Photobiological effects upon the human retina, cornea and lens are highly dependent on the optical exposure geometry as well as spectral characteristics of the exposure. The organ of sight is exquisitely sensitive to light because it performs well in very low nighttime illumination levels and yet it also must adapt to extremely bright environments where light exposures are greater by many orders of magnitude. The eye has evolved to protect itself reasonably well against excessive exposure in bright environments. The retina is minimally exposed in extremely bright environments and the cornea and lens are surprisingly well protected in harsh environments. Although these protective mechanisms are good, they are not perfect and adverse changes from both acute and chronic exposures to sunlight still exist. The geometrical protective factors must be understood and appreciated whenever assessing potential adverse effects of environmental UV radiation and light on ocular structures. These natural ocular protective factors also work with the ever-changing spectrum of sunlight and the different spectral distribution of light and UV radiation across the eye's field of view. Spectral characteristics of the ocular media are also important. One can visualize a series of intraocular color filters that progressively filter shorter wavelengths and thereby aid in color vision, reduce the impact of chromatic aberrations and significantly reduce the optical radiation hazards to the lens and retina.

The adverse effects of sunlight, from melanoma to cataracts, are well known and frequently reported (1). However, because humans evolved under sunlight, it is not surprising that there are many positive effects of light on human health. Light that reaches the human eye has two fundamental biological functions: regulation of the visual cycle and of circadian rhythm. We report here the most recent developments in both of these areas.

Ultraviolet (UV)-mediated DNA damage in various tissues has been well documented. However, research on the damaging effect of UV irradiation on the DNA of corneal epithelium is scarce, even though this is of interest because the cornea is directly exposed to damaging solar (UV) radiation. In this study, we developed a corneal epithelium Comet assay model to assess the background DNA damage (as strand breaks) in cells retrieved from different layers of the porcine corneal epithelium, and to investigate the effect of UV irradiation on DNA damage in corneal epithelial cells. Results show that the background DNA strand breaks decreased significantly (P < 0.001) toward deeper layers of the epithelium. Exposure to the same intensity (0.216 J/cm²) of UVA, UVB and UVC caused a significant (P < 0.001) increase in DNA strand breaks of deeper-layer cells: mean ± SD %DNA scores (10 gels per treatment, with 100 irradiated cells scored per gel) were 10.2% ± 1.4% for UVA, 27.4% ± 4.6% for UVB, and 14.7% ± 1.8% for UVC compared with 4.2% ± 0.5% for controls (ambient room light). This study has shown for the first time that the Comet assay for DNA strand breaks can be used successfully with corneal epithelial cells. This report will support future studies investigating environmental influences on corneal health and the assessment of possible protective strategies, and in applying DNA lesion-specific versions of the Comet assay in this corneal epithelial cell model.

Cataract of the eye lens is characterized by (1) progressive opacification or loss of transparency; (2) accumulation of molecules that absorb in the UV-visible range of the spectrum; and (3) formation of fluid-filled vacuoles, particularly in diabetics when sugar levels in the lens are high. Of the various causative factors for cataract, light is an important one. Because the level of light below 320 nm reaching the lens is quite low, photodynamic effects of endogenous compounds absorbing in the UV-A-visible region become important in this connection. In this update we summarize a list of accumulant chromophores that have been identified in aging and cataract human lenses and their roles as potential sensitizers, antioxidants or as benign filters. Because such photodynamic cataractogenesis is oxidative in nature, we also cite examples where administration of antioxidants could help delay cataract progression.

The Scan Tox™ System is a method for monitoring lens optical quality (focus or lack of focus) in culture conditions, which mimic conditions inside the eye. The ocular lens is an ideal organ for long-term culture experiments because it has no direct blood supply and no connection to the nervous system. The Scan Tox™ System makes it possible to keep lenses for long-term studies of up to a few weeks. The use of cultured lenses, mainly bovine, replaces the need for testing the effects of potentially damaging agents on live animals. This optical monitoring apparatus uses a computer-operated scanning laser beam, a video-camera system and a video frame analyzer to record the focal length and transmittance of the cultured lens. The scanner is designed to measure the focal length at points across the diameter of the lens. The lens container permits the lens to be exposed to a vertical laser beam from below. The laser source projects its light onto a plain mirror, which is mounted at 45° on a carriage assembly. The mirror reflects the laser beam directly up through the test lens. The mirror carriage is connected to a positioning motor, which moves the laser beam across the lens. The camera sees the cross section of the beams and, by examining the image at each position of the mirror, Scan Tox™ software is able to measure the quality of the lens by calculating the back vertex distance for each beam position. The cultured lenses continue to maintain their original refractive function. When foreign substances are introduced to a cultured lens, the Scan Tox™ System measures the resulting optical response. This provides a very sensitive means to follow early damage to the eye lens. Because the lens is maintained in an intact state in solutions that are similar to those inside the eye, the lens retains its normal recuperative powers. So in addition to measuring early damage, this system allows measurement of recovery from damage.

There are two different types of ocular melanocytes and melanomas. Conjunctival melanocytes are located on the surface of the eye and are exposed to visible light and UV radiation. Recently, epidemiological studies demonstrated that sunlight plays a definite role in the occurrence of conjunctival melanoma, as it does in cutaneous melanoma. Uveal melanocytes consist of the iridal melanocytes, which are exposed to visible light and UV radiation; and the ciliary body melanocytes and choroidal melanocytes, which are not exposed to light radiation. Epidemiological studies demonstrated that sunlight may play a role in the occurrence of iridal melanoma, but may not be a major factor in the etiology of ciliary body and choroidal melanomas. Uveal melanocytes differ from epidermal melanocytes in that epidermal melanocytes respond to UV radiation and skin color becomes darker after exposure to sunlight; but uveal melanocytes do not respond to UV radiation and the iris color remains stable after exposure to sunlight. Recently, in vitro studies indicate that this phenomenon is determined both by cellular factors and environmental factors.

Melanosomes were isolated from the retinal pigment epithelium (RPE), iris and choroid of mature (age >2 years) and newborn (age <1 week) bovine eyes. Scanning electron microscopy was utilized to analyze the morphology of the melanosomes, which were found to vary among different tissues and different ages. While the total content of amino acids differs slightly (ranging from 9% to 15% by mass), the distributions of the amino acids are similar. The pheomelanin content is low in the choroid and the RPE (0.1–0.5%), and moderate in the iris (<2%); therefore, the major melanin component of bovine eye melanosomes is eumelanin, independent of the shape of the melanosomes. The yields of pyrrole-2,3,5-tricarboxylic acid from melanosomes decrease in the following order: choroid > iris > RPE, and exhibit decreasing yields with age. ¹³C solid-state nuclear magnetic resonance (NMR) spectroscopic analysis of iris and choroid melanosomes indicates the same trends. These observations suggest that the 5,6-dihydroxyindole-2-carboxylic acid contents decrease in the following order: choroid > iris > RPE, and decrease with age. Moreover, the ¹³C solid-state NMR spectra show (1) for the same age samples, the CH:Cq ratio for choroid is larger than that for iris melanosomes; and (2) an increase in the concentration of carbonyl groups with age within each type of melanosome.

Bovine iris and choroid melanosomes at two ages (<1 week and >2 years) were examined by inductively coupled plasma mass spectrometry (ICP-MS), elemental analysis, infrared spectrometry (IR) and X-ray photoelectron spectrometry (XPS). When iris and choroid melanosomes at the same age were compared, the quantification of metal elements by ICP-MS revealed that choroid melanosomes had a higher binding capacity for the carboxylate-binding metal ions (e.g. Na , K , Mg² , Ca² and Zn² ). Elemental analysis showed a higher O:N ratio in choroid melanosomes. Both observations suggested that choroid melanosomes have a higher content of carboxylate-containing monomer than iris melanosomes. IR spectrometric analysis showed a red shift (∼8 cm⁻¹) of the absorption peak of aromatic C=C, C=N and C=O at ∼1630 cm⁻¹ in the IR spectrum of iris melanosomes relative to choroid melanosomes. Increased conjugation in the molecular structure of the pigment is proposed to contribute to this peak shift. It is also notable that although the elemental analysis showed different C, N and O contents in the two types of melanosomes, XPS showed almost the same elemental compositions on the surface of two types of iris and choroid melanosomes studied. When the melanosomes from the same tissues at different ages were compared, ICP-MS analysis suggested that the number of carboxylate groups in the melanosomes decreased with age. Both elemental analysis and XPS showed that C:N ratio decreased with age, which was proposed to be due to both a decrease in carboxylate groups in mature samples and to the fissure of phenol rings caused by age-associated oxidation. Such age-related oxidative damage diminishes conjugation and is manifested by blue shifts of absorption peaks for aromatic double bonds in the IR spectra of mature melanosomes. XPS analysis showed that the ratio of C–O:C=O decreased with age. These tissue-related and age-related chemical differences between samples affected the optic density and metal binding properties of melanosomes, which are believed to be closely associated with the biological functions of melanins.

Hypericin is the active ingredient of the off-the-shelf antidepressant St. John's Wort. It is an effective phototoxic agent and its systemic administration at therapeutic doses could induce particular damage in the eye due to continuous light exposure. Hypercin is strongly fluorescent and its fluorescence properties can be monitored to investigate noninvasively its localization and interactions. To this aim, time-resolved microspectrofluorimetry and fluorescence lifetime imaging were used to assess the spectral and temporal properties as well as the spatial distribution of the fluorescence emitted by retinal pigment epithelium (RPE) cells treated with Hyp at concentrations in the micromolar range (0.5–10 μM). In the presence of hypericin, the emission peaks at 600–605 nm and the fluorescence decay is best fitted with three lifetimes (5.5–7 ns, 1.9–2.5 ns and <0.8 ns). Spectral and temporal differences were observed between high (≥5 μM) and low hypericin concentrations. In particular, upon increasing concentration, the emission spectrum of the slow component broadens and its lifetime shortens. The latter change is observed also when high concentrations are reached locally, due to more efficient localization within the cell.

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.

Previous work has shown that indocyanine green (ICG)-assisted peeling of the internal limiting membrane during vitreoretinal surgery may damage the retinal pigment epithelium (RPE). The present study tested the direct toxic effects and phototoxic effects of ICG on cultured human RPE. RPE cells were exposed to ICG (0.5%, 5 min) with or without lutein (20 μM), followed by light irradiation at different doses of light energy (1.0, 3.0 and 10.0 J/cm²). After 48 h, cells were collected and stained with trypan blue to obtain the number of viable and nonviable cells in different groups. Cultures exposed to ICG without light irradiation showed a significant decrease of viable cells (−13.3%) and an increase of nonviable cells (×2.5-fold) compared with cultures not exposed to either ICG or light, indicating the presence of direct toxic effects of ICG. In cultures exposed to ICG plus light irradiation (10.0 J/cm²), viable cells decreased significantly (−45.0%) and nonviable cells increased significantly (×4.4-fold) compared with cultures exposed to ICG alone. The damage to the RPE cells depended on the dose of light (1.0–10.0 J/cm²), indicating that ICG has a phototoxic effect as well as a toxic one. Lutein, an endogenous ocular antioxidant, had a protective effect on cultures exposed to ICG and light, cells treated with leutin showed an increase of viable cells ( 74.6%) and decrease of nonviable cells (−74.4%) compared with cultures without leutin but not on cultures exposed to ICG alone. Thus, it seems that photoactivated ICG kills cells through a photoxidative mechanism. Our study suggests that preoperative oral administration of lutein may protect against the phototoxic-induced damage of ICG on the RPE cells.

The damaging effects of intense light on the rat retina are known to vary depending on the time of day of exposure. The purpose of this study was to determine if rhodopsin phosphorylation patterns, a measure of the activity of the pigment, varied in a similar manner. After 10 min in strong light (1400 lux), all six threonine and serine sites in the rat rhodopsin C-terminus were phosphorylated, with mono- to tetraphosphorylation being substantially more prominent than penta- to hexaphosphorylation. The level and multiplicity of rhodopsin phosphorylations were reduced both with the duration of light exposure and the duration of subsequent darkness. Although showing vast differences in susceptibility to light damage, rats exposed at 5 P.M. or 1 A.M. showed similar rhodopsin phosphorylation levels and patterns. These data indicate that a process controlled by circadian rhythm other than rhodopsin phosphorylation is involved either in damaging or mediating the damage evoked by intense light exposure.

In eukaryotic cell nuclei, double-stranded DNA is found in the form of chromatin, a large fiber made up of DNA complexed to histone proteins. In this article, recent studies using fluorescence techniques to look at the dynamics of chromatin, both in vivo and in vitro, are reviewed. Two-photon counterpropagating fluorescence recovery after patterned photobleaching is used to examine chromatin fluctuations on lengthscales ranging from less than 100 nm to microns. By combining in vivo studies with data on isolated nuclei and by measuring how these fluctuations depend on variables like ionic strength and photochemical cross-linking, it is demonstrated that the relatively large-scale motions of chromatin observed in vivo are consistent with smaller scale modifications of the histone–DNA interaction. This connection may provide a means to use conformational dynamics as an in vivo probe of the biochemical events involved in gene expression.

Many aspects of cellular function or physiology can be used to indicate the level of damage resulting from the application of potentially deleterious agents such as drugs, solvents or even light. The dose required to reach a specific biological endpoint will necessarily depend on the characteristics of the damage induced by the agent. By using multiple biological probes, it is possible to get a more complete description of the type of damage induced. Photodamage was induced in rat basophilic leukemia cells by either 254-nm UVC light exposure or rose bengal photosensitization. Damage was measured by three quantitative assays employing fluorescent probes: calcein, to measure nonspecific esterase activity, propidium iodide (PI), to measure loss of plasma membrane integrity, rhodamine 123 (R123) to measure mitochondrial depolarization, and the incorporation of 5′-bromodeoxyuridine (BrdU), to measure the progress of cell replication. BrdU incorporation was found to be the most sensitive indicator for both forms of photodamage. For UVC photodamage, the BrdU assay was 330 times more sensitive than the other two assays. For rose bengal photosensitization, the BrdU assay was 48 or 62 times more sensitive than either the R123 or calcein/PI assays, respectively.

Apoptosis involves a highly organized and programmed series of events aimed at maintaining genomic stability by eliminating defective host cells. The purpose of this study was to determine the threshold doses and environmental UV-A and UV-B exposure times necessary to produce apoptosis and necrosis in the normal cells of a human fibroblast cell line. Environmental UV-A and UV-B doses were measured over a 6 year period with a four-channel UV radiometer. The fibroblasts were irradiated once using an Oriel UV Solar Simulator with six doses of environmentally-based UV. Doses corresponded to 0, 11, 19, 23 and 45 min of average environmental UV-A and UV-B radiation at solar noon in Puerto Rico. The Annexin-V binding method was used to differentiate between normal fibroblasts and apoptotic or necrotic fibroblasts. The threshold dose from apoptosis to necrosis was found between 24–28 kJ/m², which corresponded to 19 and 23 min of environmental UV-A and UV-B exposure. This study provides the first data that specify the environmental threshold doses of UV-A and UV-B at which human fibroblasts undergo apoptosis and necrosis. These results may provide valuable dose–response thresholds for apoptosis and necrosis for future mechanistic studies and baseline data for skin cancer prevention programs.

This study was designed to provide more detailed information on the subcellular sites of binding of the porphycene, termed 9-capronyloxytetrakis (methoxyethyl) porphycene (CPO), with a fluorescence resonance energy transfer (FRET) technique. The proximity of CPO to two fluorescent probes was determined: nonyl acridine orange (NAO), a dye with specific affinity for the mitochondrial lipid cardiolipin, and dihexa-oxacarbocyanine iodide (DiOC₆), an agent that labels the endoplasmic reticulum (ER). FRET spectra indicated energy transfer between DiOC₆ and CPO but no significant transfer between NAO and CPO. These results confirm data obtained by fluorescence microscopy, suggesting a similar pattern of subcellular localization by CPO and DiOC₆ but not by CPO and NAO. However, when cells containing CPO were irradiated and then loaded with NAO, FRET between the two fluorophores was observed. Hence, a relocalization of CPO can occur during irradiation. These data provide an explanation for recent studies on CPO-catalyzed photodamage to both ER and mitochondrial Bcl-2.

Earlier studies have shown that on exposure to UVA, hydroperoxynaphthalene diimide (IA) generates hydroxyl radicals, induces DNA strand scission, and kills cells.Here we employed electron paramagnetic resonance (EPR) and spin trapping to investigate the free radical photochemistry of IA and that of related naphthalene diimides, which are devoid of the hydroperoxyl moiety (N,N′-bis[2-methyl]-1,4,5,8-naphthaldiimide [IB], N,N′-bis[2-thiomethyl-2-methoxyethyl]-1,4,5,8-naphthaldiimide [IC]) and therefore are unable to generate hydroxyl radicals. It is shown that on UV irradiation (>300 nm) in air-free methanol or ethanol solutions all these naphthalene diimides undergo one-electron reduction to corresponding anion radicals, positively identified by EPR. With EPR and a spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO), we found that the photogeneration of the naphthalene diimide radicals is concomitant with the formation of radicals from the solvents, presumably through electron/hydrogen atom abstraction by photoactivated diimides. Irradiation of IA, IB or IC in the presence of oxygen generates superoxide, which was detected as a DMPO adduct. The high photoreactivity of IB and IC supports the notion that hydroperoxide IA can induce oxidative damage via photoprocesses that are independent of ^•OH generation. These observations could be pertinent to the application of naphthalene diimides as selective photonucleases, PDT anticancer agents or both.

Biochemical and structural changes of the dermal connective tissue substantially contribute to the phenotype of aging skin. To study connective tissue metabolism with respect to ultraviolet (UV) exposure, we performed an in vitro (human dermal fibroblasts) and an in vivo complementary DNA array study in combination with protein analysis in young and old volunteers. Several genes of the collagen metabolism such as Collagen I, III and VI as well as heat shock protein 47 and matrix metalloproteinase-1 are expressed differentially, indicating UV-mediated effects on collagen expression, processing and degradation. In particular, Collagen I is time and age dependently reduced after a single UV exposure in human skin in vivo. Moreover, older subjects display a lower baseline level and a shorter UV-mediated increase in hyaluronan (HA) levels. To counteract these age-dependent changes, cultured fibroblasts were treated with a specific soy extract. This treatment resulted in increased collagen and HA synthesis. In a placebo-controlled in vivo study, topical application of an isoflavone-containing emulsion significantly enhanced the number of dermal papillae per area after 2 weeks. Because the flattening of the dermal–epidermal junction is the most reproducible structural change in aged skin, this soy extract appears to rejuvenate the structure of mature skin.

The fluorescence anisotropy decay dynamics of the fluorescent probe Coumarin-153 (C153) have been investigated in two neutral micelles, Triton-X-100 (TX-100) and Brij-35 (BJ-35), at different temperatures and analyzed on the basis of the well-known two-step model. Because steady-state fluorescence spectra of the above probe do not show any noticeable changes with respect to temperature, for either of the studied micelles, suggests a similar polarity in the microenvironment around the probe at all the temperatures studied. The anisotropy results indicated that, for both the micelles, the fluidity inside the Palisade layer increases with temperature. However, the temperature effect on the anisotropy decay is relatively more pronounced in TX-100 than in BJ-35. It is inferred that the temperature effect on the anisotropy decay in the BJ-35 micelle is mainly due to the thermal effect on the microviscosity in the micellar phase. In the case of TX-100, the results indicate that, along with the above thermal effect, an additional effect is observed due to the increased size and hydration of the micelle with temperature, with the result being that the fluorescence anisotropy decay in TX-100 is more sensitive to temperature than in BJ-35. In the TX-100 micelle, our studies show that with an increase in temperature, even though the micellar size increases substantially, the distance of the probe from the micellar core does not increase that significantly. Thus, with increasing temperature, the probe undergoes a relative migration toward the micellar core to avoid the increased hydration in the micellar Palisade layer.

Carp (Cyprinus carpio) were repeatedly exposed to 0, 60, 120 and 240 mJ/cm² ultraviolet B (UVB) radiation three times in 1 week (short-term exposure) or 12 times in 4 weeks (long-term exposure). The effect of UVB on the functioning of the carp immune system was studied on day 2 after the final irradiation. After short-term UVB exposure, the whole-blood respiratory burst and cytotoxic activity were markedly enhanced, with parallel responses in both the number of circulating granulocytes and in the plasma cortisol concentration of the fish. These changes were not detectable after long-term exposure. The respiratory burst by head kidney granulocytes was suppressed dose dependently after both exposures, but cytotoxic activity was not affected. Exposure to UVB also modulated lymphocyte functions: nonstimulated and PHA-stimulated proliferation of head kidney lymphocytes in vitro was enhanced by both short-term and long-term exposure. LPS-stimulated proliferation was not affected by exposure nor was the number of immunoglobulin-secreting cells in the head kidney. In long-term exposure, the highest dose reduced the level of plasma IgM. This study indicates that UVB irradiation induces immunomodulation in the blood and head kidney of the carp and that the effects of short- and long-term exposure differ from each other. The results emphasize the potentially harmful impact of increased solar UVB radiation on fish immune functions.

A network of scanning spectroradiometers has acquired a multiyear database of visible solar irradiance, covering wavelengths from 400 to 600 nm, at four sites in the high-latitude Southern Hemisphere, from 55°S to 90°S. Monthly irradiations computed from the hourly measurements reveal the character of the seasonal cycle and illustrate the role of cloudiness as functions of latitude. Near summer solstice, the combined influences of solar elevation and the duration of daylight would produce a monthly irradiation with little latitude dependence under clear skies. However, the attenuation associated with local cloudiness varies geographically, with the greatest effect at the most northern locations, Ushuaia, Argentina and Palmer Station on the Antarctic Peninsula. Near summer solstice, the South Pole experiences the largest monthly irradiation of the sites studied, where relatively clear skies contribute to this result. Scaling factors derived from radiative-transfer calculations combined with the measured 400–600 nm irradiances allow estimating irradiances integrated over the wavelength band 400–700 nm. This produces a climatology of photosynthetically active radiation for each month of the year at each site.

Photodynamic therapy removes unwanted or harmful cells by overproduction of reactive oxygen species (ROS). Fractionated light delivery in photodynamic therapy may enhance the photodynamic effect in tumor areas with insufficient blood supply by enabling the reoxygenation of the treated area. This study addresses the outcome of fractionated irradiation in an in vitro photodynamic treatment (PDT) system, where deoxygenation can be neglected. Our results show that fractionated irradiation with light/dark intervals of 45/60 s decreases ROS production and cytotoxicity of PDT. This effect can be reversed by addition of 1,3-bis-(2-chlorethyl)-1-nitrosurea (BCNU), an inhibitor of the glutathione reductase. We suggest that the dark intervals during irradiation allow the glutathione reductase to regenerate reduced glutathione (GSH), thereby rendering cells less susceptible to ROS produced by PDT compared with continuous irradiation. Our results could be of particular clinical importance for photodynamic therapy applied to well-oxygenated tumors.

The phosphorescence lifetime (τ) of tryptophan (Trp) residues in proteins in aqueous solutions at ambient temperature can vary several orders of magnitude depending on the flexibility of the local structure and the rate of intramolecular quenching reactions. For a more quantitative interpretation of τ in terms of the local protein structure, knowledge of all potential quenching moieties in proteins and of their reaction rates is required. The quenching effectiveness of each amino acid (X) side chain and of the peptide backbone was investigated by monitoring their intramolecular quenching rate (k_obs) in tripeptides of the form acetyl-Trp-Gly-X-CONH₂ (WGX), where Trp is joined to X by a flexible Gly link. The results indicate that among the various groups present in proteins only the side chains of Cys, His, Tyr and Phe are able to quench Trp phosphorescence at a detectable rate (k_obs > 40 s⁻¹), with the quenching effectiveness for rotationally unrestricted side chains ranking in the order Cys ≫ His > Tyr ≫ Phe ∼ His. For the aromatic side chains the corresponding contact rate at 20°C is estimated to be between 3–4 × 10⁹ s⁻¹ for Cys (as determined by Lapidus et al.), 0.8–8 × 10⁶ s⁻¹ for His , 0.37–3.7 × 10⁶ s⁻¹ for Tyr and 0.2–2 × 10⁵ s⁻¹ for Phe and His. In the cases of His and Tyr, k_obs drops sharply with increasing pH, with midpoint transitions about 1 pH unit above the pKa, indicating that quenching is almost exclusive to the protonated form. From the temperature dependence of the rate, obtained in 50/50 propylene glycol/water between −20°C and 20°C, the reaction is characterized by activation energies of about 5 kcal·M⁻¹ for His and Tyr and 8 kcal·M⁻¹ for Phe. An analysis of the groups in contact with Trp residues in proteins that exhibit long phosphorescence lifetimes at ambient temperature leads to the conclusion that the contact rate of the peptide group and of the remaining side chains is lower than 0.1 s⁻¹, showing that these moieties are practically inert with respect to the triplet-state lifetime. It shows further that the immobilization of the aromatic side chains within the globular fold cuts their quenching effectiveness drastically to contact rates < 2 s⁻¹, a phenomenon attributed to the low probability of forming a stacked exciplex with the indole ring. All evidence suggests that, except in the case of nearby Cys or Trp residues, whose interaction with the triplet state reaches beyond van der Waals contact, the emission of buried Trp residues is unlikely to be quenched by surrounding protein groups.

Ultraviolet-A (UVA) radiation causes significant oxidative stress because it leads to the generation of reactive oxygen species (ROS), leading to extensive cellular damage and eventual cell death either by apoptosis or necrosis. We evaluated the protective effects of cyanidin-3-O-β-glucopyranoside (C-3-G) against UVA-induced apoptosis and DNA fragmentation in a human keratinocyte cell line (HaCaT). Treatment of HaCaT cells with C-3-G before UVA irradiation inhibited the formation of apoptotic cells (61%) and DNA fragmentation (54%). We also investigated antioxidant properties of C-3-G in HaCaT cells against ROS formation at apoptotic doses of UVA; C-3-G inhibited hydrogen peroxide (H₂O₂) release (an indicator of cellular ROS formation) after UVA irradiation. Further confirmation of the potential of C-3-G to counteract UVA-induced ROS formation comes from our demonstration of its ability to enhance the resistance of HaCaT cells to the apoptotic effects of both H₂O₂ and the superoxide anion (O₂^·−), two ROS involved in UVA-oxidative stress. Furthermore, in terms of Trolox Equivalent Antioxidant Activity, C-3-G treatment led to a greater increase in antioxidant activity in the membrane-enriched fraction than in the cytosol (55% vs 19%). The protective effects against UVA-induced ROS formation can be attributed to the higher membrane levels of C-3-G incorporation. These encouraging in vitro results support further research into C-3-G (and other anthocyanins) as novel agents for skin photoprotection.

Adolescents constitute an important audience for photoprotection programs. Sun exposure and sun protection habits acquired during adolescence have a significant impact on skin cancer incidence. We administered a questionnaire to 724 students about ultraviolet radiation effects, opinions about tanning, total time of sun exposure per day, photoprotection and activities in the sun. About 90% were aware of the association between sun exposure and skin cancer, and mass media was the main source of information. However, the great majority believed that tanning improved their appearance, and that it was worth taking the risk. The most prevalent outdoor activity among boys was sports; girls preferred walks and sunbathing. Sun exposure was significantly longer in summer, when 90% of the students went to the beach. About 47% reported sunscreen use in summer and only 3% reported using sunscreen during winter. These results emphasize the need for the promotion of photoprotective habits in our population and the importance of engaging physicians and schoolteachers in developing campaigns directed at this issue to achieve effective, long-lasting results. Adolescents are aware of the effects of ultraviolet radiation on the skin but campaigns have not successfully changed their sun exposure habits.

The feasibility of potabilization of sulfurous water was investigated by photochemical oxidation processes using a batch photoreactor and a continuous-flow photoreactor, equipped with UV lamps of 1000 W and 1500 W, respectively. Additionally, two advanced processes of oxidation were applied i.e. with a use of a UV light/H₂O₂/air and UV light/H₂O₂/O₃/air. These two processes were compared for their efficiency to the direct oxidation process where ozone is used in the absence of UV light. Results obtained for both advanced processes showed better oxidation than takes place by ozone in the absence of UV light. After the photooxidation processes, different processes for the absorption or precipitation of sulfates were investigated to comply with the World Health Organization (WHO) norm that demands a limit of ≤250 mgL⁻¹ of SO₄²⁻ in drinking water. Additionally, reverse osmosis was simulated using Osmonics Inc. software to predict the feasibility of lowering the salt concentration below WHO limits.

The photorelease of a caged neurotransmitter can be used to investigate the function of neuronal circuits in tissues. We have designed and synthesized a stable, caged γ-aminobutyric acid (GABA) derivative, 4-[[(2H-1-benzopyran-2-one-7-amino-4-methoxy)carbonyl]amino] butanoic acid (BC204), that releases the neurotransmitter in physiological medium when irradiated with UV light at 300–400 nm in PBS at pH 7.4. The release of GABA occurs with the formation of the major photoproduct, 7-amino-4-(hydroxymethyl)-2H-1-benzopyran-2-one, via a solvolytic photodegradation mechanism of the coumarin moiety and was confirmed by electrospray mass spectrometry/mass spectrometry (ESI MS/MS). BC204 is chemically stable and shows no intrinsic activity after many hours under physiological dark conditions. These properties suggest that BC204 is an excellent form of caged GABA that is well suited for long-term biological studies.

The photosynthetic activity of marine phytoplankton from five algal classes (Phaeodactylum tricornutum, Skeletonema costatum, Thalassiosira oceanica, Thalassiosira weissflogii, Dunaliella tertiolecta, Mantoniella squamata, Emiliania huxleyi, Pavlova lutheri and Heterosigma akashiwo) was investigated under identical growth conditions to determine interspecies differences. Primary photochemistry and electron transport capacity of individual species were examined by pulse amplitude–modulated (PAM) fluorescence. Although few differences were found in maximal photosystem II (PSII) photochemical efficiency between various species, large differences were noticed in their PSII–photosystem I (PSI) electron transport activity. We found that species such as T. oceanica and M. squamata have much lower photochemical activity than H. akashiwo. It appeared that processes involved in electron transport activity were more susceptible to change during algal evolution compared with the primary photochemical act close to PSII. Large variations in the nonphotochemical energy dissipation event among species were also observed. Light energy required to saturate photosynthesis was very different between species. We have shown that M. squamata and H. akashiwo required higher light energy (>1300 μmol m⁻² s⁻¹) to saturate photosynthesis compared with S. costatum and E. huxleyi (ca 280 μmol m⁻² s⁻¹). These differences were interpreted to be the result of variations in the size of light-harvesting complexes associated with PSII. These disparities in photosynthetic activity might modulate algal community structure in the natural environment where light energy is highly variable. Our results suggest that for an accurate evaluation of primary productivity from fluorescence measurements, it is essential to know the species composition of the algal community and the individual photosynthetic capacity related to the major phytoplankton species present in the natural phytoplankton assemblage.

Exposure of the skin to UV radiation induces local inflammation. We hypothesized that inflammation induced by erythemal UV-B irradiation could elevate levels of serum C-reactive protein (CRP) and that suberythemal repeating doses of solar-simulating UV radiation (SSR) would produce photoadaptation to such inflammation. Separation-free high-sensitivity assays of CRP show an increase by 42% (P = 0.046) in CRP concentrations in healthy human subjects 24 h after a 3 minimal erythemal dose (MED) dose of UV-B delivered onto a 100 cm² skin area. Preceding daily suberythemal doses of whole-body SSR for 10 or 30 consecutive days completely prevented the CRP increase. UV-B–induced skin erythema was partially attenuated by 30 preceding days of SSR only (P = 0.00066). After 10 daily SSR doses, the mean baseline CRP concentrations (0.24 ± 0.21 mg/L) declined by 35% (P = 0.018). Using high-sensitivity analysis of serum CRP as the endpoint marker for cutaneous inflammation, we show that acute exposure of even a relatively small skin area to erythemal UV-B induces skin inflammation detectable also at the systemic level and that photoadaptation by preceding repeating suberythemal doses of SSR reduces signs of inflammation. Our data complement the view given by previous studies in that local photoadaptation also has systemic manifestations.

An analysis is made of measured ultraviolet erythemal solar radiation (UVER) data recorded during the year 2003 by the networks of the Catalan Weather Service and the Environment Department of Valencia (both on the Spanish Mediterranean coast). Results show a latitudinal variation at sea level, of 3–4% per degree and an increase with altitude of 10% per km. Based on these data the UV Index has been evaluated for the measuring stations. The maximum experimental value of the UV Index was around 9 during the summer, although higher values were recorded at two stations, one at the highest elevation and the other at the lowest latitude. The annual accumulated doses of irradiation on a horizontal plane have been presented as well as the evolution through the year in units of energy, Standard Erythemal Doses and Minimum Erythemal Doses according to different phototypes. Lastly, the UV Index forecast, determined with a multiple scattering radiative transfer model, has been analyzed. Total agreement or only one unit of difference between measured and modelled values was found in 94% of cloud-free cases.

The reaction center complex of heliobacteria contains three kinds of chlorophyll pigments, bacteriochlorophyll g_F (BChl g_F), its 13²-epimer BChl g_F′ and 8¹-hydroxy-chlorophyll a_F (8¹-OH-Chl a_F). Because the full stereochemistry of these naturally occurring chlorophyllous pigments has remained unknown, we determined the stereochemistry of both BChl g_F and 8¹-OH-Chl a_F extracted from Heliobacterium modesticaldum. The configurations of the specific functional groups at ring-B as well as those at ring-D and -E were investigated by use of nuclear Overhauser effect correlations in their ¹H-NMR spectra and circular dichroism spectra, as well as by modified Mosher's method in their chemical modification: (1) E-configuration was confirmed for the 8-ethylidene group at ring-B in BChl g_F, (2) R-configuration was identified for the 1-hydroxyethyl group at ring-B in 8¹-OH-Chl a_F and (3) 13²-(R)-, 17-(S)- and 18-(S)-configurations at ring-D and -E in both BChl g_F and 8¹-OH-Chl a_F were confirmed. These stereochemistries enabled us to discuss their biosynthesis and to propose possible routes for preparation of ethylidene and 1-hydroxyethyl groups at the 8-position.

There is a continuously growing interest in medical applications of ultraviolet radiation (UV-A and long-wavelength UV-B) especially for laser surgery, phototherapy and photodiagnostics of human internal organs. UV-B and UV-A radiation is potentially mutagenic, however, there has been very little information published to date concerning the significance of possible deleterious action of such photons on cells of internal tissues. The aim of this study is to compare the sensitivities of skin cells to those of internal organs upon exposure to UV-A. To assess this sensitivity we have determined the UV-A dose-dependent frequency of nuclear DNA breaks detected with the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) technique. The materials for the study were macroscopic samples of porcine skin, colon and esophagus. The UV-A dose ranged from 0.1 to 1000 mJ/cm², which is similar to doses received by cells in regions examined with laser-induced fluorescence or by cells surrounding areas subject to a laser ablation. To reduce the influence of DNA repair processes the tissue samples were kept at a low temperature during the irradiation and were deep frozen immediately after completing the irradiation procedure. The cells of the internal organs are much more susceptible to UV-A–induced breaking of DNA than the skin cells. The percentage fractions and the spatial distributions of the damaged cells and the characteristics of the UV-A dose dependence seem to vary by type of internal organ.

We report the picosecond time-scale fluorescence dynamics of a dye-labeled DNA oligonucleotide or “aptamer” designed to bind specifically to Immunoglobulin E. Comparison of the photophysics of Texas Red (TR), fluorescein and 5′-carboxytetramethylrhodamine (TAMRA)-labeled aptamers reveals surprising differences with significant implications for measurements of oligonucleotide structure and dynamics. The fluorescence decay of the TR-aptamer is a simple single exponential with a weak temperature dependence. The fluorescence decay of the fluorescein-aptamer (fl-aptamer) is pH dependent and displays a complex temperature dependence with significant changes on melting of the aptamer tertiary structure. Despite its similarities to TR, TAMRA is strongly quenched when conjugated to the aptamer and displays complex fluorescence kinetics best described by a distributed rate model. Using the maximum entropy method, we have discovered two highly temperature-dependent fluorescence lifetimes for the TAMRA-aptamer. One of these lifetimes is similar to that of free TAMRA and displays the same temperature dependence. The other lifetime is quenched and displays a temperature dependence characteristic of a charge transfer reaction. These data set TR apart as an attractive alternative to TAMRA and fluorescein for studies such as fluorescence polarization and fluorescence resonance energy transfer, where environmental sensitivity of the probe is not desired.

This study examined the nature of photoproducts after pulse laser irradiation (647.5 nm) of 5,10,15,20-tetrakis(meso-hydroxyphenyl)porphyrin (m-THPP) (10 μmol/L) in ethanol-water (1/99, vol/vol) solution. Spectroscopic measurements (UV-visible absorption and fluorescence) and mass spectrometry techniques (matrix-assisted laser desorption-ionization [MALDI] coupled with time-of-flight mass spectrometer [TOF-MS] or tandem time of flight mass spectrometer [TOF/TOF-MS]) were used to follow photomodifications. Spectroscopic measurements evidenced photomodification as the main process after m-THPP irradiation. Three oxidized photoproducts at m/z 693.25, 695.24 and 713.25 were characterized by MS. After prolonged irradiation new isotopic distributions were registered at m/z 1355.41, 2031.57, 2707.80 and 3383.98 with MALDI-TOF-MS and TOF/TOF-MS. These new photoproducts were attributed to covalent oligomeric structures as dimer, trimer, tetramer and pentamer of m-THPP.

Photophysical properties of tyrosine and its derivatives with free and blocked functional groups were studied by steady state and time-resolved fluorescence spectroscopy and global analysis in organic solvents, such as methanol, 2-propanol, tetrahydrofuran (THF), and dimethylsulfoxide (DMSO). The mono-exponential fluorescence intensity decays were observed for all tyrosine derivatives in THF and DMSO solutions, whereas in alcohols some derivatives have bi-exponential decays. The rotamer population calculated from ¹H nuclear magnetic resonance spectroscopy in DMSO does not correspond to the pre-exponential factors obtained from fluorescence spectroscopy. Moreover in the case of DMSO, the strong interaction of this solvent with the hydroxyl group of the fluorophore's phenol ring causes substantial changes in the fluorescence and nonradiative rate constants of tyrosine derivatives compared with those of tyrosine with a blocked hydroxyl group, Tyr(Me). The steady state and time-resolved fluorescence measurements in pure organic solvents and water–organic solvent mixtures indicate that the fluorescence quenching of the phenol chromophore of tyrosine by an acetyl or amide group or both depends on the polarity of the solvent used as well as the ability of the solvent to form hydrogen bonds with functional groups of tyrosine.

Synovial sarcoma (SS) is one of common malignant soft-tissue tumors and is encountered most commonly in children and young adults. It frequently involves or invades major neurovascular structures and bones, and its local recurrence rate after simple resection has been reported to be as high as up to 80%. Because major nerves and vessels, as well as an adequate amount of bone, must be preserved to restore excellent limb function in cases of SS, a surgical technique entailing a low risk of local recurrence is needed. Based on the findings of recent experimental studies conducted by us using a mouse osteosarcoma model, we developed a novel therapeutic technique for SS, consisting of reduction surgery followed by photodynamic therapy using acridine orange (AO-PDT), with or without X-ray irradiation at 5 Gy. A preliminary study revealed that low-dose X-rays also excite AO like photons. After an initial study on cell cultures, this novel technique was applied to six cases of SS. A follow-up of the subjects to determine the clinical outcome revealed that none of the cases treated by AO-PDT, including the four cases treated by additional 5 Gy irradiation and the two cases not receiving any radiation, showed any evidence of recurrence or local/systemic complications during the follow-up period of 19–51 months after the surgery. Therefore, we believe that AO-PDT with 5 Gy irradiation may be an excellent novel therapeutic modality with reduction surgery to salvage excellent limb function in SS involving major nerves and vessels or bones.