The phototoxicity of triamcinolone 16,17-acetonide has been estimated through a panel of in vitro tests. The main target involved in phototoxicity induced by triamcinolone appeared to be the cell membrane. Oxygen-independent photohemolysis was observed. A photochemical study in water and buffered solutions supported the conclusion that this is related to the action of radicals formed upon UV irradiation (in particular UV-B) by Norrish Type-I fragmentation of the C-20 ketone group. Peroxy radicals were formed in the presence of oxygen and were the active species in that case. Three photoproducts, isolated from the photodegradation of the drug, were submitted to the same toxicity tests. Two of them were proved to possess toxic or phototoxic properties on erythrocytes, primarily induced by UV-B light, and may participate in the photosensitizing activity of triamcinolone 16,17-acetonide. Our in vitro results suggest that the drug can elicit weak photosensitizing properties in vivo.

Two meso-tetraphenylporphyrin derivatives bearing adjacent: 5,10-di[4-(N-trimethylaminophenyl)-15,20-diphenylporphyrin (DADP-a) or opposite: 5,15-di[4-(N-trimethylaminophenyl)-10,20-diphenylporphyrin (DADP-o) cationic-N-(CH₃)₃ groups on two of the para-phenyl positions were examined with regard to photodynamic properties as a function of charge distribution. The two adjacent positive charges in the DADP-a structure result in a molecular distortion (asymmetry), likely from electrostatic repulsion. This could be responsible for the unusual interaction of this compound with some solvents and detergent micelles. In contrast, DADP-o is a much more symmetric molecule. In a cellular environment, fluorescence spectra of the two agents were essentially identical. Subcellular localization played a major role in photodynamic efficacy. DADP-a localized in mitochondria, and irradiation of photosensitized cells (640–650 nm) resulted in a rapid loss of the mitochondrial membrane potential (ΔΨ_m), usually a prelude to apoptotic cell death. In contrast, DADP-o localized in lysosomes, and extensive lysosomal photodamage was observed after irradiation. Both steady-state accumulation levels and absorbance spectra favored DADP-o, but the light dose required for a 90% cell kill was two-fold greater for DADP-o than for DADP-a, at a constant extracellular sensitizer concentration. These data indicate that, on a photons/cell basis, DADP-a was five-fold more efficacious. Fluorescence emission spectra in different solvents and detergents demonstrated a tendency for DADP-a association. We interpret these results to indicate partition of both drugs to membrane loci, with mitochondria being the more lethal site for photodamage.

In this work we evaluated the influence of topical application of P. umbellata root extract gel, containing 0.1% of 4-nerolidylcathecol, on the antioxidant network in UV-induced oxidative damage in hairless mouse skin. The UV-irradiation had no influence on ascorbic acid levels or on the antioxidant enzyme (superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase) activities, but topical P. umbellata treatment protected α-tocopherol from being depleted after UV-irradiation. α-Tocopherol concentration decreased significantly (≈40%, P < 0.01) in the irradiated control groups, whereas in the P. umbellata–treated group, α-tocopherol was totally preserved (≈100%, P > 0.05). These data demonstrate that P. umbellata may be successfully used as a topical photoprotective agent.

The photochemistry of 1,4-benzoquinone (BQ) and alkyl-, Cl- and related derivatives, e.g. methyl-, 2,6-dimethyl-, chloro-, 2,5-dichloro-1,4-benzoquinone, duroquinone and chloranil, was studied in nonaqueous solvents by UV–vis spectroscopy using nanosecond laser pulses at 308 nm. The reactivity of the triplet state (³Q^*) of the quinones with 2-propanol in the absence of water is largest for BQ and depends mainly on the quinone structure, whereas the rate constant of electron transfer from amines, such as triethylamine (TEA) or 1,4-diazabicyclo[2.2.2]octane, is close to the diffusion-controlled limit for BQ and most derivatives. Photoinduced charge separation after electron transfer from amines to ³Q^* and the subsequent charge recombination or neutralization are supported by time-resolved conductivity measurements. The half-life of the decay kinetics of the semiquinone radical ( ^•QH/Q^•–) depends significantly on the donor and the medium. The photoconversion into the hydroquinones was measured under various conditions, the quantum yield, λ_irr = 254 nm, increases with increasing 2-propanol and TEA concentrations. The effects of quenching of ³Q^*, the ^•QH/Q^•– radicals and the photoconversion are outlined. The mechanisms of photoreduction of quinones in acetonitrile by 2-propanol are compared with those by TEA in benzene and acetonitrile, and the specific properties of substitution are discussed.

Singlet molecular oxygen (¹O₂) is one of the major agents responsible for (photo)oxidative damage in biological systems including human skin and eyes. It has been reported that the neural hormone melatonin (MLT) can abrogate ¹O₂-mediated cytotoxicity through its purported high antioxidant activity. We studied the interaction of MLT with ¹O₂ in deuterium oxide (D₂O), acetonitrile and methanol by measuring the phosphorescence lifetime of ¹O₂ in the presence of MLT and related indoles for comparison. Rose bengal (RB) was used as the main ¹O₂ photosensitizer. The rate constant (k_q) for the total (physical and chemical) quenching of ¹O₂ by MLT was determined to be 4.0 × 10⁷ M^–1 s^–1 in D₂O (pD 7), 6.0 × 10⁷ M^–1 s^–1 in acetonitrile, and 6.1 × 10⁷ M^–1 s^–1 in methanol-d₁. The related indoles, tryptophan, 5-hydroxyindole, 5-methoxytryptamine, 5-hydroxytryptamine (5-OH-T, serotonin), 6-hydroxymelatonin (6-OH-MLT) and 6-chloromelatonin quenched ¹O₂ phosphorescence with similar k_q values. We also compared the photosensitized photobleaching rate of MLT with that of other indoles, which revealed that MLT is the most sensitive to ¹O₂ bleaching. Hydroxylation of the indole moiety in 5-OH-T and 6-OH-MLT makes them more sensitive to photodegradation. In the absence of exogenous photosensitizers MLT itself can generate ¹O₂ with low quantum yield (0.1 in CH₃CN) upon UV excitation. Thus, the processes we investigated may occur in the skin and eyes during physiological circadian rhythm (photo)signaling involving MLT and other indoles. Our results indicate that all the indoles studied, including MLT, are quite efficient yet very similar ¹O₂ quenchers. This directly shows that the exceptional antioxidant ability proposed for MLT is unsubstantiated when merely chemical mechanism(s) are considered in vivo, and it must predominantly involve humoral regulation that mobilizes other antioxidant defenses in living organisms.

Biological action spectra are commonly used to assess health and ecosystem responses to increases in spectral ultraviolet (UV) irradiances resulting from stratospheric ozone (O₃) reductions. For each action spectrum, a normalized sensitivity coefficient (the radiation amplification factor [RAF]) can be calculated as the relative increase in biologically active UV irradiance for a given relative decrease in the atmospheric O₃ column amount. We use a detailed radiative transfer model to calculate the dependence of RAF on the O₃ column amount and the solar zenith angle (and, therefore, implicitly on latitude and season) for several commonly used action spectra. A simple analytical model is used to interpret the results in terms of the semilogarithmic slope of the action spectra in the UV-B and UV-A wavelength ranges. We also show that RAF may be overestimated substantially if the UV-A portion of an action spectrum is significant but is neglected. This is illustrated using several idealized action spectra as well as published action spectra for plant responses to UV irradiation. Generally, if the portion of an action spectrum measured longward of ∼300 nm spans less than about two orders in magnitude in its sensitivity, significant errors in the estimated RAF may ensue, and the use of this action spectrum in O₃-related studies can be compromised.

The fluorescence of tryptophan, reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD) were characterized in normal human breast cells as well as in malignant human breast cells of similar and dissimilar genetic origins. Fluorescence measurements of each cell line were made over a wide range of cell concentrations, and the fluorescence per cell was determined from the slope in the linear range of the fluorescence intensity vs cell concentration plot. All of the malignant cells showed a statistically significant decrease in the tryptophan fluorescence per cell relative to that of the normal cells. No statistically significant differences were observed in the NAD(P)H or FAD fluorescence per cell between the normal and any of the malignant cell types. NAD(P)H fluorescence was also imaged from monolayers of the normal and malignant cells (of similar genetic origin) using two-photon fluorescence microscopy. A statistically significant decrease in the NAD(P)H fluorescence with malignancy was observed, suggesting that fluorescence imaging of single cells or the cell monolayer preparation may provide more contrast than volume-averaged fluorescence measurements of cells in suspension. In conclusion, the differences in normal and malignant human breast tissue fluorescence spectra may be attributed in part to differences in the intrinsic cellular fluorescence of normal and malignant breast epithelial cells.

The effects of ultraviolet (UV) radiation on chlorophyll content and accumulation of the anti-inflammatory monoterpene-indole alkaloid brachycerine in plants and calli of Psychotria brachyceras (Rubiaceae) were investigated. In this study, we also investigated a protective role for brachycerine against stress conditions. Calli and tip cuttings incubated in nutrient media were daily supplemented with 4 or 16 h of UV. High-performance liquid chromatography analyses of methanolic extracts showed only traces of brachycerine in irradiated aseptic cultures, with no alkaloid being observed in control calli. In cuttings, a 10-fold increase in brachycerine content was seen after exposure for 16 h to UV-C, whereas a 4 h daily supplementation doubled the amount of the alkaloid in leaves. Exposure to a UV-B source also doubled the alkaloid yield. In vitro brachycerine was able to quench singlet oxygen. The data indicate a potential protective role for brachycerine against UV radiation, acting as a UV filter (absorption peaks are within the UV range) and a reactive oxygen species scavenger. In addition, UV radiation may be used to increase yields of this compound of pharmaceutical interest.

Photodynamic therapy (PDT) is based on a photochemical reaction using a photosensitizer and light to produce reactive oxygen species that have biological effects. Although its application in some fields is largely based on thrombosis, in the vascular setting thrombosis must be prevented. In this study we examined the effects of PDT on the changes in activity of thrombomodulin (TM) and tissue factor (TF) as important regulators of the coagulation process of endothelial cells. Human umbilical vein endothelial cells were treated with PDT (chloro-aluminum-sulfonated phthalocyanine, λ = 630 nm) at different light-energy doses, and TM and TF levels were measured using fluorescence spectroscopy. Microparticles (MP) were analyzed using flow cytometry analysis. PDT alters the thrombogenic state of endothelial cells by causing decreased expression of TM and increased expression of functional TF in a light-energy dose–dependent way. PDT-treated endothelial cells shed large numbers of MP containing high levels of TF. TF functionality of PDT-treated cells, measured by a Factor Xa–generating assay, was high. TF was located mostly intracellularly and in MP. The disturbed anticoagulant balance described in this study may explain the occurrence of thrombosis induced by PDT and, if not contained, dispute the suitability of PDT as an adjuvant modality to treat vascular restenosis.

Photodetection (PD) and photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA)–induced protoporphyrin IX (PPIX) accumulation are approaches to detect and treat dysplasia and early cancer in the gastrointestinal tract and in the urinary bladder. Because ALA-induced PPIX production is limited, we synthesized ALA ester hydrochlorides 3–22 and tested them in two different in vitro models (gastrointestinal tract: HT29–CCD18; urinary bladder: J82–UROTSA). PPIX accumulation after incubation with 0.12 mmol/L for 3 h and PPIX accumulation as a function of different incubation times were measured using flow cytometry. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays were performed to check cellular dark toxicity. Phototoxicity after irradiation was tested. ALA nonafluorohexylester hydrochloride 11, ALA thiohexylester hydrochloride 13 and ALA dibenzyldiester dihydrochloride 19 induced appreciably increased PPIX levels and showed improved phototoxicity compared with the references ALA hydrochloride 1, ALA hexylester hydrochloride 3 and ALA benzylester hydrochloride 4. Thus, the new compounds 11, 13 and 19 are promising compounds for PD and PDT.

The core modification of expanded porphyrins has been proved to have better photochemical properties, which are favorable for photodynamic therapy (PDT) applications. In this context, this study was aimed to investigate the in vitro and in vivo photodynamic activity of one such core-modified expanded porphyrin, namely, ammonium salt of 5,10,15,20-tetrakis-(meso-p-sulfonato phenyl)-25,27,29-trithia sapphyrin. For the in vitro studies, human erythrocytes were used as a membrane semimodel system to investigate the partitioning ability and drug-uptake characteristics. The partition studies on the membrane semimodel system revealed that maximum partitioning occurs at 12 µgm/mL concentration, and from the drug-uptake studies it is observed that maximum amount of the sensitizer is bound to the erythrocyte membranes during a 45 min incubation period. Photohemolysis studies at different concentrations of the sensitizer and exposure time showed maximum damage at 5 µgm/mL and 30 min exposure time. In vivo studies were performed on 7,12-dimethylbenz-(a)nthracene–induced superficial squamous cell carcinoma on mouse skin. The sensitizer at a concentration of 2.5% in 2.0% dimethyl sulfoxide was applied topically on the tumor spot. After 1 h incubation the tumor spot was exposed to laser irradiation from Nd–YAG laser at its second harmonic wavelength of 532 nm. The photodynamic efficacy was estimated by tumor volume measurements at regular intervals after the treatment. One month after PDT exposure a 3.9-fold decrease in the tumor volume was observed with respect to the tumor volume before treatment. The treatment efficacy was further confirmed by histological and fluorescence spectroscopic evaluations of the tissue biopsy sample from the treated area. The results of our study suggest that the ammonium salt of 5,10,15,20-tetrakis-(meso-p-sulfonato phenyl)-25,27,29-trithia sapphyrin may find possible applications in the new modality of cancer treatment.

Tumor hypoxia, either preexisting or as a result of oxygen depletion during photodynamic therapy (PDT) light irradiation, can significantly reduce the effectiveness of PDT-induced cell killing. To overcome tumor hypoxia and improve tumor cell killing, we propose using supplemental hyperoxygenation during Photofrin-PDT. The mechanism for the tumor cure enhancement of the hyperoxygenation–PDT combination is investigated using an in vivo–in vitro technique. A hypoxic tumor model was established by implanting mammary adenocarcinoma in the hind legs of mice. Light irradiation (200 J/cm² at either 75 or 150 mW/cm²), under various oxygen supplemental conditions (room air, carbogen, 100% normobaric or hyperbaric oxygen), was delivered to animals that received 12.5 mg/kg Photofrin 24 h before light irradiation. Tumors were harvested at various time points after PDT and grown in vitro for colony formation analysis. Treated tumors were also analyzed histologically. The results show that when PDT is combined with hyperoxygenation, the hypoxic condition could be improved and the cell killing rate at various time points after PDT could be significantly enhanced over that without hyperoxygenation, suggesting an enhanced direct and indirect cell killing associated with high-concentration oxygen breathing. This study further confirms our earlier observation that when a PDT treatment is combined with hyperoxygenation it can be more effective in controlling hypoxic tumors.

Chromophore-modified bacteriorhodopsin (bR) analogs are prepared, to study the nature of chromophore–protein interaction as well as to develop new bR analogs that can find applications as photoactive element in molecular electronic devices. This article describes the preparation and characterization of hitherto unknown bR analogs based on diphenylpolyene chromophores. Diphenylpolyene compounds, namely, 4-[(E)-2-phenylvinyl]benzaldehyde (1), 3-methyl-5-{4-[(E)-2-phenylvinyl]phenyl}penta-2E,4E-dienal (2), 4-[4-phenylbuta-1E,3E-dienyl]benzaldehyde (3) and 3-methyl-5-{4-[4-phenylbuta-1E,3E-dienyl]phenyl}penta-2E,4E-dienal (4), have been synthesized, and their interaction with bacterioopsin (bOP) has been studied. Whereas aldehydes 2 and 4 interact with bOP and yield bR analogs bR-2 and bR-4, aldehydes 1 and 3 do not yield any pigment. Analogs bR-2 and bR-4 have been characterized for their opsin shift, competitive binding, photochemical properties and fluorescence spectral behavior.

Pharaonis phoborhodopsin (ppR, also called Natronobacterium pharaonis sensory rhodopsin II) and its transducer protein, pharaonis halobacterial transducer of ppR (pHtrII), form a signaling complex, and light signals are transmitted from the sensor to the transducer by the protein–protein interaction. A truncated pHtrII(1–159) consisting of intramembrane helices (expressing amino acid residues from the first to the 159th position) and ppR form the complex in a solution containing 0.1% n-dodecyl-β-d-maltoside. At 75–85°C, the time-dependent color loss of ppR was caused by denaturation. We found that pHtrII(1–159) retarded the denaturation rate of ppR. This increase in the thermal stability was used as a probe for the binding ability in the dark. Tyr199 of ppR and Asn74 of pHtrII(1–114) were proposed as amino acid residues interacting with each other through hydrogen bonding. Then, ppR and pHtrII(1–159) mutants at these positions were prepared to examine the effect on the binding in the dark. The wild-type and Y199F mutant can bind pHtrII(1–159), suggesting that the hydrogen bonding between these specific amino acid residues may not be the only cause of the binding, but the hydrophobic interaction via phenyl ring of ppR may contribute dominantly.

There is a growing body of evidence showing that optical spectroscopy has the potential to be a useful in vivo diagnostic tool. Yet, so far there is no definitive cellular and biochemical understanding for the differences seen in the spectra from different tissue categories and disease states. In this study, we examine the use of organotypic raft cultures as an in vitro model of in vivo tissue conditions in an attempt to overcome some of the limitations of previously used methods. Organotypic raft cultures resembling normal and dysplastic epithelial cervical tissue were constructed and grown at an air–liquid interface for 2 weeks. Raman spectra of normal as well as dysplastic raft cultures were measured and compared with in vivo spectra from the corresponding tissue type. Histologic comparisons ensured that the raft cultures had similar structure and morphology to the corresponding intact tissue types. Raman spectra were also acquired from different layers of tissue. Spectral comparisons show that the Raman spectra of the raft cultures are similar to the spectra acquired from the cervix in vivo for both normal and dysplastic tissues. These results show that organotypic raft cultures are an effective and useful tool for the cellular and biochemical analysis of tissue spectroscopy.

The absorption spectra of bacteriochlorophyll (BChl) c solutions in two mixtures of two solvents (acetonitrile with pyridine and dimethylsulfoxide with methanol) exhibiting different refractive indices were measured and deconvoluted into Gaussian components. The refractive index of mixed solvents was changed by the change in the ratio of the volumes of the liquids used in the mixture. Using the Q_y(0,0) band half widths and absorption coefficient, based on the simplified formula proposed by Knox, the dipole strengths of the Q_y(0,0) BChl c transition for various values of solvent refractive index were calculated and compared with values given by Knox and Spring. For both investigated combinations of two liquids, the dependence of Q_y(0,0) transition dipole strength of the BChl c on solvent refractive index was almost linear. The slopes of the lines obtained from the experimental absorption bands were different for two investigated solvent mixtures. More accurate linear dependence and similar slopes of lines for both solvent mixtures were obtained using half widths and absorption coefficients of the Gaussian components of Q_y(0,0) transition. It is explained by the superposition of the contributions from other electronic and vibronic transitions of BChl c monomers or possibly also from transitions of the pigments involved in some complexes with solvent molecules in the absorption region investigated. The results show that the formula proposed by Knox can be successfully applied also for BChl c, after elimination of the overlapped contributions from the other transitions, by applying Gaussian analysis to select only contribution from Q_y(0,0) pigment transition.