Photobleaching of melanosomes from retinal pigment epithelium: I. Effects on protein oxidation

Melanin in the long-lived melanosomes of the retinal pigment epithelium (RPE) may undergo photobleaching with aging, which appears to diminish the antioxidant function of melanin and could make photobleached melanosomes less efficient in protecting biomolecules from oxidative modification. Here we analyzed whether photobleaching of melanosomes affects their ability to modify the oxidation state of nearby protein. As conventional methods developed to study soluble antioxidants are not well suited for analysis of granules such as melanosomes, we developed a new analytic method to focus on particle surfaces that involves experimentally coating granules with the cytoskeletal protein beta-actin to serve as a reporter for local protein oxidation. Isolated porcine RPE melanosomes were photobleached with visible light to simulate aging, then photobleached melanosomes, untreated melanosomes and control particles (black latex beads) were actin coated and illuminated in a photosensitized cell free system. Protein was re-stripped from particles and analyzed for carbonylation by Western blotting. Quantitative densitometry showed no reproducible differences for protein associated with untreated melanosomes when compared with control particles. Melanin has both anti- and pro-oxidant functions when light irradiated, but neither of these functions predominated in the protein oxidation assay when untreated melanosomes were used. However, protein extracted from photobleached melanosomes showed markedly increased carbonylation, both of associated actin and of endogenous melanosomal protein(s), and the effect increased with extent of granule photobleaching. Photobleaching of RPE melanosomes therefore changes the oxidation state of protein endogenous to the organelle and reduces the ability of the granule to modify the oxidation of exogenous protein near the particle surface. The results support the growing body of evidence that photobleaching of RPE melanosomes, which is believed to occur with aging, changes the physicochemical properties of the organelle and reduces the likelihood that the granules perform an antioxidant function.     

Photobleaching of melanosomes from retinal pigment epithelium: II. Effects on the response of living cells to photic stress

Melanosomes of the retinal pigment epithelium (RPE) are long lived organelles that may undergo photobleaching with aging, which can diminish the antioxidant efficiency of melanin. Here, isolated porcine RPE melanosomes were experimentally photobleached with visible light to simulate aging and compared with untreated granules or control particles (black latex beads) for their effects on the survival of photically stressed ARPE-19 cultures. Particles were delivered to cultures for uptake by phagocytosis then cells were exposed to violet light and analyzed by a new live cell imaging method to identify the time of apoptotic blebbing as a dynamic measure of reduced cell survival. Results indicated that untreated melanosomes did not decrease photic injury to ARPE-19 cells when compared with cells lacking particles or with cells containing control particles, as might be expected if melanin performed an antioxidant function. Instead cells with untreated melanosomes showed reduced survival indicated by an earlier onset of blebbing and a lower fraction of surviving cells after photic stress. Cell survival was reduced even further in stressed cells containing melanosomes that were photobleached, and survival decreased with increasing photobleaching time. Photobleaching of RPE melanosomes therefore makes cells containing them more sensitive to light-induced cytotoxicity. This observation raises the possibility that aged melanosomes increase RPE cell photic stress in situ, perhaps contributing to reduced tissue function and to degeneration of the adjacent retina that the RPE supports. How melanosomes (photobleached or not) interact with their local subcellular environment to modify RPE cell survival is poorly understood and is likely determined by the physicochemical state of the granule and its constituent melanin. The live cell imaging method introduced here, which permitted detection of a graded effect of photobleaching, provides a sensitive bioassay for probing the effects of melanosome modifications.     

Effect of UV radiation and hydrogen peroxide on the antiradical and antioxidant activities of DOPA-melanin and melanosomes from retinal pigment epithelial cells

The effect of continuous UV radiation and hydrogen peroxide on destruction and antioxidant properties of synthetic DOPA-melanin (prepared by oxidation of 3,4-dihydroxyphenylalanine (DOPA)) and melanosomes isolated from cells of the retinal pigment epithelium (RPE) was investigated. The kinetics of melanin destruction was recorded based on the accumulation of fluorescent low-molecular-weight reaction products, the antiradical activity of melanin was determined by chemiluminescence method, the concentration of free radical products was measured by electron paramagnetic resonance, and the antioxidant activity of melanins was estimated by their inhibitory effect on lipid peroxidation. It was shown that UVC—UVA irradiation (up to 5 hours) of DOPA-melanin and melanosomes of retinal pigment epithelium decreased neither the latency period of luminol chemiluminescence nor the inhibitory action of pigments on Fe²⁺- and UV-induced peroxidation of cardiolipin liposomes. However, very long UV irradiation gave rise to fluorescent destruction products, decreased the concentration of paramagnetic centers in the pigment (especially light-dependent ones), and decreased the antiradical and antioxidant activities. For example, UV irradiation of DOPA-melanin during 52 h resulted in approximately a 2-fold decrease in the concentration of paramagnetic centers and decline of antiradical and antioxidant activities. However, even with such a hard irradiation the pigment retained significant inhibitory activity against lipid peroxidation. The oxidative destruction of DOPA-melanin in the presence of hydrogen peroxide in the dark resulted in complete destruction of the polymer and loss of its protective properties. It is assumed that destruction of RPE cell melanin is caused mainly by oxidative processes.     

The phototoxicity of aged human retinal melanosomes

The purpose of this study was to determine whether an age-related increase in photoreactivity of human retinal melanosomes (MS) can cause phototoxicity to retinal pigment epithelium (RPE) cells. MS were isolated post mortem from young (20-30 years, young human melanosomes [YHMs]) and old (60-90 years, old human melanosomes [OHMs]) human eyes and from young bovine eyes (bovine melanosomes [BMs]). Confluent cultured ARPE-19 cells were fed equivalent numbers of OHMs or BMs and accumulated similar amounts of melanin as determined by electron paramagnetic resonance assay. Cells with and without MS were either maintained in the dark or exposed to blue light for up to 96 h and assessed for alterations in cell morphology, cell viability and lysosomal integrity. Incubation of cells in dark in the presence of internalized MS or irradiation of cells with blue light in the absence or presence of BMs did not significantly affect cell viability. However, exposures to blue light in the presence of OHMs resulted in abnormal cell morphology, up to approximately 75% decrease in mitochondrial activity, loss of lysosomal pH and cell death. OHMs contained significantly less melanin than YHMs, supporting the hypothesis that melanin undergoes degradation during RPE aging. Our results demonstrate that aged MS can be phototoxic to human RPE cells and support a contributing role of MS in RPE aging and in the pathogenesis of age-related macular degeneration.     

Amount of Melanin Granules in Human Hair Defines the Absorption and Conversion to Heat of Light Energy in the Visible Spectrum

One of the known important functions of hair is protection from extensive sunlight. This protection is accomplished in large part due to natural hair pigmentation which is known to reflect the number of melanin granules (melanosomes) in the hair shaft, and melanin variants. Melanin takes in excessive light energy and converts it to heat in a process called absorption; heat is then dissipated into the environment as infrared radiation, thereby protecting the underlying skin. We used transmission electron microscopy (TEM) to visualize the melanosome counts in samples of human hair, and used thermal microscopy to measure the temperature changes of the samples when exposed to green and blue light lasers. In our experiments green light conversion to heat was highly correlated to the number of melanosomes, whereas blue light conversion to heat was less correlated, which may be because the reddish melanosomes it contains are less effective in absorbing energy from the blue spectrum of light. Anyway, we have shown the metals accumulation in the melanin can be easily visualized with TEM. We confirmed that the amount of melanin granules in human hair defines the conversion to heat of light energy in the visible spectrum.     

纳米管钛酸的ESR特性及其可见光照的影响

本文报道了纳米管钛酸在真空-0.1MPa、温度100℃的条件下,经过不同时间处理后的ESR特性及其可见光照的影响.发现纳米管钛酸经一定处理后,不经光照即出现g=2.003ESR信号,该信号是由捕获一个电子的氧空位(V_o)产生的,此信号随着处理时间的延长而增强;在532nm的可见光照射下,随着光照时间的延长信号强度随之增加,达到一定强度值后,不再随光照时间的延长而增加;光源关闭后,信号强度又逐渐减小,但不能恢复到原来信号强度的水平.     

Time-resolved detection of melanin free radicals quenching reactive oxygen species

Melanin, a ubiquitous, heterogeneous biological polymer composed of many different monomers, contains a population of stationary, intrinsic semiquinone-like radicals. Additional extrinsic semiquinone-like radicals are reversibly photogenerated with visible or UV irradiation. The free radical chemistry of melanin is complex and not well characterized, especially the photochemistry of melanin in the presence of oxygen. To determine directly how melanin reacts in the presence of oxygen, time-resolved electron paramagnetic resonance (TREPR) spectroscopy was used to examine melanin free radical chemistry in human retinal pigment epithelium (RPE) cells under aerobic and anaerobic conditions. A TREPR difference spectrum was used to explore the nature of melanin chemistry in the presence of oxygen. The position and symmetrical line shape of the TREPR three-dimensional difference spectrum shows that when reactive oxygen species (ROS) are scavenged, only one of the two or more chemically different melanin free radical species participates in ROS scavenging. This protective melanin radical species exists in both the extrinsic and intrinsic populations of melanin free radicals, allowing melanin to protect the RPE from toxic species in both the light and dark.     

Comparisons of the structural and chemical properties of melanosomes isolated from retinal pigment epithelium, iris and choroid of newborn and mature bovine eyes

Melanosomes were isolated from the retinal pigment epithelium (RPE), iris and choroid of mature (age >2 years) and newborn (age <1 week) bovine eyes. Scanning electron microscopy was utilized to analyze the morphology of the melanosomes, which were found to vary among different tissues and different ages. While the total content of amino acids differs slightly (ranging from 9% to 15% by mass), the distributions of the amino acids are similar. The pheomelanin content is low in the choroid and the RPE (0.1-0.5%), and moderate in the iris (<2%); therefore, the major melanin component of bovine eye melanosomes is eumelanin, independent of the shape of the melanosomes. The yields of pyrrole-2,3,5-tricarboxylic acid from melanosomes decrease in the following order: choroid > iris > RPE, and exhibit decreasing yields with age. 13C solid-state nuclear magnetic resonance (NMR) spectroscopic analysis of iris and choroid melanosomes indicates the same trends. These observations suggest that the 5,6-dihydroxyindole-2-carboxylic acid contents decrease in the following order: choroid > iris > RPE, and decrease with age. Moreover, the 13C solid-state NMR spectra show (1) for the same age samples, the CH:Cq ratio for choroid is larger than that for iris melanosomes; and (2) an increase in the concentration of carbonyl groups with age within each type of melanosome.     

The pro-oxidant effects of interactions of ascorbate with photoexcited melanin fade away with aging of the retina

Photoexcited melanin from retinal pigment epithelium (RPE) has been shown to induce photo-oxidation of ascorbate with concomitant generation of hydrogen peroxide. The aim of this study was to test whether the age-related changes in melanin content and distribution in the RPE affect the susceptibility of RPE cells to ascorbate-mediated photo-oxidation. Our results demonstrate that there is an age-dependent shift in the pathways with which ascorbate interacts in human RPE. In young RPE, melanin-ascorbate interactions may lead to pro-oxidant effects, but in the aged there is no net increase in photo-oxidation in the presence of ascorbate in comparison with samples without ascorbate. However, as ascorbate undergoes light-induced depletion and photogenerates ascorbyl free radical in the old RPE cells with initial yields similar to that observed for young RPE, an influence of ascorbate on oxidation pathways is revealed in the old RPE as well. Interestingly, the pro-oxidant effects of photoexcited melanolipofuscin-ascorbate interactions are greater than for photoexcited melanosomes when normalized to the same melanin content. The pro-oxidant effects of photoexcited melanin-ascorbate interactions are strongly dependent on the irradiation wavelength, this being the greatest for the shortest wavelength studied (340 nm) and steeply decreasing with increasing wavelength but still detectable even at 600 nm.     

ZnO-B2O3∶Tb3+长余辉玻璃的发光性质

通过还原方法制备了Tb³⁺离子掺杂的硼酸锌玻璃，并观察到在254 nm紫外光激发后有明亮的绿色长余辉发光现象，余辉时间达6 h。通过激发与发射光谱、余辉光谱、余辉衰减曲线、热释光谱、热释光释出速率衰减曲线等得到的信息，研究了Tb³⁺离子掺杂的硼酸锌玻璃的发光性质。     

Properties and function of the ocular melanin--a photobiophysical view.

This paper reviews the biosynthesis and physicochemical properties of the ocular melanin. Age-related changes of melanin granules and the corresponding formation of lipofuscin pigments in the retinal pigment epithelium (RPE) are also described. Adverse photoreactions of the eye and, in particular, light-induced damage to the RPE-retina are reviewed in relation to the ocular pigmentation. A hypothesis on the photoprotective role of the RPE melanin is presented that is based on the ability of the cellular melanin to bind redoxactive metal ions. Since bound-to-melanin metal ions are expected to be less damaging to the pigment cells, it is proposed that sequestration of heavy metal ions by the RPE melanin is an efficient detoxifying mechanism. It is postulated that oxidative degradation of RPE melanin may lower its metal-binding capability and decrease its anti-oxidant efficiency. Cellular and environmental factors that may contribute to possible oxidative damage of the RPE melanin are discussed in connection with the etiology of age-related macular degeneration.     

Melanosomal damage in normal human melanocytes induced by UVB and metal uptake--a basis for the pro-oxidant state of melanoma

Melanins are ubiquitous catecholic pigments, formed in organelles called melanosomes within melanocytes, the function of which is to protect skin against harmful effects of UV radiation. Melanosomes within melanoma cells are characteristically abnormal, with fragmented melanin and disrupted membranes. We hypothesize that the disruption of melanosomal melanin might be an early event in the etiology and progression of melanoma, leading to increased oxidative stress and mutation. In this report, we examine the effect of a combination of UV treatment and metal ion exposure on melanosomes within melanocytes, as well as their ability to act as pro-oxidants in ex situ experiments, and assay the effects of this treatment on viability and cell cycle progression. UVB exposure causes morphologic changes of the cells and bleaching of melanosomes in normal melanocytes, both significantly enhanced in Cu(II) and Cd(II)-treated cells, as observed by microscopy. The promoted bleaching by Cu(II) is due to its ability to redox cycle under oxidative conditions, generating reactive oxygen species; verified by the observed enhancement of hydroxyl radical generation when isolated melanosomes were treated with both Cu(II) ions and UVB, as assayed by DNA clipping. Single-dose UVB/Cu treatment does not greatly affect cell viability or cell cycle progression in heavily pigmented cells, but did so in an amelanotic early stage melanoma cell line.     

OXIDATION OF ASCORBIC ACID AS AN INDICATOR OF PHOTOOXIDATIVE STRESS IN THE EYE

When whole retinal pigmented epithelium (RPE) cells isolated from bovine eyes are incubated with 14C-labeled ascorbic acid and exposed to a visible laser, the ascorbic acid is oxidized to dehydro-L-ascorbic acid (DHA). The amount of ascorbic acid which is oxidized is proportional to the radiant exposure of the sample (i.e. the total amount of radiation per unit area delivered over the exposure time). Blue light is more effective than red light in driving the reaction. The amount of label appearing in the DHA fraction is increased if unlabeled DHA is present in the reaction mixture, indicating that some redox cycling of ascorbate is occurring in the RPE cells. The ascorbic acid oxidizing activity does not depend on intact cells, is not inactivated by heating the cells to 80 degrees C, and appears to reside mainly in the subcellular fraction which contains melanin pigment granules. The ascorbic acid oxidation may be caused by free radicals formed when melanin is illuminated with light. This reaction appears to be a useful method for quantifying the production of free radicals during photooxidative stress.     

Direct Measurement of the Ultraviolet Absorption Coefficient of Single Retinal Melanosomes

A novel approach to photoemission electron microscopy is used to enable the first direct measurement of the absorption coefficient from intact melanosomes isolated from bovine retinal pigment epithelial cells. The difference in absorption between newborn and adult melanosomes is in good agreement with that predicted from the relative amounts of the monomeric precursors present in the constituent melanin as determined by chemical degradation analyses. The results demonstrate that for melanosomes containing eumelanins, there is a direct relation between the absorption coefficient and the relative 5,6-dihydroxyindole: 5,6-dihydroxyindole-2-carboxylic acid (DHI:DHICA) content, with an increased UV absorption coefficient associated with increasing DHICA content.     

The role of melanin as protector against free radicals in skin and its role as free radical indicator in hair

Throughout the body, melanin is a homogenous biological polymer containing a population of intrinsic, semiquinone-like radicals. Additional extrinsic free radicals are reversibly photo-generated by UV and visible light. Melanin photochemistry, particularly the formation and decay of extrinsic radicals, has been the subject of numerous electron spin resonance (ESR) spectroscopy studies. Several melanin monomers exist, and the predominant monomer in a melanin polymer depends on its location within an organism. In skin and hair, melanin differs in content of eumelanin or pheomelanin. Its bioradical character and its susceptibility to UV irradiation makes melanin an excellent indicator for UV-related processes in both skin and hair. The existence of melanin in skin is strongly correlated with the prevention against free radicals/ROS generated by UV radiation. Especially in the skin melanin (mainly eumelanin) ensures the only natural UV protection by eliminating the generated free radicals/ROS. Melanin in hair can be used as a free radical detector for evaluating the efficacy of hair care products. The aim of this study was to investigate the suitability of melanin as protector of skin against UV generated free radicals and as free radical indicator in hair.     

Characterization of Retinal Pigment Epithelial Melanin and Degraded Synthetic Melanin Using Mass Spectrometry and In Vitro Biochemical Diagnostics

With increasing age, there is an observable loss of melanin in retinal pigment epithelial (RPE) cells. It is possible that degradation of the pigment contributes to the pathogenesis of retinal disease, as the cellular antioxidant material is depleted. Functionally, intact melanin maintains protective qualities, while oxidative degradation of melanin promotes reactive oxygen species (ROS) generation and formation of metabolic byproducts, such as melanolipofuscin. Understanding the structural and functional changes to RPE melanin with increasing age may contribute to a better understanding of disease progression and risk factors for conditions such as age‐related macular degeneration (AMD). In this study, human donor RPE melanin is characterized using MALDI mass spectrometry to follow melanin degradation trends. In vitro models using ARPE‐19 cells are used to assess photo‐reactivity in repigmented cells. Significant protection against intracellular ROS produced by blue light is observed in calf melanin‐pigmented cells versus unpigmented and black latex bead controls (P < 0.0001). UV‐B exposure to aged human melanin‐pigmented cells results in a significant increase in nitric oxide production versus control cells (P < 0.001). Peroxide‐treated synthetic melanin is characterized to elucidate degradation products that may contribute to RPE cell damage.     

Blue light irradiation inhibits the production of HGF by human retinal pigment epithelium cells in vitro

Blue visible light damage to retinal pigment epithelial cells occurs through a photooxidative mechanism and the resultant damage is hypothesized to induce or exacerbate age-related macular degeneration. The purpose of the present study was to identify changes in the cell growth and the expression of hepatocyte growth factor (HGF) in cultured human retinal pigment epithelium (RPE) cells as a result of both blue and red light irradiation. HGF is a growth factor and neurotrophic factor that stimulates growth of various ocular cells and promotes the survival of RPE and retinal neurons. Early passages of human RPE cells were exposed to blue light (460 nm) and red light (640 nm). Nonirradiated cells were used as controls. After 24 and 48 h, conditioned medium was collected and the amount of HGF was measured by ELISA. Cells were detached from the well and counted. Cell viability was evaluated by trypan-blue exclusion study. Blue light at dosage of 63 J/cm(2) significantly inhibited the growth of RPE cells without affecting of cell viability. Amounts of HGF in the culture medium were significantly inhibited by blue-light irradiation at the dosage from 32 to 63 J/cm(2). Red light at a dose of 174 J/cm(2) causes a nonsignificant inhibition of growth of RPE cells and a slight decrease of secretion of HGF. As HGF promotes survival of RPE cells and retinal neurons, the inhibition of production of HGF by visible light, especially by blue light, may enhance the phototoxic effects of visible light on the RPE and retinal neurons.     

Comparable Photoreactivity of Hair Melanosomes,Eu‐ and Pheomelanins at Low Concentrations: Low Melanin a Risk Factor for UVA Damage and Melanoma?†

Melanin is known to be photoreactive and photoprotective, but its function in skin in vivo is still debated. Data is lacking of the effects of UVA irradiation on human skin melanosomes of different pigmentation, which have not been extensively degraded by isolation procedures. We have shown previously that melanosomes isolated from human oriental and black cat hair, and synthetic eumelanins, are photoreactive producing superoxide at low concentrations when exposed to UVA irradiation comparable to UK levels of sunlight. Here we investigated the UVA-irradiation of melanosomes, isolated from different colored human hair samples, using electron spin resonance spectroscopy and spin trapping. Comparable irradiation of synthetic pheomelanins synthesized from L-dopa and L-cysteine was also studied. An alkali method (5 min NaOH at 90 degrees C) could be used to isolate oriental hair melanosomes but was not suitable for auburn and blonde hair. Dithiothreitol and proteinase K resulted in melanin release from possible over-digestion of melanosomes; however, dithiothreitol and papain resulted in no melanin release and good melanosome yields with separation from residual keratin for brown, auburn and blonde hair. Melanosomes isolated by the latter method and synthetic pheomelanins were similar in UVA-photoreactivity at low concentrations, independent of hair color, and broadly comparable to synthetic melanins. Melanosome concentration at constant fluence may be more significant with respect to photodamage and UVA photocarcinogenesis (melanoma) via superoxide radical production than pigment type.     

Antioxidant properties of melanin in retinal pigment epithelial cells

The retinal pigment epithelium (RPE) is a monolayer of highly pigmented cells lining the inner aspect of Bruch's membrane. This pigmentation is due to eumelanin and a possible antioxidant role of melanin is reported here. The photo-oxidation of A2E, a constituent of RPE lipofuscin, leads to the sequential addition of up to nine oxygen atoms and/or the addition or loss of two hydrogen atoms. These photo-oxidations were investigated in the presence and absence of either calf or human RPE melanin in A2E-laden RPE cells. It was found that calf melanin was protective against the photo-oxidation of A2E, with an inhibition of oxidation of up to 50% in the case of the addition of two oxygen atoms. Calf melanin was also protective against blue light-induced damage to RPE cells. In addition this ability appears to decrease in humans as they grow older. With aging, a melanin-lipofuscin complex called melanolipofuscin forms. It is suggested that the oxidation or photo-oxidation of A2E in vivo may contribute to the age-related deterioration of the anti-oxidant role of RPE melanin and lead to various retinal disorders, such as age-related macular degeneration.     

Retinal damage by light in the golden hamster: an ultrastructural study in the retinal pigment epithelium and Bruch's membrane.

The mechanism of the toxicity of light on the retina remains unclear despite a large number of investigations. The purpose of this study is to identify and localize the ultrastructural changes and the site of the earliest damage after intense light exposure. Nine adult Syrian golden hamsters (Mesocricetus auratus) have been maintained under constant illumination with a high-pressure mercury lamp (HQJ R 80 W Deluxe, Osram, Berlin, light intensity 1000 lx) for 12 h, followed by an additional 3 h in the dark. Light damage is assessed by light and electron microscopy. Morphological evaluation reveals focal damage to the retinal pigment epithelial (RPE) cells in close proximity to less-affected RPE cells and normal photoreceptors. Collagen fibers in Bruch's membrane lose their parallel orientation. Occasionally, fusion of cell membranes of neighboring rod outer segments (ROS) is also observed. Continuous, 12 h exposure of hamsters to intense light results in initial focal damage to some RPE cells, such that severely damaged RPE cells are found adjacent to intact RPE cells. Only slight damage to the photoreceptors is evident, suggesting that the sequence of the pathological changes resulting from light begins with damage to the RPE cells and associated Bruch's membrane.