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.     

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.     

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.     

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.     

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.     

Mitochondria-derived reactive oxygen species mediate blue light-induced death of retinal pigment epithelial cells

Throughout the lifetime of an individual, light is focused onto the retina. The resulting photooxidative stress can cause acute or chronic retinal damage. The pathogenesis of age-related macular degeneration (AMD), the leading cause of legal blindness in the developed world, involves oxidative stress and death of the retinal pigment epithelium (RPE) followed by death of the overlying photoreceptors. Evidence suggests that damage due to exposure to light plays a role in AMD and other age-related eye diseases. In this work a system for light-induced damage and death of the RPE, based on the human ARPE-19 cell line, was used. Induction of mitochondria-derived reactive oxygen species (ROS) is shown to play a critical role in the death of cells exposed to short-wavelength blue light (425 +/- 20 nm). ROS and cell death are blocked either by inhibiting the mitochondrial electron transport chain or by mitochondria-specific antioxidants. These results show that mitochondria are an important source of toxic oxygen radicals in blue light-exposed RPE cells and may indicate new approaches for treating AMD using mitochondria-targeted antioxidants.     

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.     

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.     

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.     

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.     

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.     

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.     

Long-term effects of human induced pluripotent stem cell-derived retinal cell transplantation in Pde6b knockout rats

Retinal degenerative disorders, including age-related macular degeneration and retinitis pigmentosa (RP), are characterized by the irreversible loss of photoreceptor cells and retinal pigment epithelial (RPE) cells; however, the long-term effect of implanting both human induced pluripotent stem cell (hiPSC)-derived RPE and photoreceptor for retinal regeneration has not yet been investigated. In this study, we evaluated the long-term effects of hiPSC-derived RPE and photoreceptor cell transplantation in Pde6b knockout rats to study RP; cells were injected into the subretinal space of the right eyes of rats before the appearance of signs of retinal degeneration at 2–3 weeks of age. Ten months after transplantation, we evaluated the cells using fundus photography, optical coherence tomography, and histological evaluation, and no abnormal cell proliferation was observed. A relatively large number of transplanted cells persisted during the first 4 months; subsequently, the number of these cells decreased gradually. Notably, immunohistochemical analysis revealed that the hiPSC-derived retinal cells showed characteristics of both RPE cells and photoreceptors of human origin after transplantation. Functional analysis of vision by scotopic electroretinogram revealed significant preservation of vision after transplantation. Our study suggests that the transplantation of hiPSC-derived retinal cells, including RPE cells and photoreceptors, has a potential therapeutic effect against irreversible retinal degenerative diseases.Subject terms: Translational research, Stem-cell research, Retina     

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.     

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.     

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.     

Phototoxicity in human retinal pigment epithelial cells promoted by hypericin, a component of St. John's wort

St. John's wort (SJW), an over-the-counter antidepressant, contains hypericin, which absorbs light in the UV and visible ranges. In vivo studies have determined that hypericin is phototoxic to skin and our previous in vitro studies with lens tissues have determined that it is potentially phototoxic to the human lens. To determine if hypericin might also be phototoxic to the human retina, we exposed human retinal pigment epithelial (hRPE) cells to 10(-7) to 10(-5) M hypericin. Fluorescence emission detected from the cells (lambda(ex) = 488 nm; lambda(em) = 505 nm) confirmed hypericin uptake by human RPE. Neither hypericin exposure alone nor visible light exposure alone reduced cell viability. However when irradiated with 0.7 J cm(-2) of visible light (lambda > 400 nm) there was loss of cell viability as measured by MTS and lactate dehydrogenase assays. The presence of hypericin in irradiated hRPE cells significantly changed the redox equilibrium of glutathione and a decrease in the activity of glutathione reductase. Increased lipid peroxidation as measured by the thiobarbituric acid reactive substances assay correlated to hypericin concentration in hRPE cells and visible light radiation. Thus, ingested SJW is potentially phototoxic to the retina and could contribute to retinal or early macular degeneration.     

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.     

PHOTOPHYSICAL STUDIES ON HUMAN RETINAL LIPOFUSCIN

Fluorescent material generated in the human retina accumulates within lipofuscin granules of the retinal pigment epithelium (RPE) during aging. Its presence has been suggested to contributed to various diseases including age-related macular degeneration. Because this material absorbs light at wave lengths as long as 550 nm, photophysical studies were performed to determine whether lipofuscin could contribute to light damage and to determine if its composition is similar to a synthetically prepared lipofuscin. Time-resolved experiments were performed to monitor (1) fluorescence decay, (2) the UV-visible absorption of longer-lived excited states and (3) the formation and decay of singlet oxygen at 1270 nm. Steady-state and time-resolved fluorescence studies indicate that human and synthetic lipofuscin have fluorophores in common. Time-resolved absorption experiments on human retinal lipofuscin and synthetic lipofuscin showed the presence of at least two transient species, one absorbing at 430 nm (lifetime caμs) and a second absorbing at 580 nm, which decays via second order kinetics. In addition, there is a third absorbing species stable to several hundred milliseconds. The transient species at 430 nm is quenched by oxygen, suggesting that it is a triplet state. Subsequent studies showed the formation of singlet oxygen, which was monitored by its phosphorescence decay at 1270 nm. These studies demonstrate that lipofuscin can act as a sensitizer for the generation of reactive oxygen species that may contribute to the age-related decline of RPE function and blue light damage.     

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.