The formation of ground-state complexes of methylene blue (MB) and thionine (TN) with sodium hyaluronate (NaHA) was clearly observed by means of absorption spectra in aqueous solution. Irradiation of the complexes using 313 nm light caused significant degradation of NaHA under oxygen and argon. However, the use of visible light over 400 nm, which gives the lowest excited singlet state of the cationic dyes, caused no degradation. MB and TN were more efficient sensitizers for the degradation of NaHA than rose bengal (RB), although RB is a more efficient singlet oxygen (¹O₂) sensitizer than the cationic dyes. Under similar conditions the polysaccharides with carboxyl groups, such as alginic acid and polygalacturonic acid, also photodecomposed. However, the polysaccharides without carboxyl groups, such as pullulan and methyl cellulose, did not. The irradiation of the polysaccharides in the presence of powdered titanium dioxide as a photocatalyst to generate the hydroxyl radical (^·OH) in aerated aqueous solution caused the fragmentation of all the polymers. It was confirmed that methyl viologen, an electron-accepting sensitizer, formed a charge-transfer complex with NaHA, the irradiation of which caused the efficient degradation of NaHA. In the presence of β- and γ-cyclodextrins the MB- and TN-sensitized photodegradation of NaHA was markedly suppressed. This was probably due to the formation of the inclusion complexes comprising the cationic dyes and the cyclodextrins. On the basis of the results obtained we propose that the cationic dye–sensitized degradation of NaHA involves a photoinduced electron-transfer process between the upper excited dyes and the ground-state NaHA and that ^·OH and ¹O₂ do not participate in the degradation.

Two molecules showing photochemistry but no fluorescence have been investigated at 80 K in a rigid matrix regarding the behavior of the quantum yield for bond fragmentation as a function of the vibrational/vibronic level and electronic excited state. A new equation was developed to determine the photochemical quantum yield under ambient conditions (80 K). The levels/bands involved were those within a given vibrational progression, in different progressions as well as in combination. The yield was low (Φ = 0.1) with excitation into the n = 0 level of S₁ but very rapidly increased with excitation into higher levels whether they were harmonics or combination levels. A parallel result was observed upon excitation into S₂. Vibrational relaxation/deactivation occurs only between levels of the same vibrational progression. Deactivation from the 0 level of S₂ does not occur via levels of S₁. The photochemically active modes correspond to the vibrational modes present in the region of the molecule where bond breakage occurs. These results add further proof of the complex nature and number of processes that can occur within excited states of photochemically active molecules.

Photolysis of 6-chloro-1,3-dimethyluracil and mesitylene in the presence of trifluoroacetic acid (TFA) at low temperature gave 1,3,5,7,9- and 1,3,6,8,10-pentamethylcyclooctapyrimidine-2,4-diones (1b, 1c). Sequential photoreaction of the former (1b) resulted in the formation of 9,11-diazapentacyclo[6.4.0.0^1,3.0^2,5.0^4,8]dodecane-2,4-dione (2b) by way of 9-exo-methylene derivative (7b) and cyclobutaquinazoline (8b). On the other hand, UV-irradiation of 1c led to the bond shift isomer (5c) whose photolysis in the presence of TFA gave rise to the formation of the [6.4.0.0^1,3.0^2,6.0^4,8]dodecane isomer (3c).

The ability of the DNA duplex to behave as an efficient organized medium for cis–trans isomerization induced by electron transfer (ET) has been explored. Isomerization studies, luminescence quenching and DNA photocleavage assays show that photoexcited Ru(1,10-phenanthroline [phen])₃² transfers an electron to E,Z1,4-bis[2-(1-methylpyridinium-4-yl)vinyl]benzene (E,Z pMPVB), which subsequently undergoes one-way isomerization to E,E pMPVB. The unusual feature of the system is manifested by the lack of friction that is usually imposed on the photoisomerizable ligand by highly organized media. The apparent rate of ET in DNA increases when compared with the homogeneous solution. However, after correction for the local concentration of the reagents onto the biopolymer, the rate constant becomes independent of the DNA concentration and is at least 4 × 10² times smaller than that in the homogeneous aqueous solution. Using the photoinduced isomerization system, a large enhancement in the efficiency of single-strand break formation was found in plasmid DNA over that for Ru(phen)₃ ² alone using irradiation at λ > 480 nm.

It is now well documented that chronic UVA exposure induces damage to human skin. Therefore, modern sunscreens should not only provide protection from both UVB and UVA radiation but also maintain this protection during the entire period of exposure to the sun. UVA filters, however, are rare and not sufficiently photostable. We investigated the effect of the introduction of a new UV filter, bis-ethylhexyloxyphenol methoxyphenyl triazine (Tinosorb S), in oil in water sunscreen formulations on the photostability of butyl methoxydibenzoylmethane (Avobenzone [AVB]) after irradiation with an optically filtered Xenon arc source (UV irradiance adjusted at 1 mean effective dose [MED]/min). With spectrophotometrical methods to assess the sun protection factor (SPF) and UVA ratio and chromatographical methods to determine the amount of UV filters recovered after irradiation we showed that Tinosorb S prevented the photodegradation of AVB in a concentration-dependent way, leading to a sustained SPF and UVA ratio even after irradiation with doses of up to 30 MED. Since AVB was shown to destabilize ethylhexyl methoxycinnamate (EHM) we tested the effect of Tinosorb S in sunscreens containing this UV filter combination. Here too Tinosorb S showed photoprotective properties toward both UV filters. Thus, Tinosorb S can be used successfully to improve the photostability and efficiency of sunscreens containing AVB and EHM.

Sunlight was found to strongly induce the formation of N^ε-(carboxymethyl)lysine (CML) from glycated polylysine in the presence of Fe(III) ion. The initial step of this Fe(III)-catalyzed CML formation was noted to be similar to that of blueprint photography as was confirmed by the production of Turnbull's blue in sunlight-exposed glycated human serum albumin ferricyanide solution in the presence of Fe(III). Based on this, photoinduced oxidative C–C bond cleavage of the Amadori compound was assumed to be initiated by photochemical single electron transfer from ligand to Fe(III) in the Fe(III)–Amadori compound complex affording the Fe(II)–Amadori compound radical intermediate, which eventually yields either CML or active oxygen species. CML is thus a useful oxidative stress marker. The mechanism proposed here would explain the high accumulation of CML in lens protein and skin actinic elastosis.

The daily autumn and winter ultraviolet-A (320–400 nm) (UVA) exposures and 6 min UVA irradiance data for a southern hemisphere subtropical site (Toowoomba, Australia, 27.6°S, 151.9°E) are presented. This data is used to quantify the effect of cloud on UVA using an integrated sky camera and radiation system. Additionally, an estimate of the effect of enhanced UVA exposure on humans is made. The measurement system consisted of broad-band visible–infrared and UVA sensors together with a sun tracking, wide-angle video camera. The mean daily June exposure was found to be 409 kJ m⁻². Under the constraints of the uncertainty of both the UVA measurement system and clear-sky model, one case of enhanced UVA irradiance was found. Three cases of cloud enhancement of daily UVA exposure, approaching clear-sky levels, were also determined using a calculated clear-sky envelope. It was also determined that for a fulltime outdoor worker the additional UVA exposure could approach approximately that of one third of a full winter's day. For indoor workers with an outside lunch break of 12:00–1:00 P.M. the additional UVA exposure was on an average 6.9 kJ m⁻² over three cloud-enhanced days. To the authors' knowledge this is the first paper to present some evidence of cloud-enhanced UVA human exposure.

The in vivo assessment of sunscreen protection does not include the photogenotoxicity of UVA or UVB solar radiation. Using the comet assay we have developed a simple and rapid technique to quantify sunscreen efficacy against DNA damage induced by UV light. Cutaneous human melanocytes from primary cultures were embedded in low-melting point (LPM) agarose and exposed to UVA (0.8 J/cm²) or to UVB (0.06 J/cm²) through a quartz slide covered with 10 μL volumes of sunscreens. DNA single-strand breaks induced directly by UVA at 4°C and indirectly through nucleotide excision repair by UVB following a 35 min incubation period at 37°C were quantified using the comet assay. Tail moments (TM) (tail length × %tail DNA) of 100 cells/sample were determined by image analysis. DNA damage was evaluated with a nonlinear regression analysis on the normalized distribution frequencies of TM using a χ² function. The coefficients of genomic protection (CGP) were defined as the percentage of inhibition of DNA lesions caused by the sunscreens. Twenty-one sunscreens were evaluated, and the calculated CGP were compared with the in vivo sun protective factor (SPF) and with the protection factor UVA (PFA). Nonlinear relationships were found between SPF and CGP_UVB and between PFA and CGP_UVA.

The goal of this investigation was to correlate the melanin content in human pigmentary cells with the generation of UVB-induced photoproducts and to examine the relationship between the melanin content and the removal of the photoproducts. Cultured melanocytes from light-skinned individuals synthesized less melanin and produced more cyclobutane pyrimidine dimers and 6–4 photoproducts upon UVB exposure than did melanocytes from black skin. Tyrosine-stimulated melanogenesis provided protection against DNA damage in both cell types. In another set of pigmented cell lines a ratio between eumelanin and pheomelanin was determined. The assessment of association between DNA damage induction and the quantity and quality of melanin revealed that eumelanin concentration correlated better with DNA protection than pheomelanin. Skin type–I and skin type–VI melanocytes, congenital nevus (CN)-derived cells and skin type–II melanocytes from a multiple-melanoma patient were grown in media with low or high l-tyrosine concentration. The cells were irradiated with 200 J/m² UVB, and the levels of the photoproducts were determined immediately and after 6 and 24 h. Once again the induction of the photoproducts was mitigated by increased melanogenesis, and it was inversely correlated with the skin type. No significant differences were found for the removal of photoproducts in the cultures of skin types I and VI and CN cells. No indications of a delay in the removal of photoproducts in the melanocytes from the multiple-melanoma patient were found either.

Under conditions of iron deficiency certain cyanobacteria induce a chlorophyll (Chl)-binding protein, CP43′, which is encoded by the isiA gene. We have previously suggested that CP43′ functions as a nonradiative dissipator of light energy. To further substantiate its functional role an isiA overexpression construct was introduced into the genome of a cyanobacterium Synechococcus sp. PCC 7942 (giving isiA^oe cells). The presence of functional CP43′ in isiA^oe cells was confirmed by Western blot as well as by the presence of a characteristic blueshift of the red Chl a absorption peak and a notable increase in the 77 K fluorescence peak at 685 nm. Compared to wild-type cells isiA^oe cells, with induced CP43′, had both smaller functional antenna size and decreased yields of room temperature Chl fluorescence at various light irradiances. These observations strongly suggest that isiA^oe cells, with induced CP43′, have an increased capacity for dissipating light energy as heat. In agreement with this hypothesis isiA^oe cells were also more resistant to photoinhibition of photosynthesis than wild-type cells. Based on these results we have further strengthened the hypothesis that CP43′ functions as a nonradiative dissipator of light energy, thus protecting photosystem II from excessive excitation under iron-deficient conditions.

The properties of a negative transient signal (negative peak) observed during the first seconds of the induction of the photoacoustic (PA) signal in dark-adapted barley leaves treated with methyl viologen (MV) and diuron and then exposed to high temperatures have been examined. Under those conditions no electron donation from photosystem II (PSII) occurred, and electron flow through PSI could be supported only by soluble reductants located in the chloroplast stroma. The negative peak was observed only if the PA signal had been monitored at low, and not high, frequencies. The peak obviously originated from the oxygen consumption by PSI. The size of the peak increased as the temperature of preheating was raised from 39 to 45°C. The size of the peak decreased exponentially with a half-time of 3.7 s during illumination under low light. This decrease was found to be much faster under strong light. The recovery of the peak during dark acclimation required several minutes. It is concluded that the negative peak reflects the oxygen consumption supported by stromal reductants, their pool being rapidly exhausted under light in the presence of MV. The maximal size of the pool was calculated as 140 eq:P700 in dark-adapted leaves.

Carotenoids were isolated from the cells of Rhodobium marinum, and their structures were determined by mass spectrometry and ¹H nuclear magnetic resonance spectroscopy; the carotenoids include lycopene, rhodopin, anhydrorhodovibrin, rhodovibrin and spirilloxanthin. Time-dependent changes in the carotenoid composition in the reaction center (RC) and the light-harvesting complex 1 (LH1) were traced by high-performance liquid chromatography analysis of the extracts. The carotenoid composition changed according to the spirilloxanthin biosynthetic pathway. However, spirilloxanthin having the longest conjugated chain was always preferentially bound to the RC, and anhydrorhodovibrin and other precursors to the LH1.

A noninvasive in situ fluorescence-based method for the quantification of the photosensitizer chloroaluminum disulfonated phthalocyanine was compared to the highly accurate but nonreal time ex vivo spectrofluorometry method. Our in vivo fluorescence technique is designed to allow real-time assessment of photosensitizer in tumor and normal tissues and therefore temporally optimal light delivery. Laser-induced fluorescence was used to measure photosensitizer concentration from multiple microscopic regions of tissue. Ex vivo chemical extraction was used to quantify photosensitizer concentration in the same volume of tissue. The amount of photosensitizer in the vascular and/or parenchymal compartments of skeletal muscle and liver was determined by quantifying fluorescent signal in vivo, ex vivo and after blood removal. Confocal microscopy was used to spatially document photosensitizer localization 30 min and 24 h after delivery. While a linear correlation can exist between the fluorescence intensity measured by our fiber-optic bundle system and actual tissue concentration, temporal changes to this calibration line exist as the photosensitizer changes its partitioning fraction between the blood (vasculature) and the tissue parenchyma. In situ photosensitizer fluorescence microsampling (dosimetry) systems can be performed in real time and linearly correlated to actual tissue concentration with minimal intertissue variance. Tissue-specific differences may require temporal alterations in the calibration.

The effect of virus inactivation by 1,9-dimethylmethylene blue (DMMB) phototreatment, methylene blue (MB) phototreatment or heat on the activities of antioxidant systems of stroma-free hemoglobin (SFH) was studied. DMMB photoinactivated human immunodeficiency virus by >3.69 log₁₀ under conditions that inactivated 3.33 log₁₀ of vesicular stomatitis virus (VSV). Under conditions which inactivated VSV by 6.10 log₁₀ (1.37 J/cm² irradiation and 2 μM DMMB), there was little change in the methemoglobin (Met-Hb) formation, concentration of reduced glutathione (GSH), or superoxide dismutase (SOD), catalase (CAT) or glutathione peroxidase (GPX) activities. However, the activity of glutathione reductase (GR) was decreased by 77%. Under conditions that inactivated VSV by 5.69 log₁₀ (1.37 J/cm² irradiation and 24 μM MB) there was little effect of MB phototreatment on SOD, CAT, GPX and GSH activities. However, GR activity was decreased by 74% and Met-Hb content reached 3.98%. Under conditions that inactivated VSV by more than 6.20 log₁₀ (60°C for 2 min), virucidal heat treatment resulted in 27% Met-Hb formation and decreased GPX activity by 43%. No significant decline in SOD, CAT or GR activities or GSH concentration was observed. These results suggest that, compared with heat treatment and MB phototreatment, virucidal DMMB treatment preserves not only the oxidative state of hemoglobin but also the antioxidant systems against superoxide and hydrogen peroxide, although the reduced GR activity may limit the quenching capacity of antioxidants in DMMB-treated SFH.

Isoflavones derived from many edible plants have been reported to possess significant antioxidant, estrogenic and tyrosine kinase inhibitory activity. Genistein has been found previously to provide protection from oxidative damage induced by UV radiation both in vitro and following dietary administration. We have therefore examined the potential of a number of isoflavones from red clover (Trifolium pratense) and some metabolically related compounds to offer protection from UV irradiation in hairless mice by topical application after UV exposure. We show that whereas the primary isoflavones, daidzein, biochanin A and formononetin, were inactive, 20 μM lotions of genistein and the metabolites equol, isoequol and the related derivative dehydroequol had powerful potential to reduce the inflammatory edema reaction and the suppression of contact hypersensitivity induced by moderate doses of solar-simulated UV radiation. For equol the protection was concentration dependent and 5 μM equol markedly reduced the UV-induced inflammation but abrogated the UV-induced immunosuppression. Equol protected similarly from immunosuppression induced by the putative epidermal mediator, cis-urocanic acid (UCA), indicating a potential mechanism of action involving inactivation of this UV-photoproduct. Since immunosuppression induced by both UV radiation and by cis-UCA appears to be an oxidant-dependent response our observations support the actions of these topically applied isoflavones and their metabolites as antioxidants. They also indicate that lotions containing equol, unlike topical UV sunscreens, more readily protect the immune system from photosuppression than from the inflammation of the sunburn reaction, even when applied after exposure, and thus such compounds may have a future role as sun-protective cosmetic ingredients.

People who vacation in sunny places are exposed to the sun on multiple occasions at least on a daily basis. The clinical assessment of sun exposure is erythema in the first 48 h after exposure and pigmentation at times greater than 3–5 days. The purpose of this investigation was to determine the extent to which consecutive erythemogenic exposures result in additive erythema responses. Studies were conducted in which volunteers were first exposed to a graded series of fluences of UVB radiation and then on subsequent days (1–3 days) the same sites along with the surrounding unexposed skin were challenged with varying fluences of UVB radiation. The erythema reactions were assessed clinically and were objectively documented with diffuse reflectance spectroscopy. The sites that received two exposures always showed a reduced erythema response compared to a single erythemogenic exposure. The suppression of erythema was more pronounced when the second exposure was given 48 h after the first. The erythema suppression was maximal when the first exposure was at 1.3 minimum erythema dose (MED). The pigment response to the first exposure was completely suppressed for fluences less than 1.5 MED. We thus provide evidence for a decoupling of the classical sequence of erythema–pigmentation response. We also show that the erythema induced by a second exposure may be substantially suppressed by an earlier exposure, and that this cannot be due to melanin photoprotection or due to substantial thickening of the stratum corneum. We propose that the cause may be some diffusible element of yet unknown origin.

Alpha-crystallin possesses a molecular chaperone-like activity that prevents proteins from aggregating; however, the mechanism of this activity is not well known. Here we have taken gamma-irradiated alpha-crystallin and studied the relationship between the decrease in chaperone-like activity and the modifications such as oxidation, isomerization and racemization of amino acids in this molecule. We found that the chaperone-like activity of alpha-crystallin decreased with increasing gamma irradiation. After 4000 Gy gamma irradiation the activity of alpha-crystallin was reduced to 40% of the level of nonirradiated, native alpha-crystallin. The circular dichroism spectrum showed that the secondary structure of the irradiated alpha-crystallin had not changed. However, its tertiary structure appeared to change following more than 1000 Gy irradiation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis also indicated that cross-linking of alpha-crystallin increased with increasing radiation doses. Irradiated and nonirradiated alpha-crystallin was subjected to trypsin digestion and peptide analysis by reverse-phase high-performance liquid chromatography and mass and sequence analysis. Depending on the radiation dose, Met-1 of alpha A-crystallin was oxidized to methionine sulfoxide. In addition, Asp-151 of alpha A-crystallin was isomerized to the beta-Asp form after irradiation, and racemization of Asp-151 decreased. Thus, the loss of the chaperone-like activity of alpha-crystallin is related to changes in its isomerization, oxidation and racemization.

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 (τ ∼ 14 μs), weak absorption was observed. This species is quenched by oxygen (k = 2 × 10⁹ 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.

Pharaonis phoborhodopsin (ppR) (also pharaonis sensory rhodopsin II) is a receptor of the negative phototaxis of Natronobacterium pharaonis. ppR forms a complex with its pharaonis halobacterial transducer (pHtrII), and this complex transmits the light signal to the sensory system in the cytoplasm. The expressed C-terminal-His tagged ppR and C-terminal-His tagged truncated pHtrII (t-Htr) in Escherichia coli (His means the 6× histidine tag) form a complex even in the presence of 0.1% of n-dodecyl-β-d-maltoside, and the M-decay of the complex became about twice slower than that of ppR alone. The photocycling rates under varying concentration ratios of ppR to t-Htr in the presence of detergent were measured. The data were analyzed on the following assumptions: (1) the M-decay of both ppR alone and the complex followed a single exponential decay with different time constants; and (2) the M-decay under varying concentration ratios of ppR to t-Htr, therefore, followed a biexponential decay function which combined the decay of the free ppR and that of the complex as photoreactive species. From these analyses we estimated the dissociation constant (15.2 ± 1.8 μM) and the number of binding sites (1.2 ± 0.08).

The enthalpy (ΔH) and structural volume changes (ΔV) associated with the formation and decay of the early intermediate K₆₀₀ in the photocycle of Natronobacterium pharaonis halorhodopsin (pHR), an inward-directed anion pump, were obtained by laser-induced optoacoustic spectroscopy. A large expansion is associated with K₆₀₀ formation, its value depending on the medium and on the anion (Cl⁻, NO₃⁻, Br⁻, I⁻). A smaller expansion is associated with K₆₀₀ decay to L₅₂₀. A contraction is found for the same step in the case of the azide-loaded pHR which is an efficient outward-directed proton pump. Thus, the conformational changes in L₅₂₀ determine the direction and sign of charge translocation. The linear correlation between ΔH and ΔV for chloride-loaded pHR observed upon mild medium variations is attributed to enthalpy–entropy compensation effects and allows the calculation of the free-energy changes, ΔG_K = (97 ± 16) kJ/mol and ΔG_KL = −(2 ± 2) kJ/mol. Different from other systems, ΔS correlates negatively with ΔV in the first steps of the pHR photocycle. Thus, the space around the anion becomes larger and more rigid during each of these two steps. The photocycle quantum yield was 0.52 for chloride-pHR as measured by laser flash photolysis.