Diffuse Reflectance Spectroscopy Versus Mexameter® MX18 Measurements of Melanin and Erythema in an African Population

Melanin provides protection against excess exposure to solar ultraviolet radiation (UVR) and related adverse health effects. Diffuse reflectance spectroscopy (DRS) can be used to calculate cutaneous melanin and erythema, but this is complex and has been mostly used for light‐to‐medium pigmented skin. Handheld reflectance spectrophotometers, such as the Mexameter^® MX18, can also be used. We compared DRS‐calculated melanin and erythema values with Mexameter melanin and erythema index values to understand how these techniques/measurements correlate in an African population of predominantly deeply pigmented skin. Five hundred and three participants comprised 68.5% self‐identified Black African, 9.9% Indian/Asian, 18.4% White and 2.9% Colored. The majority of Black African (45%), Indian/Asian (34%) and Colored (53%) participants self‐identified their skin as being “brown.” Measured melanin levels increased with darker self‐reported skin color. DRS‐calculated and Mexameter melanin values demonstrated a positive correlation (Spearman rho = 0.87, P < 0.001). The results from both instruments showed erythema values were strongly correlated with their own melanin values. This finding is considered spurious and may result from the complexity of separating brown and red pigment when using narrowband reflectance techniques. Further work is needed to understand melanin, erythema and color in Black skin given sun‐related health risks in vulnerable groups in Africa.     

Lipofuscin and A2E Accumulate with Age in the Retinal Pigment Epithelium of Nrl−/− Mice†

Lipofuscin is a fluorescent material with significant phototoxic potential that accumulates with age in the retinal pigment epithelium (RPE) of the eye. It is thought to be a factor in retinal degeneration diseases. The most extensively characterized lipofuscin component, N‐retinylidene‐N‐retinylethanolamine (A2E), has been proposed to be a byproduct of reactions involving the visual pigment chromophore. To examine the impact of the visual pigment and photoreceptor cell type on lipofuscin accumulation, we analyzed the RPE from Nrl^?/? mice of various ages for lipofuscin fluorescence and A2E levels. The photoreceptor cells of the Nrl^?/? retina contain only cone‐like pigments, and produce cone‐like responses to photostimulation. The cone‐like nature of these cells was confirmed by the presence of RPE65. Lipofuscin was measured with fluorescence imaging, whereas A2E was quantified by UV/VIS absorbance spectroscopy coupled to HPLC. The identity of A2E was corroborated with tandem mass spectrometry. Lipofuscin and A2E accumulated with age, albeit to lower levels compared with wild type mice. The emission spectra of RPE lipofuscin granules from Nrl^?/? mice were similar to those from wild type mice, with λ_maxca 610 nm. These results demonstrate that cone visual pigments can contribute to the production of lipofuscin and A2E.     

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.     

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.     

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.     

RPE65 and the Accumulation of Retinyl Esters in Mouse Retinal Pigment Epithelium

The RPE65 protein of the retinal pigment epithelium (RPE) enables the conversion of retinyl esters to the visual pigment chromophore 11‐cis retinal. Fresh 11‐cis retinal is generated from retinyl esters following photoisomerization of the visual pigment chromophore to all‐trans during light detection. Large amounts of esters accumulate in Rpe65^?/? mice, indicating their continuous formation when 11‐cis retinal generation is blocked. We hypothesized that absence of light, by limiting the conversion of esters to 11‐cis retinal, would also result in the build‐up of retinyl esters in the RPE of wild‐type mice. We used HPLC to quantify ester levels in organic extracts of the RPE from wild‐type and Rpe65^?/? mice. Retinyl ester levels in Sv/129 wild‐type mice that were dark adapted for various intervals over a 4‐week period were similar to those in mice raised in cyclic light. In C57BL/6 mice however, which contain less Rpe65 protein, dark adaptation was accompanied by an increase in ester levels compared to cyclic light controls. Retinyl ester levels were much higher in Rpe65^?/? mice compared to wild type and kept increasing with age. The results suggest that the RPE65 role in retinyl ester homeostasis extends beyond enabling the formation of 11‐cis retinal.     

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.     

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.     

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.     

Melanin from epidermal human melanocytes: Study by pyrolytic GC/MS

Pigmentation of human skin is determined by the presence of melanin, the polymeric pigment that is produced in melanocytes and transferred to adjacent keratinocytes. Epidermal melanocytes produce two distinct types of melanin pigments: eumelanin, composed mainly of indole-type monomers, and pheomelanin that contains benzothiazine-type backbone. Eumelanin protects skin against UV-induced damages, whereas pheomelanin is believed to act as a potent UV photosensitizer and promote carcinogenesis. In this study, pyrolysis in combination with gas chromatography and mass spectrometry (Py-GC/MS) was applied for structural studies of the epidermal pigment isolated from the cultured human melanocytes. The analysis was preceded by investigations of DOPA-originated synthetic eumelanin and pheomelanin standards. This allowed determination of pyrolytic markers for both types of melanin pigments. To obtain additional information on the natural pigment structure, the samples were thermally degraded in the presence of tetramethylammonium hydroxide as the derivatizing agent. It was shown that the analyzed pigment from normal human epidermal melanocytes derived from moderately pigmented skin is of eumelanin type with little incorporation of a pheomelanin component. The results indicate that Py-GC/MS is a rapid and efficient technique for the differentiation of epidermal melanin types and may be an alternative to commonly used methods based on chemical degradation.     

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     

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.     

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.     

Photo‐Modification of Melanin by a Mid‐infrared Free‐electron Laser

Melanin is rigidly constructed by several nitrogen‐containing aromatic rings, and its excess accumulation in skin tissue is closely associated with melanosis. Although visible lasers (wavelength: 600–1000 nm) are conventionally used for the photo‐thermolysis of melanocyte, several pigmented nevi are difficult to be treated. Here, we propose an alternate method for targeting the molecular structure of melanin using an infrared free‐electron laser (FEL) tuned to 5.8 μm that corresponds to the stretching vibrational mode of carboxylate group. A drastic morphological change on the black‐colored surface of melanin powder was observed after the pulse irradiation with power energy of 500 mJ cm^?2, and the minimum irradiation time for damage to the morphology was 1.4 s. Analyses by mass spectroscopy, infrared spectroscopy, and ¹³C‐nuclear magnetic resonance implied that a pyrrole group was removed by the FEL irradiation. In addition, the FEL irradiation dispersed almost all of the melanoma cells from a culture solution without any influence on other ingredients in the medium, and one‐cell analysis by infrared microscopy showed that the structure of melanoma could be substantially damaged by the irradiation. This study proposes the potency of intense mid‐infrared laser as novel alternative way to reduce melanin.     

Photodynamic Effects of Hypericin on Lipid Peroxidation and Antioxidant Status in Melanoma Cells

Photodynamic-induced cytotoxicity by hypericin (HYP) was studied on three human melanoma cell lines: one pigmented cell line (G361) and two amelanotic cell lines (M18 and M6). No significant variation in the rate of uptake and in the maximum level of HYP incorporation for the different cells was observed. In the dark, no cytotoxicity was observed in the range0–10^?6M HYP for the three cell lines. Amelanotic cells were found to be more sensitive than pigmented cells to irradiation of HYP with visible light (Λ > 590 nm). In addition, for the three cell lines HYP-induced photocytotoxicity was found to be drug-dose and light-dose dependent. Under the conditions used, thiobarbituric acid-reacting substances (TBARs) were significantly increased in amelanotic cells after irradiation (P < 0.0001). By contrast, the amount of TBARS remained unchanged in pigmented cells. Antioxidant defenses including enzymes and glutathione (GSH) were assayed before and after HYP photosensitization. Significantly increased total SOD activity was observed after photosensitization for amelanotic cells (P < 0.05), while glutathione peroxidase (GSHPx) and catalase (Cat) activities but also GSH levels were significantly decreased (P < 0.01). In pigmented cells a significantly increased Cat activity was found (P < 0.05), whereas GSHPx was unaffected after irradiation. It can be inferred that (a) HYP may be an effective PDT agent for melanoma and (b) there is a relationship between melanin content and sensitivity to HYP phototoxicity in human melanoma cells.     

Cell Shape Normalization,Dendrite Orientation,and Melanin Production of Normal and Genetically Altered (Haploinsufficient NF1)‐Melanocytes by Microstructured Substrate Interactions

Little is known about how functional regulation failure in genetically altered cells is influenced by topographical confinement of cells, a situation often present in tissues in vivo. We used cultured melanocytes derived from human skin samples as a model system for such investigations. Normal melanocytes have a very well defined shape consisting of a cell body and two dendrites arranged 180° relative to each other. In contrast, neurofibromin 1‐melanocytes (NF1‐melanocytes) have up to a 50 % reduction of neurofibromin 1, which results in an altered morphology that can be easily measured. NF1‐melanocytes deviate from the defined structure of normal melanocytes by forming more than two dendrites per cell. We show that morphology consequences of genetically altered melanocytes can be canceled if cells interact with substrates microstructured by stripes that apply mechanophysical signals in the form of physical topography. The strength of the mechanophysical signal was varied systematically by increasing the height of the microstructures. Melanocytes respond to surface topographical features that are larger than 50 nm and have lateral confinements smaller 4 μm. The response of normal and NF1‐melanocytes to different topographies was analyzed quantitatively by determining density distributions for the number of dendrites per cell, the angles between dendrites, and the orientation imprinted in the substrate. The synthesis of melanin, a pigment produced by melanocytes, differs in the case of genetically altered NF1‐ and normal melanocytes. In both cases, the interaction with microstripes enhanced melanin production significantly. This enhanced melanin production is speculated to be caused by the mechanical stabilization of the dendrites by substrate guidance.     

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.     

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.