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.     

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.     

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.     

Oxidation of A2E results in the formation of highly reactive aldehydes and ketones

It has been reported that the photo-oxidation of A2E, a component of human retinal lipofuscin, leads to products that are toxic to cells via dark reactions. Because these compounds have been implicated in the development of various maculopathies such as age-related macular degeneration (AMD), it is important to determine the structures of those deleterious compounds. Both the photo-oxidation and auto-oxidation of A2E lead to the same complex mixture of products, some of which have lower molecular weights than the staring material. Because A2E is homologous to beta-carotene, it was hypothesized that its oxidation would lead to products analogous to those found in oxidized beta-carotene, namely, a series of cleavage products along the acyclic chain with the concomitant formation of aldehydes. This was found to be the case based upon 1) the formation of all of the aldehydes predicted from the oxidation of beta-carotene, 2) the loss of 28 amu (carbonyl moiety) from the molecular ion, 3) the facile reaction of the aldehydes with nitrophenylhydrazines to form nitrophenylhydrazones and 4) the subsequent MS/MS cleavage of those derivatives at the N-N bond. If formed in vivo, these aldehydes would have toxic effects on any cell. Finally, the similarity in product mixtures from both the photo-oxidation and auto-oxidation strongly suggests that the intermolecular photo-oxidation of A2E results primarily from a radical process without the involvement of singlet oxygen. Any formation of singlet oxygen most likely arises from sensitization by the aldehyde oxidation products, as this process is well known for aldehydes, in general, and retinal, specifically.     

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.     

OT-674 suppresses photooxidative processes initiated by an RPE lipofuscin fluorophore

The pathological processes involved in age-related macular degeneration (AMD) include retinal pigment epithelial (RPE) cell degeneration; oxidative mechanisms likely contribute to the demise of these cells. Indeed, RPE cells may be particularly susceptible to photooxidative mechanisms since they accumulate retinoid-derived photoreactive compounds that constitute the lipofuscin of the cell. Thus we undertook to test the capacity of OT-674, the reduction product (Tempol-H) of the nitroxide Tempol, to suppress photooxidative processes initiated by the RPE lipofuscin fluorophore A2E. Accordingly, when ARPE-19 cells that had accumulated A2E were irradiated at 430 nm, pretreatment with OT-674 (0.01-10 mM) was found to confer a resistance to cell death. Monitoring by quantitative HPLC also showed that OT-674 reduced A2E photooxidation in a cell-free system. Moreover, when presented with a singlet oxygen generator, OT-674 served as a quencher of singlet oxygen that was more effective than Trolox and alpha-tocopherol. We conclude that OT-674 is a potent antioxidant that suppresses photooxidative processes generated in cultured RPE cells by the lipofuscin fluorophore A2E. As oxidative damage to RPE cells is considered to be a risk factor for AMD, antioxidant therapy with OT-674 may serve a protective role.     

Photoprotection by porcine eumelanin against singlet oxygen production

Melanin, a major pigment found in retinal pigment epithelium (RPE) cells, is considered to function in dual roles, one protective and one destructive. By quenching free radical species and reactive oxygen species (ROS) melanin counteracts harmful redox stress. However, melanin is also thought to be capable of creating ROS. In this destructive role, melanin increases redox strain in the cell. This study uses readily available eumelanin extracted from porcine RPE cells as a more authentic model than synthetic melanin to determine specific mechanisms of melanin activity with regard to singlet oxygen in the presence and absence of rose bengal, a singlet-oxygen photosensitizer. Optical detection of singlet-oxygen was determined by monitoring the bleaching of p-nitrosodimethylaniline in the presence of histidine. Production of singlet oxygen in aqueous oxygen-saturated solutions of rose bengal without eumelanin was readily accomplished. In contrast, detection of singlet oxygen in oxygen-saturated solutions of eumelanin without rose bengal failed, consistent with results of others. However, a significant decrease in singlet oxygen production by rose bengal was observed in the presence of eumelanin. After correction for light absorption and chemical bleaching of eumelanin, the results show that eumelanin also provides a photoprotective mode arising from chemistry, that is, not just the physical process of light absorption followed by energy dissipation as heat.     

Anthocyanins protect against A2E photooxidation and membrane permeabilization in retinal pigment epithelial cells

The pyridinium bisretinoid A2E, an autofluorescent pigment that accumulates in retinal pigment epithelial cells with age and in some retinal disorders, can mediate a detergent-like perturbation of cell membranes and light-induced damage to the cell. The photodynamic events initiated by the sensitization of A2E include the generation of singlet oxygen and the oxidation of A2E at carbon-carbon double bonds. To assess the ability of plant-derived anthocyanins to modulate adverse effects of A2E accumulation on retinal pigment epithelium (RPE) cells, these flavylium salts were isolated from extracts of bilberry. Nine anthocyanin fractions reflecting monoglycosides of delphinidin, cyanidin, petunidin and malvidin were obtained and all were shown to suppress the photooxidation of A2E at least in part by quenching singlet oxygen. The anthocyanins tested exhibited antioxidant activity of variable efficiency. The structural characteristics relevant to this variability likely included the ability to form a stable quinonoidal anhydro base at neutral pH, a conjugated diene structure in the C (pyrane) ring, the presence of hydroxyl groups on the B (benzene) ring and the relative hydrophobicity conferred by the arrangement of substituents on the B ring. Cells that had taken up anthocyanins also exhibited a resistance to the membrane permeabilization that occurs as a result of the detergent-like action of A2E.     

A new approach to measuring the action spectrum for singlet oxygen production by human retinal lipofuscin

The human retinal pigment epithelial (RPE) layer contains a complex mixture of components called lipofuscin; this mixture forms with age and with various genetic disorders such as Stargardt's disease. Its presence may contribute to retinal deterioration via several mechanisms including photochemical processes. In the lipofuscin mixture, both type I and II mechanisms have been identified, with the latter consisting of the generation of singlet oxygen. Several components of that mixture have been identified, most notably a bis-retinoid pyridinium compound called A2E and its derivatives. Photo-oxidative studies on the compound A2E have revealed that its dominant photochemical mechanism is via free radical or type I processes. Because singlet oxygen is an important photooxidative intermediate in tissue, its generation in the RPE may contribute to retinal maculopathies. It is therefore necessary to determine which specific component(s) in the lipofuscin mixture produce singlet oxygen upon excitation with light. This was ascertained by evaluating the action spectrum for singlet oxygen production for the whole lipofuscin mixture using time-resolved spectroscopy. Singlet oxygen was generated by excitation of the sample at different wavelengths while maintaining a constant beam energy, and was directly detected by its phosphorescence decay at 1270 nm using a Ge photodiode. The action spectrum for singlet oxygen sensitization by the organic soluble portion of lipofuscin had an absorption maximum at ca 380 nm, which is to the blue of A2E (maximum at 430 nm). Compounds with a similar absorption maximum eluted in the HPLC earlier than A2E and were detected in human lipofuscin. The concentration of this component apparently increased in concentration in human RPE lipofuscin mixture as a function of age up to 90 years old.     

Effects of Light Exposure and Use of Intraocular Lens on Retinal Pigment Epithelial Cells In Vitro

To investigate the effect of a blue light-filtering intraocular lens (IOL) and a UV-absorbing IOL on light-induced damage to retinal pigment epithelial (RPE) cells laden with the lipofuscin fluorophore N -retinylidene- N -retinylethanolamine (A2E), A2E-laden RPE cells were exposed to white light which was filtered by either a blue light-filtering IOL or a UV-absorbing IOL. After 30 min of illumination the cell viability and the level of reactive oxygen species (ROS), free glutathione (GSH), vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) were determined. In the absence of an IOL, the white light exposure decreased cell viability to 37.2% of the nonirradiated control. The UV-absorbing IOL tended to reduce light-induced cell death; however, the decrease was not significant. The blue light-filtering IOL significantly attenuated light-induced cell damage, increasing cell viability to 79.5% of the nonirradiated control. The presence of the blue light-filtering IOL significantly increased GSH and PEDF levels, and decreased ROS and VEGF levels. This study suggests that a blue light-filtering IOL may be more protective against A2E-induced light damage and inhibit more light-induced ROS and VEGF production than a conventional UV-absorbing IOL.     

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.     

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.     

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.     

Interaction of pyridinium bis-retinoid (A2E) with bilayer lipid membranes

The accumulation of lipofuscin granules within the retinal pigment epithelium (RPE) cells is correlated with the progression of age-related macular degeneration. One of the fluorophores contained in lipofiscin granules is pyridinium bis-retinoid (A2E). To test its membrane-toxic effect, the interaction of A2E with bilayer lipid membranes (BLM) was studied. The incorporation of charged A2E molecules into the membranes has been detected as a change of either zeta-potential of multilayer liposomes or boundary potential of BLM. It was shown that the presence of up to 25mol% of A2E did not destabilize the bilayers made of saturated phosphatidylcholine (PC). However, the destabilizing effect became very significant when BLM contained negatively charged lipids such as cardiolipin or phosphatidylserine. The electrical breakdown measurements revealed that the A2E-induced decrease of BLM stability was primarily associated with the growing probability of lipid pore formation. It was found from the measurements of boundary potential of BLM that exposure of A2E to light initiates its transformation into at least two products. One of them is epoxy-A2E, which, being hydrophilic, moves from the membrane into water solution. The other product is a non-identified hydrophobic substance. Illumination of A2E-containing BLM made from unsaturated PC by visible light caused the membrane damage presumably due to oxidation of these lipids by singlet oxygen generated by excited A2E molecules. However, this effect was very weak compared to the effect of known photosensitizers. The illumination of BLM with A2E also leads to the damage of gramicidin incorporated into the membrane, as was detected by measuring the conductance of channels formed by this peptide.     

A2E: a component of ocular lipofuscin

The presence of lipofuscin in postmitotic cells is considered a hallmark of the aging process. In the retinal pigment epithelium (RPE), lipofuscin is found as micrometer-sized spherical particles and characterized by its yellow autofluorescence when exposed to blue light. This exposure to light is also known to produce reactive oxygen intermediates (ROI), but the particular molecular constituent(s) responsible for this phototoxicity have yet to be completely identified. Resulting mostly from the autophagocytosis of intracellular organelles, the composition of lipofuscin is poorly defined but known to contain protein, lipids and several fluorophores. The subsequent identification of one of the fluorophores in lipofuscin, A2E, generated much interest and resulted in a variety of studies to understand its potential role in the phototoxicity of lipofuscin. Several modes of toxicity have been suggested through which A2E can affect the health of RPE cells. These modes include photoinduced production of ROI, which places additional oxidative stress on RPE cells, the disruption of membrane integrity through its natural role as an amphiphilic detergent and inhibition of key cellular functions. This article presents the current understanding of the photochemistry of A2E and its involvement as a phototoxic agent in RPE cells.     

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.     

Enzymatic degradation of A2E, a retinal pigment epithelial lipofuscin bisretinoid

Some forms of blinding macular disease are associated with excessive accumulation of bisretinoid lipofuscin in retinal pigment epithelial (RPE) cells of the eye. This material is refractory to lysosomal enzyme degradation. In addition to gene and drug-based therapies, treatments that reverse the accumulation of bisretinoid would be beneficial. Thus, we have examined the feasibility of degrading the bisretinoids by delivery of exogenous enzyme. As proof of principle we report that horseradish peroxidase (HRP) can cleave the RPE bisretinoid A2E. In both cell-free and cell-based assays, A2E levels were decreased in the presence of HRP. HRP-associated cleavage products were detected by ultraperformance liquid chromatography (UPLC) coupled to electrospray ionization mass spectrometry, and the structures of the aldehyde-bearing cleavage products were elucidated by 18O-labeling and 1H NMR spectroscopy and by recording UV?vis absorbance spectra. These findings indicate that RPE bisretinoids such as A2E can be degraded by appropriate enzyme activities.     

The Anthocyanins,Oenin and Callistephin,Protect RPE Cells Against Oxidative Stress

The retinal pigment epithelium (RPE) is a highly metabolic layer of postmitotic cells lining Bruch's membrane in the retina. While these cells contain endogenous photosensitizers that mediate blue light‐induced damage, it has also been shown that blue light exposure damages mitochondrial DNA in RPE cells resulting in mitochondrial dysfunction and unregulated generation of reactive oxygen species (ROS). As RPE cells are postmitotic, it is imperative to decrease oxidative stress to these cells and preserve function. Dietary plant‐derived antioxidants such as anthocyanins offer a simple and accessible solution for decreasing oxidative stress. The anthocyanins malvidin‐3‐O‐glucoside (oenin) and pelargonidin‐3‐O‐glucoside (callistephin) were tested for their ability and efficacy in decreasing ROS generation and preserving mitochondrial redox activity in blue light‐irradiated ARPE‐19 cells. A significant decrease in intracellular ROS with concurrent increase in mitochondrial redox activity was observed for tested concentrations of oenin, while callistephin was beneficial to stressed cells at higher concentrations. These findings suggest anthocyanins are effective antioxidants in blue light‐stressed RPE cells in vitro. Additionally, oxidation products of these anthocyanins were examined using LC/MS and findings suggest the possibility of multiple oxidation sites for these compounds.     

Effects of photodegradation on the physical and antioxidant properties of melanosomes isolated from retinal pigment epithelium

Melanosomes of the retinal pigment epithelium (RPE) are relatively long-lived organelles that are theoretically susceptible to changes induced by exposure to visible light. Here melanosomes were isolated from porcine RPE cells and subjected to high intensity visible light to determine the effects of illumination on melanosome structure and on the content and antioxidant properties of melanin. As compared to untreated melanosomes, illuminated granules showed morphologic changes consistent with photodegradation, which included variable reductions in electron density demonstrated by transmission electron microscopy (TEM), and particle fragmentation and surface disruption revealed by scanning electron microscopy (SEM) and atomic force microscopy. Illuminated melanosomes had lower melanin content, indicated by measures of absorbance and electron spin resonance (ESR) signal intensity, and reduced ability to bind iron, shown by chemical and ESR analyses. Compared to untreated melanosomes, ESR-spin trapping analyses further indicated that illuminated melanosomes show increased photogeneration of superoxide anion and reduced ability to inhibit the iron ion-catalyzed free radical decomposition of hydrogen peroxide. It appears therefore that visible light irradiation can disrupt the structure of RPE melanosomes and reduce the amount and antioxidant properties of melanin. Some of these changes occur in human RPE melanosomes with aging and the results obtained here suggest that visible light irradiation is at least partly responsible. The consequence of light-induced changes in RPE melanosomes may be a diminished capacity of melanin to help protect aged cells from oxidative damage, perhaps increasing the risk of diseases with an oxidative stress component such as age-related macular degeneration.