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.     

Comparison of structural and chemical properties of black and red human hair melanosomes

Melanosomes in black and red human hair are isolated and characterized by various chemical and physical techniques. Different yields of 4-amino-hydroxyphenolanaline by HI hydrolysis (a marker for pheomelanin) and pyrrole-2,3,5-tricarboxylic acid by KMnO(4)/H(+) oxidation (a marker for eumelanin) indicate that the melanosomes in black hair are eumelanosomes, whereas those in red hair are mainly pheomelanosomes. Atomic force microscopy reveals that eumelanosomes and pheomelanosomes have ellipsoidal and spherical shapes, respectively. Eumelanosomes maintain structural integrity upon extraction from the keratin matrix, whereas pheomelanosomes tend to fall apart. The black-hair eumelanosomes have an average of 14.6 +/- 0.5% amino acids content, which is attributed to the internal proteins entrapped in the melanosomes granules. The red-hair melanosomes contain more than 44% of amino acid content even after extensive proteolytic digestion. This high content of amino acids and the poorly reserved integrity of red-hair melanosomes suggest that some proteins are possibly covalently bonded with the melanin constituents in addition to those that are entrapped inside the melanin species. Soluene solubilization assay indicates the absorbance of melanin per gram of sample, adjusted for the amino acid content, is a factor of 2.9 greater for the black-hair melanosomes than the red-hair melanosomes. Metal analysis reveals significant amounts of diverse heavy metal ions bound to the two types of melanosomes. The amount of Cu(II) and Zn(II) are similar but Fe(III) content is four times higher in the red-hair melanosomes. (13)C solid-state nuclear magnetic resonance spectra and infrared spectra are presented and are shown to be powerful techniques for discerning differences in the amino acid contents, the 5,6-dihydroxyindole-2-carboxylic acid:5,6-dihydroxyindole ratio, and the degree of cross-linking in the pigment. Excellent agreement is observed between these spectral results and the chemical degradation data.     

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.     

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.     

Structural Effects in Visible‐Light‐Responsive Metal–Organic Frameworks Incorporating ortho‐Fluoroazobenzenes

The ability to control the interplay of materials with low‐energy photons is important as visible light offers several appealing features compared to ultraviolet radiation (less damaging, more selective, predominant in the solar spectrum, possibility to increase the penetration depth). Two different metal–organic frameworks (MOFs) were synthesized from the same linker bearing all‐visible ortho‐fluoroazobenzene photoswitches as pendant groups. The MOFs exhibit different architectures that strongly influence the ability of the azobenzenes to isomerize inside the voids. The framework built with Al‐based nodes has congested 1D channels that preclude efficient isomerization. As a result, local light–heat conversion can be used to alter the CO₂ adsorption capacity of the material on exposure to green light. The second framework, built with Zr nodes, provides enough room for the photoswitches to isomerize, which leads to a unique bistable photochromic MOF that readily responds to blue and green light. The superiority of green over UV irradiation was additionally demonstrated by reflectance spectroscopy and analysis of digested samples. This material offers promising perspectives for liquid‐phase applications such as light‐controlled catalysis and adsorptive separation.     

Insights into melanosomes and melanin from some interesting spatial and temporal properties

Melanosomes are organelles found in a wide variety of tissues throughout the animal kingdom and exhibit a range of different shapes: spheres of up to approximately 1 mum diameters and ellipsoids with lengths of up to approximately 2 mum and varying aspect ratios. The functions of melanosomes include photoprotection, mitigation of the effects of reactive oxygen species, and metal chelation. The melanosome contains a variety of biological molecules, e.g., proteins and lipids, but the dominant constituent is the pigment melanin, and the functions ascribed to melanosomes are uniquely enabled by the chemical properties of the melanins they contain. In the past decade, there has been significant progress in understanding melanins and their impact on human health. While the molecular details of melanin production and how the pigment is organized within the melanosome determine its properties and biological functions, the physical and chemical properties of the surface of the melanosome are central to their range of ascribed functions. Surprisingly, few studies designed to probe this biological surface have been reported. In this article, we discuss recent work using surface-sensitive analytic, spectroscopic, and imaging techniques to examine the structural and chemical properties of many types of natural pigments: sepia melanin granules, human and bovine ocular melanosomes, human hair melanosomes, and neuromelanin. N 2 adsorption/desorption measurements and atomic force microscopy provide novel insights into surface morphology. The chemical properties of the melanins present on the surface are revealed by X-ray photoelectron spectroscopy and photoemission electron microscopy. These technologies are also applied to elucidate changes in surface properties that occur with aging. Specifically, studies of the surface properties of human retinal pigment epithelium melanosomes as a function of age are stimulating the development of models for their age-dependent behaviors. The article concludes with a brief discussion of important unanswered research questions in this field.     

Spectrophotometric methods for quantifying pigmentation in human hair-influence of MC1R genotype and environment.

Eumelanin (brown/black melanin) and pheomelanin (red/yellow melanin) in human hair can be quantified using chemical methods or approximated using spectrophotometric methods. Chemical methods consume greater resources, making them less attractive for epidemiological studies. This investigation sought to identify the spectrophotometric measures that best explain the light-dark continuum of hair color and the measure that is best able to distinguish red hair from nonred hair. Genetic analysis was performed on these two measures to determine the proportion of genetic and environmental influences on variation in these traits. Reflectance curves along the visible spectrum and subjective ratings of hair color were collected from 1730 adolescent twin individuals. Discriminant class analyses were performed to determine the spectrophotometric measure that could best proxy for eumelanin and pheomelanin quantities. The ratio of light reflected in the green portion of the spectrum to that reflected in the red portion of the spectrum was best able to distinguish red hair from nonred hair. Melanocortin 1 receptor (MC1R) genotype explained some, but not all, variation in this measure. Light absorbed in the red portion of the spectrum was best able to explain the light-dark continuum of hair color. Variance components analysis showed that there were qualitatively different genetic influences between males and females for the light-dark continuum of hair. Our results show that spectrophotometric measures approximating variation in eumelanin and pheomelanin may be considered as an alternative to chemical methods in larger epidemiological studies.     

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.     

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.     

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.     

Melanin/polydopamine-based nanomaterials for biomedical applications

The natural melanin is one kind of ubiquitous biological pigments, which is produced in melanosomes and widely distributed in living organisms. The synthetic melanin, often known as polydopamine(PDA), has similar chemical compositions and physical properties to natural melanin. In recent years, both natural melanin and PDA have aroused increased research interests in biomedical fields owing to their inherent biocompatibility, antioxidant activity, free-radical scavenging, metal ion chelation,strong near-infrared absorption and high photothermal conversion efficiency. Inspired by these fascinating properties, melanin and PDA have been widely used as building blocks for the construction of multifunctional nanoplatforms for various biomedical applications. This review focuses on the state-of-the-art progress in melanin/PDA-based nanomaterials, which covers from their preparation methods to biomedical applications including bioimaging, treatment, theranostics, antibacterial, UV/radiation protection, biosensor and tissue engineering. Moreover, the current trends and the future prospects of melanin/PDA-based nanomaterials are also discussed.     

Age-dependent photoionization thresholds of melanosomes and lipofuscin isolated from human retinal pigment epithelium cells

Melanosomes and lipofuscin were isolated from 14-, 59-, and 76-year-old, human retinal pigment epithelium specimens and examined. The morphological features of these samples were studied by scanning electron microscopy and atomic force microscopy, and the photoionization properties were examined by photoelectron emission microscopy. Ovoid- and rod-shaped melanosomes were observed. The size of the granules and the distribution between the two shapes show no significant age-dependent change. However, there is a higher occurrence of irregularly shaped aggregates of small round granules in older samples which suggests degradation or damage to melanosomes occurs with age. The melanosomes from the 14-year-old donor eye are well characterized by a single photoionization threshold, 4.1 eV, while the two older melanosomes exhibit two thresholds around 4.4 and 3.6 eV. Lipofuscin from both young and old cells show two thresholds, 4.4 and 3.4 eV. The similarity of the potentials observed for aged melanosomes and lipofuscin suggest that the lower threshold in the melanosome sample reflects lipofuscin deposited the surface of the melanosome. The amount, however, is not sufficient to alter the density of the melanosome, and therefore these granules do not separate in a sucrose gradient at densities characteristic of the typical melanolipofuscin granule. These data suggest that thin deposits of lipofuscin on the surface of retinal pigment epithelium melanosomes are common in the aged eye and that this renders the melanosomes more pro-oxidant.     

The UV‐Absorption Spectrum of Human Iridal Melanosomes: A New Perspective on the Relative Absorption of Eumelanin and Pheomelanin and its Consequences†

Photoemission electron microscopy is used to measure the absorption coefficients, εc, of intact iridal stroma melanosomes isolated from dark brown and blue–green human irides for the spectral range λ = 244–310 nm. These iridal stroma melanosomes were chosen because different colored irides produce organelles of varying eumelanin:pheomelanin ratios with similar size and morphology. Similar absorption spectra are found for the two types of melanosomes. The experimental spectra measured within are compared with both the extinction coefficient spectra obtained on soluble synthetic model systems and the monomeric precursors to each pigment.     

ELECTRON SPIN RESONANCE OF MELANIN FROM HAIR. EFFECTS OF TEMPERATURE, pH AND LIGHT IRRADIATION

Abstract— The variation with temperature, pH and light of the ESR signal of hydrated melanin powders from Japanese black hair has been studied. An explanation of the results is proposed on the basis of quinhydrone type complexes and of acid-base equilibria of melanin and its semiquinone radicals. During exposure to light of wavelengths 254–600 nm, both stable and unstable radicals have been observed. The action spectrum for the formation of stable melanin radicals has been determined.     

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.     

AGE-RELATED CHANGES IN THE FLUORESCENCE OF MELANIN and LIPOFUSCIN GRANULES OF THE RETINAL PIGMENT EPITHELIUM: A TIME-RESOLVED FLUORESCENCE SPECTROSCOPY STUDY

The photophysical properties of purified populations of melanin and lipofuscin granules from human retinal pigment epithelium, and their changes with donor age, have been investigated using high-sensitivity time-resolved fluorescence spectroscopy techniques with picosecond gating capabilities. The overall fluorescence intensity of both melanin and lipofuscin granules clearly increased with increasing donor age, the increase being most marked for melanin. In all granule populations the fluorescence decays were multiexponential with subnanosecond and nanosecond decay components. The resultant time-integrated and time-gated spectra also exhibited marked age-variations for each type of granule. Young melanin showed spectral patterns similar to those of bovine melanin, while a yellow-orange fluorescence band appeared in melanin samples from older age groups. Lipofuscin granules exhibited a blue, a yellow and an orange band whose relative amounts were age-related. The results demonstrate the potential of time-resolved techniques for discriminating fluorophores in vitro and in situ, and have confirmed results previously obtained using extraction techniques. Furthermore, the ability of this technique to identify and quantify individual fluorophores within granules may provide an important insight into the origin and development of lipofuscin within the retinal pigment epithelium and ultimately into the mechanisms of age-related retinal diseases.     

Biphasic fluence-response curves and derived action spectra for light-induced absorbance changes in Phycomyces mycelium

The photoresponses of Phycomyces, including phototropism and photocontrol of sporangiophore development, are mediated primarily by blue and UV light. Recent results on these two responses indicated a subsidiary role for green light. We have measured in vivo light-induced absorbance changes (LIAC) in mycelial samples of a caroteneless (carB) strain to compare the effectiveness of UV, blue, and green light. In the dual-wavelength kinetic mode of the spectrophotometer, measuring wavelengths of 445 and 470 nm were chosen, because green light produces substantial absorbance changes between these two wavelengths. Fluence-response curves were measured for 13 wavelengths between 365 and 530 nm, and for variable exposure times between 0.5 and 320 s. With one exception (365 nm), the curves were biphasic. The low fluence component was generally sigmoidal with an abrupt rise. The high fluence component failed to reach saturation for the fluences tested (less than 70 μmol m⁻² s⁻¹). Using the inferred threshold fluences of these two components as criterion effects, we obtained two action spectra. For the low fluence component, the action spectrum showed major peaks at 394, 450, and 530 nm and a minor peak at 416 nm. The high fluence component action spectrum showed very little sensitivity in the blue region. The major sensitivity was in the near UV, and a relatively small peak appeared in the green part of the spectrum at 507 nm. The biphasic character of the fluence-response curves suggests that two photosystems are responsible for the absorbance changes. The low fluence photosystem is sensitive mainly to blue and UV light and may thus represent a physiological blue-light photoreceptor. The high fluence photosystem is clearly not of this type. It (and perhaps the low fluence system as well) may mediate some of the subsidiary physiological effects of green light.     

Analyses of structure of photoreceptor organelle and blepharismin-associated protein in unicellular eukaryote Blepharisma

In the ciliated protozoan Blepharisma, step-up photophobic response is believed to be mediated by a novel type of photosensory pigment known as "blepharismins" (BL) that are contained in the pigment granules located just beneath the plasma membrane. We examined the ultrastructure of the pigment granules by freeze-fracture and thin-section electron microscopy and proposed a schematic diagram showing the granules' three-dimensional inner membranous structure. Some of the BL are suggested to be associated with 200 kDa membrane protein. High-pressure liquid chromatography analysis of pigment species associated with 200 kDa protein obtained from blue forms of Blepharisma (oxyblepharisma) revealed that the 200 kDa protein was associated with five types of oxyblepharismin. The fluorescence intensity was increased when the pigments were dissociated from the 200 kDa protein. The result supports the hypothesis that the pigment-200 kDa complex is able to transduce light energy into signals mediating the photobehavior of Blepharisma.     

氧化银纳米粒子的制备及其动态受激荧光

采用真空蒸发沉积法和辉光放电氧化法,制备了粒径在5～30 nm之间的可控、空间分布均匀、高纯度的氧化银纳米薄膜,使薄膜成岛状生长和避免光照是制备过程中的两个关键问题.用XPS分析了Ag 3d和O 1s轨道的结合能,计算了银和氧的原子比,证明其成分为Ag2O,用XRD确定了氧化银纳米薄膜最强的衍射峰分别对应Ag2O的（111）、（110）、（200）、（211）晶面族.研究了这种薄膜在可见光波段的光吸收,计算得氧化银的禁带宽度为2.8 eV.在蓝光持续照射几分钟激活后,观察到了其中纳米粒子在蓝光激发下发黄光和绿光,在绿光激发下发红光的现象,这种光致发光具有动态“闪烁”的特点.提出了氧化银光分解引入缺陷能级（如Ag3O, Ag2＋O和Ag3＋O）新的理论模型并对此现象进行定性的解释.     

Role of ocular melanin in ophthalmic physiology and pathology

The mammalian eye consists of several layers of pigmented tissues that contain melanin. The eye is a unique organ for pigment cell research because one can isolate and compare melanosomes from different tissues and embryonic origins. Retinal, iris and ciliary pigment epithelial cells are derived from the neural ectoderm, more specifically from the extremity of the embryonic optical cup, which is also the origin of the retina. In contrast, the pigment-generating cells in the choroid and in the stroma of the iris and ciliary body, uveal melanocytes, are developed from the neural crest, the same origin as the melanocytes in skin and hair. This review examines the potential functions of ocular melanin in the human eye. Following a discussion of the role of melanins in the pigment epithelium and uveal melanocytes, three specific topics are explored in detail-photo-screening protective effects, biophysical and biochemical protective effects, and the biologic and photobiologic effects of the two main classes of melanins (generally found as mixtures in ocular melanosomes)--eumelanin and pheomelanin.