidiation, while weakly photobleached melanosomes were actually better antioxidant and photoprotective agents. The observed changes could be attributed to two effects - modification of the melanosome morphology and oxidative degradation of the melanin functional groups induced by different degree of photobleaching. While the former increases the accessibility of melanin nanoaggregates to reagents, the latter reduces the efficiency of melanin to interact with chemical and physical agents.
authors.affiliation: Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland.
authors.forename: Andrzej
authors.initials: A
authors.lastname: Zadlo
authors.affiliation: Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland.
authors.forename: Grzegorz
authors.initials: G
authors.lastname: Szewczyk
authors.affiliation: Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland.
authors.forename: Michal
authors.initials: M
authors.lastname: Sarna
authors.affiliation: Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Kozinska
authors.affiliation: Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Pilat
authors.affiliation: Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland.
authors.forename: Patrycja
authors.initials: P
authors.lastname: Kaczara
authors.affiliation: Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland. Electronic address: tadeusz.sarna@uj.edu.pl.
authors.forename: Tadeusz
authors.initials: T
authors.lastname: Sarna
mesh_terms: D000818:Animals; D002417:Cattle; D015227:Lipid Peroxidation; D008543:Melanins / Q000378:metabolism; D020460:Melanosomes / Q000528:radiation effects / Q000648:ultrastructure*; D008751:Methylene Blue / Q000494:pharmacology; D010101:Oxygen Consumption; D038761:Photobleaching; D055213:Retinal Pigment Epithelium / Q000502:physiology / Q000528:radiation effects / Q000648:ultrastructure
chemical_list: D008543:Melanins; D008751:Methylene Blue
keywords: Melanin granules; Melanin nanoaggregates; Melanin photobleaching; Melanin radicals; Photoprotection; Photosensitized oxidation; Retinal pigment epithelium; Surface morphology