Melanin photoprotection in the human retinal pigment epithelium and its correlation with light-induced cell apoptosis

Abstract

Time-resolved electron paramagnetic resonance (TREPR) spectroscopy was used to study melanin free radicals in human retinal pigment epithelium (RPE) cells and tyrosine-derived synthetic melanin. TREPR signal traces from RPE cells reveal in vivo light-induced melanin free radical photochemistry in more detail than previously known. Electron spin polarization reflecting a non-Boltzmann population within the energy levels of the spin system is observed in RPE cells as the result of the triplet state photoproduction and subsequent disappearance of free radicals in the melanin polymer. In a set of RPE cells cultured from individual sources, differences in optical absorption, continuous wave EPR spectra, and TREPR signals were correlated with apoptosis assays performed by flow cytometry. Continuous wave EPR spectra of RPE cells and TREPR of acidified synthetic melanin suggest that increased melanin aggregation provides an increase in photoprotection in the RPE cells that are relatively less susceptible to blue light-induced apoptosis.continuous wave EPR ͉ electron spin polarization ͉ time-resolved EPR I n the eye, retinal pigment epithelium (RPE) is a monolayer of cuboidal cells between the photoreceptors and choriocapillaris of the eye and is specialized to uptake, phagocytize, and recycle the outer segment of photoreceptors and retinaldehyde, the chromophore of rhodopsin (1). RPE cell death plays a major role in the pathogenesis of age-related macular degeneration, the leading cause of blindness in the population Ͼ60 years of age in the developed world (2). Possible reasons given for the degeneration of RPE cells are their exposure to high oxidative stress and damaging irradiation (2-6).The pigment melanin, found in RPE cells, is a heterogeneous polymer consisting of various monomers believed to be oxidation products of dopa (dihydroxyphenylalinine) derived from tyrosine (7). Melanin contains intrinsic, indolesemiquinone-like doublet-state radicals (8, 9). Extrinsic indolesemiquinone-like radicals are reversibly photogenerated under visible or UV irradiation (9). RPE melanin serves a photoprotective role by absorbing radiation and scavenging free radicals and reactive oxygen species (ROS) (8-11). Evidence also exists for a phototoxic role for melanin in RPE cells, especially in aged cells, including measurable ROS photoproduction (8,(12)(13)(14)(15)(16)(17)(18). Electron paramagnetic resonance (EPR) spectroscopy enables a sensitive and nondestructive analysis and differentiation of natural melanin (9,19,20). The photophysical, redox, and aggregation properties of melanin as well as its ability to bind metals, proteins, and drugs have stimulated numerous studies in chemical, physical, and medical research (8, 9). The diversity and interrelations of its properties and the lack of chemical structures have hampered a clear understanding of the roles of melanin in RPE cells (8,9). Nanosecond time-resolved (TR) EPR studies have not been reported despite the relevance of TREPR to the study of melanin photochemistry (9). El...

Melatonin and amfenac modulate calcium entry, apoptosis, and oxidative stress in ARPE-19 cell culture exposed to blue light irradiation (405 nm)

“…Blue light has been shown to induce production of reactive oxygen species (ROS) in RPE cells, 7 triggering apoptosis. 8 The indole melatonin (N-acetyl-5-methoxytryptoamine) is a neurohormone that has crucial roles in the regulation of many physiological events. 9 It is a highly lipophilic molecule that can easily pass through the cell membranes and reach subcellular compartments.…”

Photoprotection of human retinal pigment epithelium cells against blue light-induced apoptosis by melanin free radicals from Sepia officinalis

“…Metabolically, RPE cells are highly active and contain large numbers of mitochondria (1). Blue light induces the production of reactive oxygen species (ROS) in the mitochondria of RPE cells (15) and has been shown to lead to cell apoptosis (14), potentially triggered by ROS damage to mtDNA (16). It has also been suggested that the accumulation of ROS-induced damage during one's lifetime contributes not just to RPE loss but also to the overall aging process (17).…”

“…Blue light is especially harmful (9) and produces light-induced apoptosis of rat RPE cells by a free radical mechanism (10). Although lipofuscin involvement in blue light-induced RPE loss could be important in aged RPE (11)(12)(13), fetal RPE are also susceptible to blue light-induced apoptosis (BLIA) (14). Metabolically, RPE cells are highly active and contain large numbers of mitochondria (1).…”

“…Melanin monomers are cross-linked into planar oligomers of Ϸ4-6 monomers, and several planar oligomers are assembled into successively larger aggregates by -stacking and van der Waals interactions (26)(27)(28)(29). These -stacked aggregates of indolequinone-like radicals may be ideal for free radical migration (14). Atomic force microscopy and scanning electron microscopy have been successfully used to visualize melanin ultrastructure, revealing that aggregates of native RPE eumelanin are composed of regular 150-nm substructures, which are themselves composed of Ϸ10-nm constituent aggregates (28)(29)(30).…”

“…However, it is also believed that RPE melanin serves a protective role in vision by absorbing light and scavenging free radicals and ROS (12,18,(46)(47)(48). We recently used TREPR to account for the ability of RPE melanin to serve as an efficient light absorber (14) and to directly detect melanin free radicals scavenging ROS in RPE cells (48).…”