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.     

Environmental effects on the photochemistry of A2-E, a component of human retinal lipofuscin

Several retinal dystrophies are associated with the accumulation of lipofuscin, a pigment mixture, in the retinal pigment epithelium (RPE). One of the major fluorophores of this mixture has been identified as the bis-retinoid pyridinium compound, A2-E. Because this compound absorbs incident radiation that is transmitted by the anterior segment of the human eye, photophysical and photochemical studies were performed to determine if A2-E could photosensitize potentially damaging reactions. Steady-state fluorescence measurements indicate that the fluorescence emission maximum and quantum yield are very sensitive to the chemical environment and a correlation between these two parameters and the solvent dielectric constant is observed. Time-resolved absorption experiments of A2-E in pure organic solvents showed no formation of transient species on the timescale of our experiments. However, when these measurements were repeated for A2-E in Triton X-100 micelles, a short-lived (tau approximately 14 microseconds), weak absorption was observed. This species is quenched by oxygen (k = 2 x 10(9) M-1 s-1) and by the addition of the antioxidants, cysteine and N,N,N',N'-tetramethylphenylenediamine. Quenching of this species by 2,3,5-trimethylhydroquinone results in the formation of the 2,3,5-trimethylsemiquinone free radical and an increase in yield of the A2-E-derived species. Sensitization of the A2-E triplet excited state indicates that the species observed in micelles upon direct excitation is not consistent with the triplet excited state. Based on these data we tentatively assign this absorption to a free radical. In the RPE these initial processes can ultimately lead to damage to the tissue through the formation of peroxides and other oxidized species.     

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

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

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.     

Photophysical Studies of A2-E, Putative Precursor of Lipofuscin, in Human Retinal Pigment Epithelial Cells

With age, human retinal pigment epithelial cells accumulate lipofuscin that can absorb photons in the visible range leading to light-induced damage and impaired vision. A putative precursor of lipofuscin, 2-[2,6-dimethyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E, 5E,7E- octatetraenyl]-1-(2-hydroxyethyl)-4-[4-methyl-6-(2,6,6-trimethyl-1 - cyclohexen-1-yl)-1E,3E,5E-hexatrienyl]-pyridinium (A2-E), has recently been isolated and characterized from aged human retinal pigment epithelial cells. We have found that A2-E inhibits the growth of human retinal pigment epithelial cells at concentrations greater than 1 microM. Time-resolved fluorescence measurements of 1 microM A2-E in solution, performed under 413 nm excitation, showed that fluorescence wave forms integrated across the spectrum (450-600 nm) were best-fitted with three decay times in the nanosecond and subnanosecond time scale: 6.6, 1.9 and 0.33 ns. Untreated retinal pigment epithelial cells were characterized by three fluorescence lifetimes: 6.3, 1.7 and 0.35 ns. In retinal pigment epithelial cells treated with 1 microM A2-E, the fluorescence decay was significantly faster, with the marked presence (approximately equal to 30%) of a fourth short lifetime (0.12 ns). These fluorescence decay times for A2-E bound to human retinal pigment epithelial cells are similar to those of lipofuscin granules isolated from aged human retinal pigment epithelial cells. This similarity supports the hypothesis that A2-E is a precursor of lipofuscin and suggests that A2-E may play a role in the overall light damage associated with age-related retinal diseases.     

A2E: a component of ocular lipofuscin

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

Compositional studies of human RPE lipofuscin: mechanisms of molecular modifications

The accumulation of lipofuscin has previously been implicated in several retinal diseases including Best's macular dystrophy, Stargardt's disease and age-related macular degeneration (AMD). Previously one of the major fluorophores of lipofuscin was identified as a bis-retinoid pyridinium salt called A2E, which is known to photochemically cause damage. In addition to A2E, there are numerous components in RPE lipofuscin that are unidentified. These compounds were determined to be structurally related to A2E by their fragmentation pattern with losses of 106, 190, 174 and/or 150 amu from the parent ion and the formation of fragments of ca 592 amu. The vast majority consists of relatively hydrophobic components corresponding to derivatized A2E with molecular weights in discrete groups of 800-900, 970-1080 and > 1200 m/z regions. In order to determine the mechanism of these modifications, A2E was chemically modified by; (1) the formation of specific esters, (2) reaction with specific aldehydes and (3) spontaneous auto-oxidation. The contribution of ester formation to the naturally occurring components of lipofuscin was discounted since their fragmentation patterns were different to those found in vivo. Alternatively, reactions with specific aldehydes result in nearly identical products as those found in vivo. Artificial aging of RPE lipofuscin gives a complex mixture of structurally related components. This results from the auto- and/or photooxidation of A2E to form aldehydes, which then back react with A2E giving a series of higher molecular weight products. The majority of these modifications result in compounds that are much more hydrophobic than A2E. These higher molecular weight materials have increased values of log P compared to A2E. This increase in hydrophobicity most likely aids in the sequestering of A2E into granules with the concomitant diminution of its reactivity. Therefore, these processes may serve as protective mechanisms for the RPE.     

Photochemistry of single wall carbon nanotubes embedded in a mesoporous silica matrix

By embedding single wall carbon nanotubes in a mesoporous silica matrix (SWNT@SiO2) the photochemical properties have been measured upon laser excitation at 266 nm; the SWNT@SiO2 exhibits long-lived emission (lambda em = 400 nm, tau = 0.95 microsecond), transient absorption (lambda max = 390 nm, tau = 11 microseconds) and is able to generate singlet oxygen in D2O.     

Transmission of light to the young primate retina: possible implications for the formation of lipofuscin

The purpose of this study was to determine the transmission properties of the anterior segment of young primate eyes and potentially relate those changes to photochemical processes in the retina that lead to the early, rapid formation of lipofuscin. A simple method has been developed to determine the optical properties of the anterior segment of the intact eye. Using this technique, the transmission/absorption properties of primate cadaver eyes were determined. A young primate anterior segment has a maximum absorption at 365nm due to the presence of the O-β-glucoside of 3-hydroxykynurenine in the lens. This is synthesized in the last trimester of gestation. Although this compound filters out most of the UV light from reaching the retina, there is a small window of transmission centered on an absorption minimum at 320nm. This closes by the second decade of life. The window of transmission of UV light to the primate retina may explain the initial accelerated formation of lipofuscin in the young human retina by a photochemical process. This would be exacerbated by any decrease in the ozone layer with concomitant increase in UV-B reaching the earth's surface.     

THE PHOTOCHEMICAL YIELD OF SINGLET OXYGEN FROM PORPHYRINS IN DIFFERENT STATES OF AGGREGATION

Abstract— Aqueous solutions of hematoporphyrin and hematoporphyrin derivatives were exposed to light. When present in such solutions tryptophan is degraded by a singlet oxygen mechanism. This is true for excitation at 396 nm, where porphyrin monomers have their absorption maximum, as well as for excitation at 360 nm, where porphyrin aggregates seem to absorb strongly. The quantum yield of singlet oxygen production is similar within 25% for excitation at 396 and 360 nm while the fluorescence quantum yield is more than a factor 2 lower for excitation at 360 nm than for excitation at 396 nm. Photoexcitation of the clinically used hematopotophyrin derivatives photofrin I and photofrin II produces singlet oxygen with significantly smaller yields than photoexcitation of hematoporphyrin. Thus, the aggregates present in solutions of photofrin I and photofrin II are of a different nature than those present in aqueous solutions of hematoporphyrin.     

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.     

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

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

Different molecular constituents in pheomelanin are responsible for emission, transient absorption and oxygen photoconsumption

Steady-state absorption and emission spectroscopies, oxygen activation and transient spectroscopy on a single sample of synthetic pheomelanin are compared. The absorption, emission and excitation spectra of pheomelanin depend on the molecular weight (MW) of the dissolved pigment constituents. While weakly-depending on MW, the maximum of the emission excitation spectrum is approximately 400 nm. The electron paramagnetic resonance oximetry measurements show a clear increase in oxygen uptake between 338 and 323 nm, and also reveal a local enhancement around approximately 370 nm. Pump-probe absorption spectroscopy reveals that photoexcitation of pheomelanin by UVA light generates a transient absorption peak in the visible and UV regions within the instrument response. The action spectrum for the formation of the 780 nm transient species peaks at approximately 360 nm. While both transient absorption bands show the same ultrafast decay component, the 780 nm peak completely vanishes on the 10s of picosecond time scale, but the UV band shows a kinetic evolution to a subsequent intermediate. While in a similar wavelength range, the maximum of the action spectrum derived from the transient data, the emission excitation spectrum and the action spectrum for photoconsumption all differ from one another, suggesting that the chromophore responsible for each is not that same. This raises concern about comparing the results from different photochemical methodologies for melanin, which is a specific case of comparing data on systems where molecular constituents are not well defined.     

Photobleaching of hypericin bound to human serum albumin,cultured adenocarcinoma cells and nude mice skin

Hypericin is a promising photosensitizer for photodynamic therapy (PDT) characterized by a high yield of singlet oxygen. Photobleaching of hypericin has been studied by means of absorption and fluorescence spectroscopy in different biological systems: in human serum albumin solution, in cultured human adenocarcinoma WiDr cells and in the skin of nude mice. Prolonged exposure to light (up to 95 min, 100 mW/cm2) of wavelength around 596 nm induced fluence-dependent photobleaching of hypericin in all studied systems. The photobleaching was not oxygen dependent, and singlet oxygen probably played no significant role. Emission bands in the spectral regions 420-560 nm and above 600 nm characterize the photoproducts formed. An emission band at 615-635 nm was observed after irradiation of cells incubated with hypericin or of mouse skin in vivo but not in albumin solution. The excitation spectrum of these products resembled that of hypericin. Hypericin appears to be more photostable than most sensitizers used in PDT, including mTHPC and Photofrin.     

Photochemistry and phototoxicity of aloe emodin

Photochemical pathways leading to the phototoxicity of the aloe vera constituent aloe emodin were studied. The results indicate a photochemical mechanism involving singlet oxygen to be the most likely pathway responsible for the observed phototoxicity. Aloe emodin was found to efficiently generate singlet oxygen when irradiated with UV light (phidelta = 0.56 in acetonitrile). The survival of human skin fibroblast cells in the presence of aloe emodin was found to decrease upon irradiation with UV light. A further decrease in cell survival was observed in D2O compared with H2O, suggesting the involvement of singlet oxygen as the primary pathway. Laser flash photolysis experiments were also carried out on aloe emodin alone and in the presence of various biological substrates. Aloe emodin proved to be relatively photostable (phi = 1 x 10(-4)) and a poor photo-oxidant (E*red = +1.02 V). Only absorption bands caused by the triplet state of aloe emodin (lambdamax = 480 nm) and the aloe emodin conjugate base (lambdamax = 520 nm) were observed in the transient spectra.     

Comparison of the aerobic photoreactivity of A2E with its precursor retinal

A2E (2-[2,6-dimethyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E, 3E,5E,7E-octatetraenyl]-1-(2-hydroxyethyl)-4-[4-methyl-6(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E-hexatrienyl]pyridinium) is a blue-absorbing molecular constituent of human ocular lipofuscin and contributes to the golden-yellow emission of this pigment. Lipofuscin photoproduces toxic reactive oxygen intermediates (ROI), but the specific molecular components responsible for this phototoxicity remain unidentified. In this article the aerobic photoreactivity of A2E is quantified by comparison with its biosynthetic precursor, all-trans-retinal, and with other appropriate standards. Under blue-light exposure the efficacies for formation of cholesterol (Ch) hydroperoxides and the superoxide radical anion (O2*-) were determined using high-pressure liquid chromatography with electrochemical detection and electron spin resonance oximetry and spin trapping, respectively. Photogeneration of singlet oxygen after blue-light excitation of A2E was demonstrated unambiguously by the Ch peroxidation assay. After blue-light irradiation of A2E, O2*- were detected, but the concentration was insufficient to account for the measured production of O2*- by the solvent extract of lipofuscin granules. The collective data support the conclusion that A2E does not produce sufficient concentrations of ROI to be the primary phototoxic constituent of lipofuscin.     

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

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

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

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

Singlet oxygen generation via two-photon excited FRET

A new approach to two-photon excited photodynamic therapy has been developed. A dendritic array of eight donor chromophores capable of two-photon absorption (TPA) was covalently attached to a central porphyrin acceptor. Steady-state fluorescence measurements demonstrated that the donor chromophores transfer excited-state energy to the porphyrin with 97% efficiency. Two-photon excitation of the donor chromophores at 780 nm resulted in a dramatic increase in porphyrin fluorescence relative to a porphyrin model compound. Enhanced singlet oxygen luminescence was observed from oxygen-saturated solutions of the target compound under two-photon excitation conditions.     

One- and two-photon singlet oxygen generation with new fluorene-based photosensitizers.

The spectral properties of new fluorene-based photosensitizers for efficient singlet oxygen production are investigated at room temperature and 77 K. Two-photon absorption (2PA) cross-sections of the fluorene derivatives are measured by the open aperture Z-scan method. The quantum yields of singlet oxygen generation under one- and two-photon excitation (phi(delta) and 2PAphi(delta), respectively), are determined by the direct measurement of singlet oxygen luminescence at approximately 1270 nm. The values of phi(delta) are independent of excitation wavelength, ranging from 0.6-0.9. The singlet oxygen quantum yields under two-photon excitation are 2PAphi(delta) approximately 1/2 phi(delta), indicating that the two processes exhibit the same mechanism of singlet oxygen production, independent of the mechanism of photon absorption.