Retinal Pigment Epithelium Pigment Granules: Norms, Age Relations and Pathology.

The retinal pigment epithelium (RPE), which ensures the normal functioning of the neural retina, is a pigmented single-cell layer that separates the retina from the Bruch's membrane and the choroid. There are three main types of pigment granules in the RPE cells of the human eye: lipofuscin granules (LG) containing the fluorescent "age pigment" lipofuscin, melanoprotein granules (melanosomes, melanolysosomes) containing the screening pigment melanin and complex melanolipofuscin granules (MLG) containing both types of pigments simultaneously-melanin and lipofuscin. This review examines the functional role of pigment granules in the aging process and in the development of oxidative stress and associated pathologies in RPE cells. The focus is on the process of light-induced oxidative degradation of pigment granules caused by reactive oxygen species. The reasons leading to increased oxidative stress in RPE cells as a result of the oxidative degradation of pigment granules are considered. A mechanism is proposed to explain the phenomenon of age-related decline in melanin content in RPE cells. The essence of the mechanism is that when the lipofuscin part of the melanolipofuscin granule is exposed to light, reactive oxygen species are formed, which destroy the melanin part. As more melanolipofuscin granules are formed with age and the development of degenerative diseases, the melanin in pigmented epithelial cells ultimately disappears.

Understanding the Mechanism of Light-Induced Age-Related Decrease in Melanin Concentration in Retinal Pigment Epithelium Cells.

It is known that during the process of aging, there is a significant decrease in the number of melanosomes in the retinal pigment epithelium (RPE) cells in the human eye. Melanosomes act as screening pigments in RPE cells and are fundamentally important for protection against the free radicals generated by light. A loss or change in the quality of melanin in melanosomes can lead to the development of senile pathologies and aggravation in the development of various retinal diseases. We have previously shown that the interaction between melanin melanosomes and superoxide radicals results in oxidative degradation with the formation of water-soluble fluorescent products. In the present study, we show, using fluorescence analysis, HPLC, and mass spectrometry, that visible light irradiation on melanolipofuscin granules isolated from RPE cells in the human eye results in the formation of water-soluble fluorescent products from oxidative degradation of melanin, which was in contrast to lipofuscin granules and melanosomes irradiation. The formation of these products occurs as a result of the oxidative degradation of melanin by superoxide radicals, which are generated by the lipofuscin part of the melanolipofuscin granule. We identified these products both in the composition of melanolipofuscin granules irradiated with visible light and in the composition of melanosomes that were not irradiated but were, instead, oxidized by superoxide radicals. In the melanolipofuscin granules irradiated by visible light, ions that could be associated with melanin oxidative degradation products were identified by applying the principal component analysis of the time-of-flight secondary ion mass spectrometry (ToF-SIMS) data. Degradation of the intact melanosomes by visible light is also possible; however, this requires significantly higher irradiation intensities than for melanolipofuscin granules. It is concluded that the decrease in the concentration of melanin in RPE cells in the human eye with age is due to its oxidative degradation by reactive oxygen species generated by lipofuscin, as part of the melanolipofuscin granules, under the action of light.

Lipofuscin-Mediated Photic Stress Induces a Dark Toxic Effect on ARPE-19 Cells.

Lipofuscin granules from retinal pigment epithelium (RPE) cells contain bisretinoid fluorophores, which are photosensitizers and are phototoxic to cells. In the presence of oxygen, bisretinoids are oxidized to form various products, containing aldehydes and ketones, which are also potentially cytotoxic. In a prior study, we identified that bisretinoid oxidation and degradation products have both hydrophilic and amphiphilic properties, allowing their diffusion through the lipofuscin granule membrane into the RPE cell cytoplasm, and are thiobarbituric acid (TBA)-active. The purpose of the present study was to determine if these products exhibit a toxic effect to the RPE cell also in the absence of light. The experiments were performed using the lipofuscin-fed ARPE-19 cell culture. The RPE cell viability analysis was performed with the use of flow cytofluorimetry and laser scanning confocal microscopy. The results obtained indicated that the cell viability of the lipofuscin-fed ARPE-19 sample was clearly reduced not immediately after visible light irradiation for 18 h, but after 4 days maintaining in the dark. Consequently, we could conclude that bisretinoid oxidation products have a damaging effect on the RPE cell in the dark and can be considered as an aggravating factor in age-related macular degeneration progression.

Water-Soluble Products of Photooxidative Destruction of the Bisretinoid A2E Cause Proteins Modification in the Dark.

Aging of the retina is accompanied by a sharp increase in the content of lipofuscin granules and bisretinoid A2E in the cells of the retinal pigment epithelium (RPE) of the human eye. It is known that A2E can have a toxic effect on RPE cells. However, the specific mechanisms of the toxic effect of A2E are poorly understood. We investigated the effect of the products of photooxidative destruction of A2E on the modification of bovine serum albumin (BSA) and hemoglobin from bovine erythrocytes. A2E was irradiated with a blue light-emitting diode (LED) source (450 nm) or full visible light (400-700 nm) of a halogen lamp, and the resulting water-soluble products of photooxidative destruction were investigated for the content of carbonyl compounds by mass spectrometry and reaction with thiobarbituric acid. It has been shown that water-soluble products formed during A2E photooxidation and containing carbonyl compounds cause modification of serum albumin and hemoglobin, measured by an increase in fluorescence intensity at 440-455 nm. The antiglycation agent aminoguanidine inhibited the process of modification of proteins. It is assumed that water-soluble carbonyl products formed as a result of A2E photodestruction led to the formation of modified proteins, activation of the inflammation process, and, as a consequence, to the progression of various senile eye pathologies.

Mitigating effects of sublethal and lethal whole-body gamma irradiation in a mouse model with soluble melanin.

The field of radiation countermeasures is growing, however, currently there are no effective and non-toxic compounds which could be administered orally to the individuals post exposure to high doses of ionising radiation. The pigment melanin is ubiquitous through all kingdoms of life and provides selective advantage under radiation stress through its role as a chemical and physical shield, and its capacity to respond and react to exposures. Soluble allomelanin was administered to mice following whole-body exposure to lethal or sublethal doses of gamma radiation to determine its capacity to mitigate the effects of acute radiation syndrome, and its utility as a radiation countermeasure. Allomelanin has shown a trend to improve survival post an 8 Gy sublethal radiation exposure when administered up to 48 h post-irradiation. Furthermore, it improved median and overall survival to a 10 Gy lethal radiation exposure, specifically when administered at 24 h post-irradiation. Histological analysis on the jejunum region of the small intestine of this treatment group indicated that alterations of the mucosal and submucosal architecture, and disruption of the lymphatic system associated with lethal radiation exposure were mitigated when allomelanin was administered at 24 h post-irradiation. Based on this work soluble allomelanin derived from a fungal source could serve as an easily sourced, cost-effective, and viable countermeasure to accidental radiation exposure and merits further investigation.

Novel Extract from Beetle Ulomoides dermestoides: A Study of Composition and Antioxidant Activity.

A biologically active extract from the darkling beetle Ulomoides dermestoides was obtained using the electro-pulse plasma dynamic extraction method. The beetle water extract contained a complex of antioxidant substances such as antioxidant enzymes and nonprotein antioxidants, as well as a complex of heat shock antistress proteins. This determines the rather high antioxidant activity of the aqueous extract of the beetle, i.e., 1 mg of dry matter/mL of the extract has an equivalent antioxidant activity to 0.2 mM Trolox (a water-soluble analog of vitamin E). It was shown that the beetle extract can lead to a 25-30% increase in the average lifespan of nematode Caenorhabditiselegans, under normal conditions, and a 12-17% increase under conditions of oxidative stress (with paraquat), and significantly inhibits the fructosylation reaction of serum albumin. Therefore, the beetle aqueous extract shows promise as a biologically active complex exhibiting antioxidant activity.

Lipofuscin Granule Bisretinoid Oxidation in the Human Retinal Pigment Epithelium forms Cytotoxic Carbonyls.

Age-related macular degeneration (AMD) is the primary cause of central blindness among the elderly. AMD is associated with progressive accumulation of lipofuscin granules in retinal pigment epithelium (RPE) cells. Lipofuscin contains bisretinoid fluorophores, which are photosensitizers and are phototoxic to RPE and neuroretinal cells. In the presence of oxygen, bisretinoids are also oxidized, forming various products, consisting primarily of aldehydes and ketones, which are also potentially cytotoxic. In a prior study, we identified that in AMD, bisretinoid oxidation products are increased in RPE lipofuscin granules. The purpose of the present study was to determine if these products were toxic to cellular structures. The physicochemical characteristics of bisretinoid oxidation products in lipofuscin, which were obtained from healthy donor eyes, were studied. Raman spectroscopy and time-of-flight secondary ion mass spectrometry (ToF-SIMS) analysis identified the presence of free-state aldehydes and ketones within the lipofuscin granules. Together, fluorescence spectroscopy, high-performance liquid chromatography, and mass spectrometry revealed that bisretinoid oxidation products have both hydrophilic and amphiphilic properties, allowing their diffusion through lipofuscin granule membrane into the RPE cell cytoplasm. These products contain cytotoxic carbonyls, which can modify cellular proteins and lipids. Therefore, bisretinoid oxidation products are a likely aggravating factor in the pathogenesis of AMD.

Antioxidative Properties of Melanins and Ommochromes from Black Soldier Fly Hermetia illucens.

A comparative study of melanin and ommochrome-containing samples, isolated from the black soldier fly (BSF) by enzymatic hydrolysis, alkaline and acid alcohol extraction or by acid hydrolysis, was carried out. Melanin was isolated both as a melanin-chitin complex and as a water-soluble melanin. Acid hydrolysis followed by delipidization yielded a more concentrated melanin sample, the electron spin resonance (ESR) signal of which was 2.6 × 1018 spin/g. The ommochromes were extracted from the BSF eyes with acid methanol. The antiradical activity of BSF melanins and ommochromes was determined by the method of quenching of luminol chemiluminescence. It has been shown that delipidization of water-soluble melanin increases its antioxidant properties. A comparison of the antioxidant activity of BSF melanins and ommochromes in relation to photoinduced lipid peroxidation was carried out. The ESR characteristics of native and oxidized melanins and ommochromes were studied. It is assumed that H. illucens adult flies can be a useful source of natural pigments with antioxidant properties.

Lipofuscins prepared by modification of photoreceptor cells via glycation or lipid peroxidation show the similar phototoxicity.

AIM: To investigate the effect of two ways of lipofuscin production (lipid peroxidation and glycation) on lipofuscin fluorescence characteristics and phototoxicity and to compare them with the properties of natural lipofuscin. METHODS: Model lipofuscins were prepared on the basis of bovine photoreceptor outer segments (POS) with bisretinoid A2E addition. One set of samples was prepared from POS modified by lipid peroxidation, while another set from POS modified by glycation with fructose. Fluorescent properties and kinetics of photoinduced superoxide generation of model lipofuscins and human retinal pigment epithelium (RPE) lipofuscin were compared. The fluorescence spectra of samples were measured at 365 nm excitation wavelength and 380-650 emission wavelength. RESULTS: The fluorescence spectra of model lipofuscins are almost the same as the spectrum of natural lipofuscin. Visible light irradiation of both model lipofuscins and natural lipofuscin isolated from RPE cells leads to decrease of a fluorescence maximum at 550 nm and to appearance of a distinct, new maximum at 445-460 nm. The rate of photogeneration of reactive oxygen forms by both model lipofuscins was almost the same and approximately two times less than that of RPE lipofuscin granules. CONCLUSION: These data suggest that fluorescent characteristics and phototoxicity of lipofuscin granules depend only to an insignificant degree on the oxidative modification of POS proteins and lipids, and generally are defined by the bisretinoid fluorophores contained in them.