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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.

The pigment melanin, which is believed to play a photoprotective role, was quantified here in human RPE cells from donors of different age. Electron spin resonance (ESR) spectroscopy was shown to provide a quantitative measure of melanin and was used as a non-destructive measure of melanin content. Results indicated an age-related melanin loss in RPE cells, with melanin content diminishing 2.5-fold between the first and the ninth decade of life. To determine whether photo-oxidation may contribute to age-related changes in RPE melanin, RPE in human eyecups, isolated human and bovine RPE cells, purified melanin granules, or synthetic dopa melanin were irradiated with various wavelengths and intensities of visible light. Samples were analysed for changes in melanin content by ESR spectroscopy, and by absorption and emission spectrophotometry. The concentration of hydrogen peroxide was measured in some samples, and some human eyecups were examined by transmission electron microscopy. Irradiation of RPE in eyecups with intense visible light was found to produce a time-dependent photobleaching of melanosomes that was accompanied by the formation of hydrogen peroxide. Photobleaching of isolated RPE melanosomes and synthetic dopa melanin resulted in enhanced melanin fluorescence, as previously shown for melanin from aged donors by others, and significantly reduced ESR signal intensity, resembling the changes in melanin with aging observed here. We conclude that the content of melanin in RPE cells undergoes an age-related change to which photo-oxidation may contribute. This observation raises the question of whether age-related changes in melanin reduce the photoprotective role of the pigment in aging RPE cells.

The binding of tetra(4-N,N,N,N-trimethylanilinium)porphyrin (TAP) to melanins quenches the porphyrin emission. Time-resolved femtosecond absorption spectroscopy reveals that the mechanism behind this quenching is ultrafast nonradiative energy transfer ((tau)ET < 100 fs) from electronically excited TAP to melanin. Similar dynamics are observed for both dopa and cysteinyldopa melanins. Steady-state emission studies demonstrate that the emission from melanin increases upon excitation of bound TAP, thereby confirming that rapid energy transfer occurs. These results are consistent with previous photoacoustic studies, which revealed that the TAP-melanin complex behaves like a supermolecular system liberating heat as a whole.

Although 8-methoxypsoralen (8-MOP) has been successfully used in extracorporeal photochemotherapy (ECP) of several T cell-mediated diseases, the exact mechanism of the drug therapeutic action has not been established. We have studied in vitro apoptotic activity of 8-MOP, and for comparison of 4,6,4'-trimethylangelicin (TMA) and chlorpromazine (CPZ) as alternative photosensitizers for potential use in photopheresis. However, while 8-MOP and CPZ are known for their immune suppressive activity, TMA does not exhibit such an activity in an animal model for ECP. Apoptosis and necrosis were measured in both Jurkat cells and primary rat leukocytes under conditions comparable to those used in the animal model to suppress contact hypersensitivity (CHS). Cells were irradiated with UVA (200 kJ/m(2)) after treatment with 8-MOP, CPZ or TMA (300 ng/ml). Flow cytometric analysis (annexin-V-FLUOS/propidium iodide) and fluorescence microscopy examinations, using acridine orange/propidium iodide, indicated that the number of cells undergoing apoptosis or necrosis increased significantly after 24 h following treatment. Similar results were observed irrespective of the cell type and photosensitizer used. The results of the present study, combined with previous observations with the animal model for ECP, suggest that apoptosis is not likely to be a critical step in the cascade of events leading to immunosuppression.

The action spectra for the photoconsumption of oxygen by lipofuscin isolated from human retinal pigment epithelium cells and liposomal suspensions containing extracts of lipofuscin are reported. The lipofuscin and lipofuscin extract action spectra are similar, demonstrating the phototoxic constituents of lipofuscin are present in the lipofuscin solvent extract. 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 (A2E), present in both intact granules and the solvent extract, has been invoked as an important contributor to the phototoxicity of lipofuscin. The action spectrum for oxygen photoconsumption by A2E follows its absorption spectrum but does not resemble the action spectrum for photoconsumption of oxygen by lipofuscin granules or lipofuscin extract. These results combined with recently reported experimental studies on the aerobic photoreactivity of A2E indicate that it is not a major contributor to the phototoxicity of lipofuscin.

Purpose: To determine whether aging is accompanied by changes in aerobic photoreactivity of retinal pigment epithelial (RPE) melanosomes isolated from human donors of different ages, and to compare the photoreactivity of aged melanosomes with that of RPE lipofuscin., Methods: Human RPE pigment granules were isolated from RPE cells pooled into groups according to the age of the donors. Photoreactivity was determined by blue-light-induced oxygen uptake and photogeneration of reactive oxygen species. Short-lived radical intermediates were detected by spin-trapping, hydrogen peroxide by an oxidase electrode, singlet oxygen by cholesterol assay, and lipid hydroperoxides by iodometric assay., Results: Blue-light photoexcitation of melanosomes resulted in age-related increases in both oxygen uptake and the accumulation of superoxide anion spin adducts. The efficiencies of these processes, however, were still significantly lower than that induced by photoexcited lipofuscin. During irradiation of melanosomes, a substantial amount of oxygen was converted into hydrogen peroxide, whereas for lipofuscin, hydrogen peroxide accounted for not more than 3% of oxygen consumed. In contrast to lipofuscin, photoexcited melanosomes did not substantially increase the rate of oxidative reactions in the presence of polyunsaturated lipids or albumin. However, oxygen uptake was significantly elevated in the presence of ascorbate. Thus, the rate of photo-induced oxygen uptake in samples containing both ascorbate and melanosomes approached that observed in lipofuscin samples., Conclusions: Blue-light-induced photoreactivity of melanosomes increases with age, perhaps providing a source of reactive oxygen species and leading to depletion of vital cellular reductants, which, together with lipofuscin, may contribute to cellular dysfunction.

Urocanic acid, UCA, is characterized by two electronic transitions in the UV-B (280-320 nm) which comprise its broad absorption spectrum and give rise to wavelength-dependent isomerization quantum yields. The absorption spectrum of UCA extends into the UV-A (320-400 nm). Given the UV-A component of sunlight is significantly greater than the UV-B component it is hypothesized even weak UV-A photochemistry of UCA could be important for in vivo responses to UV radiation. Degenerate pump-probe experiments performed on t-UCA at several wavelengths in the UV-A reveal an excited-state absorption that undergoes a rapid, approximately 1 ps decay. Photoacoustic experiments performed on both the cis and trans isomers reveal the formation of a long-lived intermediate following UV-A excitation. The efficiency and action spectra for this latter photoactive process are presented and are similar for both isomers of UCA. Cholesterol hydroperoxide assays designed to investigate the nature of the UV-A photoreactivity of t-UCA confirm the production of reactive oxygen species. The bimolecular rate constant for the quenching of singlet oxygen by t-UCA is determined to be 3.5 x 10(6) M(-1) s(-1). Taking into consideration recent theoretical calculations and jet expansion studies of the electronic structure of gas-phase t-UCA, a model is proposed to explain the isomerization and photoreactivity of t-UCA in solution over the UV-A region.

Lipofuscin accumulation in the retinal pigment epithelium is associated with the onset of age-related macular degeneration. Lipofuscin is phototoxic and affects cellular function through the photochemical generation of reactive oxygen intermediates. Mass spectral analysis of solvent extracts of human retinal lipofuscin granules reveals the presence of retinyl palmitate, the substrate for the enzymatic regeneration of 11-cis-retinal. Retinyl palmitate has an appreciable binding constant for phosphatidylcholine liposomes, and based on the glycophospholipids present in lipofuscin, retinal palmitate likely accumulates within the lipid content of the granule. Photochemical oxidation of retinal palmitate generates anhydroretinol, an intracellular signaling retinoid in the signal transduction cascade from the plasma membrane that causes apoptosis by generating reactive oxygen intermediates. These data are used to propose a model for the phototoxicity of lipofuscin., (Copyright 2001 Academic Press.)

The emission spectra of single lipofuscin granules are examined using spectrally resolved confocal microscopy and near-field scanning optical microscopy (NSOM). The emission spectrum varies among the granules examined revealing that individual granules are characterized by different distributions of fluorophores. The range of spectra observed is consistent with in vivo spectra of human retinal pigment epithelium cells. NSOM measurements reveal that the shape of the spectrum does not vary with position within the emissive regions of single lipofuscin granules. These results suggest that the relative distribution of fluorophores within the emissive regions of an individual granule is homogeneous on the spatial scale approximately 150 nm.

Using scanning tunneling microscopy (STM), we have imaged two types of mildly-bleached, synthetic tyrosine-derived melanins for comparison with the unbleached melanin from which they were prepared. These mildly-bleached melanins were generated by mild oxidation of the unbleached melanin, using either basic hydrogen peroxide or air/light. The unbleached melanin, and two mildly-bleached melanins, were independently deposited from very dilute tetrahydrofuran (THF) solutions onto highly oriented pyrolytic graphite (HOPG) substrate for STM imaging. Lateral dimensions (23 A, average of two directions) of structures from each of the three samples showed no differences. However, structures from both mildly-bleached melanins showed similar dramatic decreases (from approximately 15 A to approximately 5 A) in their STM-measured apparent heights, compared with structures from the unbleached melanin sample. These STM observations are compatible with structural models for unbleached and mildly-bleached melanins, incorporating a three-dimensional structure for unbleached melanin composed of multi-layered, pi-pi-stacked, carboxylic and amino variants of polyaromatic polymeric sheets. The STM-observed decrease in apparent heights after mild oxidation, which we associate with a change in stack height, has been confirmed by experiments using tapping mode atomic force microscopy (TM-AFM) for the unbleached and mildly-hydrogen-peroXide-bleached melanins (from approximately 14 A to approximately 6 A). In these TM-AFM experiments, the melanins were deposited directly onto magnesium cation-treated glass substrates in contact with methanolic solutions of each of the melanins. We interpret our mild-bleaching results as an oxidative conversion of the multi-layered, stacked sheets of mainly carboxylic and amino variants of polyquinhydrone-like moieties, to largely de-stacked, mildly-bleached melanin sheets. These oxidized and, hence, electron-deficient sheets should not readily form multi-layered, pi-pi interacting stacks, but instead appear to be either single-layer polyquinone sheets or, at most, double-layer polyquinhydrone sheets. The effects of such de-stacking on in vivo melanin photoprotection, and structural similarities between melanin derived from natural sources and the synthetic melanin samples used in this work are discussed.

To determine the role of major chromophores of the human retinal pigment epithelium (RPE) in photooxidation of ascorbate, we monitored spectrophotometrically rates of ascorbate depletion, induced by blue light, in suspensions of human RPE melanin, melanolipofuscin and lipofuscin and in preparation of pigmented and nonpigmented bovine RPE cells. The results clearly show that melanin is the key retinal pigment responsible for the photosensitized oxidation of exogenous ascorbate. To elucidate the mechanism of the photooxidation process, we used purified RPE melanin granules and synthetic dopa (dihydroxyphenylalanine) melanin and employed electron spin resonance (ESR) spectroscopy, ESR oximetry and oxidase electrode. Our data indicate that photoinduced melanin radicals oxidize ascorbate via one-electron transfer reaction. The reduced melanin is reoxidized by molecular oxygen with the formation of superoxide anion and hydrogen peroxide, while the ascorbate radicals decay by disproportionation. Because in the absence of oxygen, no measurable oxidation of ascorbate is observed, it can be concluded that melanin acts as an electron transfer agent. Biological implications of this study remain unclear; however, the formation of oxygen-reactive species that accompany melanin-mediated photooxidation of ascorbate may represent a potential risk to the RPE that should be minimized by yet unknown cellular mechanisms.

Neuromelanin is an amorphous pigment of the catecholamine origin that accumulates in certain dopaminergic neurons of the substantia nigra of human brain. In Parkinson's disease, there appears to be selective degeneration of the most heavily pigmented neurons of the substantia nigra, and this process has been linked to the presence of neuromelanin. It has been postulated that neuromelanin could increase the risk of oxidative stress reactions. On the other hand, melanin is usually considered to be an efficient antioxidant. Here we analyze experimental conditions that stimulate, or inhibit, antioxidant properties of neuromelanin. Using electron spin resonance (ESR)--spin trapping technique and salicylate hydroxylation assay, we monitored the formation of free hydroxyl radicals generated by a Fenton system in the presence of varying concentration of dopamine-melanin, a synthetic model for neuromelanin. Our data clearly indicate that the antioxidant action of neuromelanin is predominantly due to its ability to sequester redox-active metal ions such as iron. Using direct ESR spectroscopy, we have shown that ferric complexes with neuromelanin are resistant to reduction by mild biological reductants such as ascorbate. We have demonstrated that dopamine-melanin saturated with ferric ions, could enhance the formation of free hydroxyl radicals by redox activation of the ions. Thus, under the conditions that stimulate the release of accumulated metal ions, neuromelanin may actually become an efficient prooxidant. It is conceivable that neuromelanin, which normally is able to protect pigmented dopaminergic neurons against metal-ion related toxicity, could under extreme conditions have a cytotoxic role.

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 application of recent developments of EPR oximetry to photosynthetic systems is described and used to study rapid processes in isolated thylakoid membranes from spinach and in intact photoautotrophic soybean cells. Using the peak heights of 15N perdeuterated Tempone and two microwave power levels oxygen evolution and consumption were measured. The method measured time-resolved oxygen concentration changes in the micromolar range. Oxygen evolution was linearly proportionate to the chlorophyl concentration of thylakoid membrane over the range studied (0-2 mg/ml). Oxygen evolution associated with single turnover light pulses was consistent with the four state model. The time (t1/2) to reach equilibrium of oxygen concentrations after a single turnover pulse was 0.4-0.5 ms, indicating that the evolution of oxygen coupled to the S4-S0 transition may be shorter than reported previously. The time for equilibrium of oxygen after single turnover pulses in soybean cells was relatively long (400 ms), which suggests that there are significant barriers to the free diffusion of oxygen in this system. The method also was used to study oxygen consumption by the electron transport chain of photosystem I and photosystem II. We conclude that EPR oximetry can provide quantitative and time-resolved data on oxygen concentrations with a sensitivity that is useful for studies of such systems.

Pulse radiolysis studies of anisyl-3,4-semiquinone, formed in the metabolic activation of 4-hydroxyanisole, a possible melanocytotoxic drug under current assessment as a treatment for malignant melanoma, have shown this semiquinone to be unreactive towards oxygen (k less than or equal to 10(5) M-1 s-1), although the reverse reaction of O2- with anisyl-3,4-quinone is very rapid (k = 8.7 x 10(8) M-1 s-1). Since 1,4 benzoquinone is also unreactive towards anisyl-3,4-semiquinone (k less than or equal to 10(5) M-1 s-1), the one-electron reduction potential, E17 (anisyl-3,4-quinone/anisyl-3,4-semiquinone), is likely to be considerably more positive than 0.1 V. This suggests that the cytotoxicity mechanism does not involve the generation of O2- and possible subsequent production of H2O2 and/or OH., leading to lipid peroxidation, as previously proposed, but rather involves as yet unknown reactions of anisyl-3,4-quinone. This quinone is unstable in water and its absorption spectrum was measured immediately (less than 0.1 s) following disproportionation of anisyl-3,4-semiquinone, before significant decay of the quinone had occurred.

The oxidation of four catechol(amine)s by tyrosinase has been studied by electron spin resonance and optical methods. Rates of oxygen consumption and of dopaquinone and dopachrome formation during the oxidation of dopa have been measured, and compared with rates of dopasemiquinone production measured using spin-stabilization procedures. In the presence of spin-stabilizing metal ions, production of semiquinone is approximately quantitative. Time-dependent ESR spectra obtained from dopa and dopamine show a slow regeneration of semiquinone, suggesting that a semiquinone precursor is slowly reformed. In contrast, time-dependent spectra for 4-methylcatechol and N-acetyldopamine show decay of the primary semiquinone together with buildup of a secondary semiquinone apparently derived from the corresponding 6-hydroxy-catechol(amine). Thus, catecholamines that give rise to a cyclizable quinone show a pattern of behavior that differs from those that produce a non-cyclizable quinone. These results are discussed in terms of their possible significance to melanogenesis and the toxicity of catechol(amine)s, which has been attributed to production of semiquinones and/or other oxygen radicals.

The nature and origin of a g = 1-98 signal observed in ESR investigations of tissue have been established. It is due to paramagnetic chromium which is introduced into tissues during sample handling. This characteristic signal could be used to measure chromium levels in tissues.

A new method for the unambiguous identification of melanin in biological materials has been developed. It may also be used to differentiate between melanins from various tissues and with various properties. The method is based on the detection and characterization of the free radicals in melanin by Electron Spin Resonance Spectroscopy. Applications of this approach include the identification of microscopically undetectable melanin in amelanotic melanomas and identification of the nature of the pigment in the Dubin-Johnson Syndrome.