The skin is the only tissue yet known in which the complete ultraviolet-B (UV-B)–induced pathway from 7-dehydrocholesterol to hormonally active calcitriol (1α,25-dihydroxyvitamin D₃) occurs under physiological conditions. Epidermal synthesis of calcitriol could be of fundamental relevance because calcitriol regulates important cellular functions in keratinocytes and immunocompetent cells. Because of their antiproliferative and prodifferentiating effects, calcitriol and other vitamin D analogs are highly efficient in the treatment of psoriasis vulgaris. The known antipsoriatic effect of UV-B light could, at least in part, be mediated via UV-B–induced synthesis of calcitriol. In addition, mounting evidence indicates that cutaneous vitamin D₃ synthesis is of high importance for the prevention of a broad variety of diseases, including various malignancies. New but controversially discussed sun-protection guidelines were established for the prevention of internal cancers. A better understanding of the metabolism of vitamin D in the skin opens new perspectives for therapeutic applications of vitamin D analogs.

Most common diseases appear to result from complex, poorly understood interactions between genetic and environmental factors. Relatively few factors have been unequivocally linked with disease risk or outcome. Evidence from various studies using different experimental approaches has been interpreted as showing that, apart from its harmful effects on the pathogenesis of the common skin cancers, ultraviolet radiation (UVR) may exert a beneficial effect on development of various internal cancers and other pathologies. This concept is supported by parallel studies showing that hypovitaminosis D is linked with increased risk of various diseases including insulin resistance and multiple sclerosis. These findings suggest that, first, host factors such as skin pigmentation that affect UVR-induced synthesis of vitamin D and, second, polymorphism in genes that mediate the effectiveness of vitamin D action are susceptibility candidates for a variety of diseases. Collectively, these data suggest the hypothesis that, via effects on vitamin D synthesis, UVR exposure has beneficial effects on susceptibility and outcome to a variety of complex diseases. We describe evidence from studies in various diseases, but mainly from prostate cancer patients, that supports this hypothesis, but we emphasize that, although supportive data are available, the concept is unproven. Indeed, other explanations are possible. However, given the potentially important public health implications of the hypothesis and the potential for the development of novel therapeutic modalities, we believe the concept is worthy of further investigation.

This paper reviews briefly the evidence for an association between various measures of UV radiation (UVR) exposure and the development of cancer. Issues such as data quality, study design, measurement variation, comparability of studies, and quantification of UV exposure in relation to skin cancer are discussed. A range of exposure, based on skin type, might be appropriate: from 5 min a day three times a week for light-skinned individuals and 10 min a day three times a week for darker-skinned individuals. These exposures translate into 13 h per year, for a light-skinned individual, leading to 650 h of exposure from birth to age 50.

We review the evidence indicating a possible beneficial role for UVR on three Th1-mediated autoimmune diseases: multiple sclerosis, type 1 diabetes and rheumatoid arthritis in relation to recent developments in photoimmunology. Recent work suggests that UVR exposure may be one factor that can attenuate the autoimmune activity leading to these three diseases through several pathways involving UVB and UVA irradiation, UVR-derived vitamin D synthesis and other routes such as α-melanocyte-stimulating hormone, calcitonin gene related peptide and melatonin. Ecological features, particularly a gradient of increasing prevalence of multiple sclerosis and type 1 diabetes with higher latitude, provide some support for a beneficial role of UVR. Analytical studies provide additional support, particularly as low vitamin D has been prospectively associated with disease onset for all three diseases, but are not definitive. Randomized controlled trial data are required. Further, we discuss how associated genetic studies may assist the accumulation of evidence with regard to the possible causal role of low UVR exposure and/or low vitamin D status in the development of these diseases.

Vitamin D sufficiency is required for optimal health, and solar ultraviolet B (UVB) irradiance is an important source of vitamin D. UVB and/or vitamin D have been found in observational studies to be associated with reduced risk for over a dozen forms of cancer, multiple sclerosis, osteoporotic fractures, and several other diseases. On the other hand, excess UV irradiance is associated with adverse health outcomes such as cataracts, melanoma, and nonmelanoma skin cancer. Ecologic analyses are used to estimate the fraction of cancer mortality, multiple sclerosis prevalence, and cataract formation that can be prevented or delayed. Estimates from the literature are used for other diseases attributed to excess UV irradiation, additional cancer estimates, and osteoporotic fractures. These results are used to estimate the economic burdens of insufficient UVB irradiation and vitamin D insufficiency as well as excess UV irradiation in the United States for these diseases and conditions. We estimate that 50 000–63 000 individuals in the United States and 19 000–25 000 in the UK die prematurely from cancer annually due to insufficient vitamin D. The U.S. economic burden due to vitamin D insufficiency from inadequate exposure to solar UVB irradiance, diet, and supplements was estimated at $40–56 billion in 2004, whereas the economic burden for excess UV irradiance was estimated at $6–7 billion. These results suggest that increased vitamin D through UVB irradiance, fortification of food, and supplementation could reduce the health care burden in the United States, UK, and elsewhere. Further research is required to confirm these estimates.

Vitamin D production in human skin occurs only when incident UV radiation exceeds a certain threshold. From simulations of UV irradiances worldwide and throughout the year, we have studied the dependency of the extent and duration of cutaneous vitamin D production in terms of latitude, time, total ozone, clouds, aerosols, surface reflectivity and altitude. For clear atmospheric conditions, no cutaneous vitamin D production occurs at 51 degrees latitude and higher during some periods of the year. At 70 degrees latitude, vitamin D synthesis can be absent for 5 months. Clouds, aerosols and thick ozone events reduce the duration of vitamin D synthesis considerably, and can suppress vitamin D synthesis completely even at the equator. A web page allowing the computation of the duration of cutaneous vitamin D production worldwide throughout the year, for various atmospheric and surface conditions, is available on the Internet at  http://zardoz.nilu.no/~olaeng/fastrt/VitD.html and  http://zardoz.nilu.no/~olaeng/fastrt/VitD-ez.html. The computational methodology is outlined here.

Cryptochromes are flavoproteins that exhibit high sequence and structural similarity to the light-dependent DNA-repair enzyme, photolyase. Cryptochromes have lost the ability to repair DNA; instead, they use the energy from near-UV/blue light to regulate a variety of growth and adaptive processes in organisms ranging from bacteria to humans. The photocycle of cryptochrome is not yet known, although it is hypothesized that it may share some similarity to that of photolyase, which utilizes light-driven electron transfer from the catalytic flavin chromophore. In this review, we present genetic evidence for the photoreceptive role of cryptochromes and discuss recent biochemical studies that have furthered our understanding of the cryptochrome photocycle. In particular, the role of the unique C-terminal domain in cryptochrome phototransduction is discussed.

The presence of the regenerable visual pigment rhodopsin has been shown to be primarily responsible for the acute photodamage to the retina. The photoexcitation of rhodopsin leads to isomerization of its chromophore 11-cis-retinal to all-trans-retinal (ATR). ATR is a potent photosensitizer and its role in mediating photodamage has been suspected for over two decades. However, there was lack of experimental evidence that free ATR exists in the retina in sufficient concentrations to impose a risk of photosensitized damage. Identification in the retina of a retinal dimer and a pyridinium bisretinoid, so called A2E, and determination of its biosynthetic pathway indicate that substantial amounts of ATR do accumulate in the retina. Both light damage and A2E accumulation are facilitated under conditions where efficient retinoid cycle operates. Efficient retinoid cycle leads to rapid regeneration of rhodopsin, which may result in ATR release from the opsin “exit site” before its enzymatic reduction to all-trans-retinol. Here we discuss photodamage to the retina where ATR could play a role as the main toxic and/or phototoxic agent. Moreover, we discuss secondary products of (photo)toxic properties accumulating within retinal lipofuscin as a result of ATR accumulation.

To assist standardization of procedures, facilitate comparisons, and help guide research efforts to optimally inform development of appropriately targeted interventions, there is a need to review methods used to quantify child and adolescent solar ultraviolet radiation (UV) exposure, related outdoor activities and sun-protective practices. This holistic approach is essential for comprehensive research that will provide all-inclusive, informative and meaningful messages for preventive measures of harmful UV exposure. Two databases were searched and 29 studies were retrieved, and these studies report measurement or assessment techniques documenting UV exposure patterns and related outdoor activities. Polysulfone film badges were the main measurement instrument used in 10 studies, with questionnaire, survey data, observation, a model, electronic dosimeters, biological dosimeters, colorimeter and UV colouring labels used in the remaining studies. Methods used to record activities included self-report, parental report, a logbook and observation. Measurement duration and unit of UV exposure varied in most studies, but a method common to 15 studies was measured UV exposure as a percentage of ambient UV. The studies reviewed do not provide sufficient information for the development and evaluation of targeted youth sun protection programs. Studies are required which document precise UV exposure, concurrent activities and sun protection usage for children and adolescents.

Circular dichroism (CD) was used to study the structure of oxyblepharismin (OxyBP), the photoreceptor chromophore for the photophobic response of the blue form of Blepharisma japonicum. Both the chromophore associated to its native protein and the free chromophore in ethanol solution were investigated. CD spectra in the far-UV range indicate that OxyBP induces a slight increase in the α-helix content of the protein matrix. CD spectra in the near-UV and visible region of the spectrum show that OxyBP adopts a chiral conformation with a preferential geometry not only when associated to its protein matrix, but also when isolated and dissolved in ethanol. This experimental result is related to the existence of a high-energy interconversion barrier between two enantiomeric structures of the molecule and discussed on the basis of an asymmetric biosynthesis of its precursor, blepharismin.

The human retinal pigment epithelial (RPE) layer contains a complex mixture of components called lipofuscin; this mixture forms with age and with various genetic disorders such as Stargardt's disease. Its presence may contribute to retinal deterioration via several mechanisms including photochemical processes. In the lipofuscin mixture, both type I and II mechanisms have been identified, with the latter consisting of the generation of singlet oxygen. Several components of that mixture have been identified, most notably a bis-retinoid pyridinium compound called A2E and its derivatives. Photooxidative studies on the compound A2E have revealed that its dominant photochemical mechanism is via free radical or type I processes. Because singlet oxygen is an important photooxidative intermediate in tissue, its generation in the RPE may contribute to retinal maculopathies. It is therefore necessary to determine which specific component(s) in the lipofuscin mixture produce singlet oxygen upon excitation with light. This was ascertained by evaluating the action spectrum for singlet oxygen production for the whole lipofuscin mixture using time-resolved spectroscopy. Singlet oxygen was generated by excitation of the sample at different wavelengths while maintaining a constant beam energy, and was directly detected by its phosphorescence decay at 1270 nm using a Ge photodiode. The action spectrum for singlet oxygen sensitization by the organic soluble portion of lipofuscin had an absorption maximum at ca 380 nm, which is to the blue of A2E (maximum at 430 nm). Compounds with a similar absorption maximum eluted in the HPLC earlier than A2E and were detected in human lipofuscin. The concentration of this component apparently increased in concentration in human RPE lipofuscin mixture as a function of age up to 90 years old.

This study was designed to demonstrate that bioluminescence imaging (BLI) can be used as a new tool to evaluate the effects of low-level laser therapy (LLLT) during in vivo inflammatory process. Here, the efficacy of LLLT in modulating inducible nitric oxide synthase (iNOS) expression using different therapeutic wavelengths was determined using transgenic animals with the luciferase gene under control of the iNOS gene expression. Thirty transgenic mice, FVB/N-Tg(iNOS-luc)Xen, were allocated randomly to one of four experimental groups treated with different wavelengths (λ = 635, 785, 808 and 905 nm) or a control group (nontreated). Inflammation was induced by intra-articular injection of zymosan A in both knee joints. Laser treatment (25 mW cm⁻², 200 s, 5 J cm⁻²) was applied to the knees 15 min after inflammation induction. Measurements of iNOS expression were performed at various times (0, 3, 5, 7, 9 and 24 h) by measuring the bioluminescence signal using a highly sensitive charge-coupled device (CCD) camera. The results showed a significant increase in BLI signal after irradiation with 635 nm laser when compared to the nonirradiated animals and the other LLLT-treated groups, indicating wavelength dependence of LLLT effects on iNOS expression during the inflammatory process, and thus demonstrating an action spectrum of iNOS gene expression following LLLT in vivo that can be detected by BLI. Histological analysis was also performed and demonstrated the presence of fewer inflammatory cells in the synovial joints of mice irradiated with 635 nm compared with nonirradiated knee joints.

In bicelle bacteriorhodopsin (bcbR) crystals, the protein has a different structure from both native bacteriorhodopsin (bR) and in-cubo bR (cbR) crystals. Recently, we studied the ability of bcbR crystals to undergo the photocycle upon laser excitation, characterized by the appearance of the M intermediate by single crystal resonance Raman spectroscopy. Calculation of the M lifetime by flash photolysis experiments demonstrated that in our bcbR crystals, the M rise time is much faster than in the native or cbR crystals, with a decay time that is much slower than these other two forms. Although it is now known that the bcbR crystals are capable of photochemical deprotonation, it is not known whether photochemical deprotonation is the only way to create the deprotonated Schiff base in the bcbR crystals. We measured both the visible and Raman spectra of crystals dried under ambient lighting and dried in the dark in order to determine whether the retinal Schiff base is able to thermally deprotonate in the dark. In addition, changes in the visible spectrum of single bcbR crystals under varying degrees of hydration and light exposure were examined to better understand the retinal binding environment.

Employing a luminescence spectrometer the usefulness of light-induced delayed luminescence (DL) for the detection of aflatoxinB1 (AfB1) contamination in whole peanut was studied. Peanut was artificially contaminated with Aspergillus flavus (7.5 × 10⁵conidia/mL) and incubated for 0–72 h. The DL and fluorescence spectra of contaminated peanut were obtained by a luminescence spectrometer. The correlation between the spectra and contamination levels was established. The DL and fluorescence intensity has a negative correlation with the AfB1 concentration. Our results suggested that the DL technique might be useful for the rapid and noninvasive evaluation of AfB1 contamination levels in whole peanut.

Immunosuppressive doses of solar-simulated UV radiation activate lymph node B cells that can suppress primary immunity by inhibiting the function of dendritic cells. The aim of this study was to determine the waveband responsible for activation of these suppressor B cells. We exposed C57BL/6 mice to various doses of either UVA or UVB radiation and analyzed the number and activation state of lymph node antigen-presenting cells (APC). Immunosuppressive doses of UVB but not UVA activated B cells as assessed by major histocompatibility complex II (MHC II) expression and doubled their numbers in draining lymph nodes. Higher doses of UVA that were not immunosuppressive actually suppressed B cell activation. Our results show that UVA and UVB suppress systemic immunity via different mechanisms. Lymph node B cells are activated in response to immunosuppressive doses of UVB but not UVA. Thus, the activation state of lymph node APC appears to be important for UV immunomodulation.

The molecular structure of 1,4,6,8-tetramethylfuro[2,3-h]quinolin-2(1H)-one (FQ), a recent furocoumarin-like photosensitizer, has been modified with the aim of reducing its strong genotoxicity, by replacing the methyl group at 4 position with a hydroxymethyl one, and so obtaining 4-hydroxymethyl-1,6,8-trimethylfuro[2,3-h]quinolin-2(1H)-one (HOFQ). This modification gave rise to a strong reduction of lipophilicity and dark interaction with DNA. The formation of monoadducts (MA) was deeply affected, whereas the induction of bifunctional adducts between DNA and proteins (DPC_L>0) was replaced by an efficient production of DNA–protein cross-links at zero length (DPC_L=0), probably via guanine damage. Because of its angular molecular structure, HOFQ does not form interstrand cross-links (ISC): therefore, DPC_L=0 and MA represent the main lesions induced by HOFQ in DNA. In comparison with FQ (which induces MA and DPC_L>0) and 8-methoxypsoralen (8-MOP) (MA, ISC, DPC_L>0), HOFQ seems to be a more selective agent. In fact, contrary to FQ and 8-MOP, HOFQ, together with a noticeable antiproliferative activity, shows low levels of point mutations in bacteria and of clastogenic effects in mammalian cells. HOFQ is also an efficient apoptosis inducer, especially in comparison with 8-MOP, when tested at equitoxic experimental conditions; this property might be correlated with the complete HOFQ inability of inducing skin erythemas, a well-known side effect of classic furocoumarin photosensitization.

Photodynamic therapy (PDT) is frequently accompanied by induction of systemic immunosuppression. Photochemical mechanisms underlying this effect are not completely understood. Here, we demonstrate the immunosuppressive activity of photooxidation products of protoporphyrin IX dimethyl ester (PPIX) in a murine model of contact hypersensitivity (CHS) to 2,4-dinitrofluorobenzene (DNFB). Intravenous injection of the preirradiated solution of PPIX to mice resulted in fluence-dependent suppression of the CHS. The samples of photodecomposed PPIX with suppressive effect on the CHS contained chlorin-type products, namely, two isomers of photoprotoporphyrin (pPP1 and pPP2) as main photoproducts. Concentration-dependent suppression of the CHS was also induced when purified pPP1 or pPP2 were injected to mice intravenously. These purified photoproducts exerted equal immunosuppressive activity. The highest suppression of the CHS was induced when pPP1 was injected 20 h before sensitization with DNFB. The lowest suppression was at its injection time 24 h before challenge. The pPP1-induced suppression of the CHS was adoptively transferable and was associated with generation of cells with suppressive functions. These suppressor cells inhibited the efferent phase of the CHS. Our results strongly indicate that induction of systemic immunosuppression by PDT with PPIX may proceed through photobleaching of photosensitizer and generation of photoprotoporphyrins, which can affect T cell immunity.

We report measurements of fluorescence resonance energy transfer (FRET) for glucose sensing in an established concanavalin A–dextran affinity system using frequency-domain lifetime spectroscopy. A dextran (MW 2 000 000) labeled with a small fluorescent donor molecule, Alexa Fluor 568, was used to competitively bind to a sugar-binding protein, concanavalin A, labeled with acceptor molecule, Alexa Fluor 647, in the presence of glucose. The FRET-quenching kinetics of the donor were analyzed from frequency-domain measurements as a function of both glucose and acceptor-protein concentrations using a Förster-type decay kinetics model. The results show that the frequency-domain measurements and donor decay kinetics can quantitatively indicate changes in the competitive binding of 0.09 μM dextran to labeled concanavalin A at a solution concentration of 10.67 μM in the presence of glucose at concentrations ranging from 0 to 224 mg/dL.

By means of UV-VIS absorption and fluorescence spectroscopy, we demonstrate that the photosensitizer hypericin (Hyp) interacts nonspecifically with low-density lipoproteins (LDL), most probably with the lipid fraction of LDL. The molar ratio of monomeric Hyp binding to nonoxidized LDL and mildly oxidized LDL is 30:1. Increasing the Hyp concentration further leads to the formation of Hyp aggregates inside the LDL molecule. We also demonstrate that photoactivated Hyp oxidizes LDL in a light dose and excitation wavelength dependent manner. The level of oxidation of LDL depends on the amount of Hyp inside the LDL molecule. The maximum of the photosensitized oxidation of the LDL by Hyp is achieved for a 30:1 molar ratio, which corresponds to the maximum concentration of monomeric form of Hyp in LDL.

The effects of pH and ultraviolet-B (UV-B) irradiation on the secondary structure of human serum albumin (HSA) in the absence or presence of captopril were investigated by an attenuated total reflection (ATR)/Fourier transform infrared (FTIR) spectroscopy. The UV-B exposure affecting the stability of captopril before and after captopril–HSA interaction was also examined by using confocal Raman microspectroscopy. The results indicate that the transparent pale-yellow solution for captopril–HSA mixture in all pH buffer solutions, except pH 5.0∼7.0, changed into a viscous form then a gel form with UV-B exposure time. The secondary structural transformation of HSA in the captopril–HSA mixture with or without UV-B irradiation was found to shift the maxima amide I peak in IR spectra from 1652 cm⁻¹ assigned to α-helix structure to 1622 cm⁻¹ because of a β-sheet structure, which was more evident in pH 3.0, 8.0 or 9.0 buffer solutions. The Raman shift from 1653 cm⁻¹ (α-helix) to 1670 cm⁻¹ (β-sheet) also confirmed this result. Captopril dissolved in distilled water with or without UV-B irradiation was determined to form a captopril disulfide observed from the Raman spectra of 512 cm⁻¹, which was exacerbated by UV-B irradiation. There was little disulfide formation in the captopril–HSA mixture even with long-term UV-B exposure, but captopril might interact with HSA to change the protein secondary structure of HSA whether there was UV-B irradiation or not. The pH of the buffer solution and captopril–HSA interaction may play more important roles in transforming the secondary structure of HSA from α-helix to β-sheet in the corresponding captopril–HSA mixture than UV-B exposure. The present study also implies that HSA has the capability to protect the instability of captopril in the course of UV-B irradiation. In addition, a partial unfolding of HSA induced by pH or captopril-HSA interaction under UV-B exposure is proposed.

The present study demonstrates the in vitro effect of hypericin-mediated PDT with fractionated light delivery. Cells were photosensitized with unequal light fractions separated by dark intervals (1 or 6 h). We compared the changes in viability, cell number, survival, apoptosis and cell cycle on HT-29 cells irradiated with a single light dose (12 J/cm²) to the fractionated light delivery (1 11 J/cm²) 24 and 48 h after photodynamic treatment. We found that a fractionated light regime with a longer dark period resulted in a decrease of hypericin cytotoxicity. Both cell number and survival were higher after light sensitization with a 6-h dark interval. DNA fragmentation occurred after a single light-dose application, but in contrast no apoptotic DNA formation was detected with a 6-h dark pause. After fractionation the percentage of cells in the G1 phase of the cell cycle was increased, while the proportion of cells in the G2 phase decreased as compared to a single light-dose application, i.e. both percentage of cells in the G1 and G2 phase of the cell cycle were near control levels. We presume that the longer dark interval after the irradiation of cells by first light dose makes them resistant to the effect of the second illumination. These findings confirm that the light application scheme together with other photodynamic protocol components is crucial for the photocytotoxicity of hypericin.

Ketoprofen is an important anti-inflammatory drug, but its dermal application is limited because of the photosensitizing properties causing phototoxic reactions of the skin when exposed to UV light. We have recently investigated the peroxide formation of ketoprofen in solutions of linoleic acid during UV irradiation. To continue these studies and focus on UV-induced changes in membrane integrity and barrier function we established an in vitro model using two biosensor techniques simultaneously. Support-fixed bilayers were irradiated with different doses of UV-B up to damaging intensities with or without ketoprofen. Cyclic voltammetry was carried out to detect alterations in membrane permeability; quartz crystal microbalance (QCM) measurements were helpful in analyzing whether a permeability increase was caused by depletion of membrane components. In absence of ketoprofen, increasing UV-B doses induce membrane permeabilities of both unsaturated and saturated bilayers. QCM measurements could not reveal a significant loss of membrane components as a reason for the permeability. In contrast, 0.3 mM ketoprofen induced a dose-dependent increase in membrane permeability. QCM results indicated a mass loss. Although this model does not explain all molecular mechanisms of membrane damage by ketoprofen, the combined application of both QCM and CV is a novel and powerful tool to investigate functional mechanisms of UV-induced membrane damages.

The protozoan ciliate Paramecium bursaria exhibits membrane hyperpolarization in response to photostimulation, accompanied with an increased swimming speed. The external addition of cyclic nucleotide phosphodiesterase (PDE) inhibitors, either theophylline (1,3-dimethylxanthine) or 3-isobutyl-1-methylxanthin (IBMX), increased in both amplitudes of the membrane hyperpolarization and the increase in swimming speed. Moreover, the addition of membrane permeable cyclic nucleotide analogs, either 8-bromo-adenosine 3′,5′-cyclic monophosphate (Br-cAMP) or 8-Br-guanosine 3′,5′-cyclic monophosphate (Br-cGMP), increased these amplitudes. On the other hand, the addition of l-cis-diltiazem, known to block the conductance of cyclic nucleotide–gated channels, partially decreased both amplitudes of the membrane hyperpolarization and the increase in swimming speed. An enzyme immunoassay of cellular cyclic nucleotide contents showed that photostimulation induced a rapid increase in adenosine 3′,5′-cyclic monophosphate (cAMP), but little increase in guanosine 3′,5′-cyclic monophosphate (cGMP), raising the possibility that a rapid increase in cAMP mediates the light-induced hyperpolarization in P. bursaria.

The conceptual basis for the development of mitochondrial targeting as a novel therapeutic strategy for both chemotherapy and photochemotherapy of neoplastic diseases rests on the observation that enhanced mitochondrial membrane potential is a common tumor cell phenotype. The potential of this strategy is highlighted by the fact that the toxic effects associated with a number of cationic dyes known to localize in energized cell mitochondria are much more pronounced in tumor cells than in normal cells. Here we evaluate the phototoxic properties of four bromine derivatives of rhodamine-123 toward human uterine sarcoma (MES-SA) and green monkey kidney (CV-1) cells and compare the degrees of tumor cell selectivity associated with these dyes with those associated with two model mitochondrial triarylmethanes (crystal violet and ethyl violet). Selective phototoxicity toward tumor cells was found to be highly dependent upon the lipophilic/hydrophilic character of the cationic photosensitizer. Our experimental data have indicated that the probability of success of mitochondrial targeting in (photo)chemotherapy of neoplastic diseases is higher when the octan-1-ol/water partition coefficient of the drug candidate falls within approximately two orders of magnitude from that of the prototypical mitochondria-specific dye rhodamine-123.

The scope of photobiological processes that involve absorbers within a protein matrix may be limited by the vulnerability of the peptide group to attack by highly reactive redox centers consequent upon electronic excitation. We have explored the nature of this vulnerability by undertaking comprehensive product analyses of aqueous photolysates of 12 N-p-toluenesulfonyl peptides with systematically selected structures. The results indicate that degradation includes a major pathway that is initiated by intramolecular electron transfer in which the peptide bond serves as electron donor, and the data support the likelihood of a relay process in dipeptide derivatives.

Lichens synthesize and accumulate photoprotective compounds against possible damage induced by UV radiation in the photobiont. A biological model has been recently formulated that allows the use of lichens to evaluate changes at different UV radiation levels. The thermodynamics, photophysical and photochemical properties of lobaric acid were studied in acetonitrile, ethanol and Brij 35(3%) micelles at different pH values. Also the sun protector factor (SPF) was determined by in vitro methods. Lobaric acid was extracted from Stereoculon alpinum Laur. and characterized by means of standard procedures. Solutions were irradiated in oxygen and under nitrogen conditions with a UV medium pressure lamp. Lobaric acid absorbs at 287, 303 nm, and no fluorescence emission was observed. The maximum value of the molar extinction coefficient (5479.6 M⁻¹ cm⁻¹) was obtained in Brij 35 at pH 12. Solubility is pH dependant and is highest in Brij 35 at pH 12 (4.45 × 10⁻⁴ M). Photoconsumption quantum yields ranged between 10⁻⁴ and 10⁻⁵ in aerobic and anaerobic experimental conditions. Lobaric acid SPF was very low (0.5) compared with homosalate (4.0), (reference solar filter). Two pKa values, 5.05 (carboxylic acid group deprotonation) and 9.75 (phenolic OH deprotonation), were determined.

This study is aimed at establishing optimal conditions for the use of 2,2′-[1,3-propanediylbis[(dimethyliminio)-3,1-propanediyl-1(4H)-pyridinyl-4-ylidenemethy-lidyne]]bis[3-methyl]-tetraiodide (BOBO-1) as a fluorescent probe in the characterization of lipid/DNA complexes (lipoplexes). The fluorescence spectra, anisotropy, fluorescence lifetimes and fluorescence quantum yields of this dimeric cyanine dye in plasmid DNA (2694 base pairs) with and without cationic liposomes (1,2-dioleoyl-3-trimethylammonium-propane [DOTAP]), are reported. The photophysical behavior of the dye in the absence of lipid was studied for several dye/DNA ratios using both supercoiled and relaxed plasmid. At dye/DNA ratios (d/b) below 0.01 the fluorescence intensity increases linearly, whereas lifetime and anisotropy values of the dye are constant (τ̄ ∼ 2.5 ns and <r> = 0.20). By agarose gel electrophoresis it was verified that up to d/b = 0.01 DNA conformation is not considerably modified, whereas for d/b = 0.05–0.06 a single heavy band appears on the gel. For these and higher dye/DNA ratios the fluorescence intensity, anisotropy and average lifetime values decrease with an increase in BOBO-1 concentration. When cationic liposomes are added to the BOBO-1/DNA complex, an additional effect is noticed: The difference in the environment probed by BOBO-1 bound to DNA leads to a decrease in quantum yield and average lifetime values, and a redshift is apparent in the emission spectrum. For fluorescence measurements including energy transfer (FRET), a d/b ratio of 0.01 seems to be adequate because no considerable change on DNA conformation is detected, a considerable fluorescent signal is still measured after lipoplex formation, and energy migration is not efficient.

We present a quantitative framework to model a Type II photodynamic therapy (PDT) process in the time domain in which a set of rate equations are solved to describe molecular reactions. Calculation of steady-state light distributions using a Monte Carlo method in a heterogeneous tissue phantom model demonstrates that the photon density differs significantly in a superficial tumor of only 3 mm thickness. The time dependences of the photosensitizer, oxygen and intracellular unoxidized receptor concentrations were obtained and monotonic decreases in the concentrations of the ground-state photosensitizer and receptor were observed. By defining respective decay times, we quantitatively studied the effects of photon density, drug dose and oxygen concentration on photobleaching and cytotoxicity of a photofrin-mediated PDT process. Comparison of the dependences of the receptor decay time on photon density and drug dose at different concentrations of oxygen clearly shows an oxygen threshold under which the receptor concentration remains constant or PDT exhibits no cytotoxicity. Furthermore, the dependence of the photosensitizer and receptor decay times on the drug dose and photon density suggests the possibility of PDT improvement by maximizing cytotoxicity in a tumor with optimized light and drug doses. We also discuss the utility of this model toward the understanding of clinical PDT treatment of chest wall recurrence of breast carcinoma.

This study examines the effects of natural solar radiation on the metal-binding capacity of dissolved organic matter (DOM). Newington Bog water (35.5 mg L⁻¹ dissolved organic carbon [DOC]) was irradiated for 20 days under UV-B lamps in the laboratory and under natural solar radiation. In the presence of irradiated DOM, IC₅₀ (contaminant concentration required to reduce algal growth by 50%) was significantly decreased with UV-B treatment for four metals: Pb, 64%; Cu, 63%; Ni, 35% and Cd, 40%. Solar radiation also significantly decreased IC₅₀ of Pb (58%) and Cu (49%), DOC concentration (11%), DOM fluorescence (DOMFL, 33%) and DOC-specific UV absorbance. Further experiments on Raisin River water (20.7 mg DOC L⁻¹) exposed to 20 days of artificial UV-A and UV-B radiation produced significant decreases in IC₅₀ for Cu (48%) with UV-A and for Pb (43%) with UV-B. DOC concentration was decreased 20% by UV-B and 24% by UV-A. DOMFL decreased 51.5% in the first 5 days of UV-A exposure, an effect that was not observed with the UV-B treatment. The UV-A treatment decreased UV absorbance more at longer wavelengths and over a broader wavelength band than did the UV-B treatment. Change in toxicity with UV irradiation was inconsistent among the metals tested in this study, indicating that some organic metal-binding ligands were more quickly removed or altered than others. The DOM remaining after irradiation appears to be qualitatively different from the unirradiated DOM. The much greater irradiance of UV-A makes its contribution to the removal and/or alteration of DOM at least as important as the influence of higher energy UV-B.

The second cyanobacterial phytochrome Cph2 from Synechocystis sp. PCC 6803 was suggested as a part of a light-stimulated signal transduction chain inhibiting movement toward blue light. Cph2 has the two bilin binding sites, cysteine-129 and cysteine-1022, that might be involved in sensing of red/far-red and blue light, respectively. Here, we present data on wavelength dependence of the phototaxis inhibition under blue light, indicating that Cph2 itself is the photoreceptor for this blue light response. We found that inhibition of blue-light phototaxis in wild-type cells occurred below the transition point of about 470 nm. Substitution of cysteine-1022 with valine led to photomovement of the cells toward blue light (cph2⁻ mutant phenotype). Analysis of mutants lacking cysteine-129 in the N-terminal chromophore binding domain indicated that this domain is also important for Cph2 function or folding of the protein. Furthermore, putative blue-light and phytochrome-like photoreceptors encoded by the Synechocystis sp. PCC 6803 genome were inactivated in wild-type and cph2 knockout mutant background. Our results suggest that none of these potential photoreceptors interfere with Cph2 function, although inactivation of taxD1 as well as slr1694 encoding a BLUF protein led to cells that reversed the direction of movement under blue light illumination in mutant strains of cph2.

Ratiometric photonic explorers for bioanalysis with biologically localized embedding (PEBBLE) nanoprobes have been developed for singlet oxygen, using organically modified silicate (ORMOSIL) nanoparticles as the matrix. A crucial aspect of these ratiometric singlet-oxygen fluorescent probes is their minute size. The ORMOSIL nanoparticles are prepared via a sol-gel–based process and the average diameter of the resultant particles is about 160 nm. These sensors incorporate the singlet-oxygen–sensitive 9,10-dimethyl anthracene as an indicator dye and a singlet-oxygen–insensitive dye, octaethylporphine, as a reference dye for ratiometric fluorescence-based analysis. We have found experimentally that these nanoprobes have much better sensitivity than does the conventional singlet-oxygen–free dye probe, anthracene-9,10-dipropionic acid disodium salt. The much longer lifetime of singlet oxygen in the ORMOSIL matrix, compared to aqueous solutions, in addition to the relatively high singlet oxygen solubility because of the highly permeable structure and the hydrophobic nature of the outer shell of the ORMOSIL nanoparticles, results in an excellent overall response to singlet oxygen. These nanoprobes have been used to monitor the singlet oxygen produced by “dynamic nanoplatforms” that were developed for photodynamic therapy. The singlet oxygen nanoprobes could potentially be used to quantify the singlet oxygen produced by macrophages.

Monomolecular multimodal imaging agents (MOMIAs) are able to provide complementary diagnostic information of a target diseased tissue. We developed a convenient solid-phase approach to construct two pro-MOMIAs (before incorporating radiometal) derived from 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and cypate, a near-infrared (NIR) fluorescent dye analogous to indocyanine green (ICG). The possible interaction between d orbitals of transition metal DOTA complexes or free metals and the p orbitals of cypate chromophore could quench the fluorescence of pro-MOMIAs. However, we did not observe significant changes in the spectral properties of cypate upon conjugation with DOTA and subsequent chelation with metals. The fluorescence intensity of the chelated and nonmetal-chelated PRO-MOMIAs remained fairly the same in dilute 20% aqueous dimethylsulfoxide (DMSO) solution (1 × 10⁻⁶ M). Significant reduction in the fluorescence intensity of pro-MOMIAs occurred in the presence of a large excess of metal ions (>1 molar ratio for indium and 20-fold for a copper relative to pro-MOMIA). This study suggests the feasibility of using MOMIAs for combined optical and radioisotope imaging.

In the present study, photodynamic activity of a novel photosensitizer (PS), Chlorin e₆-2.5 N-methyl-d-glucamine (BLC 1010), was evaluated using the chorioallantoic membrane (CAM) as an in vivo model. After intravenous (i.v.) injection of BLC 1010 into the CAM vasculature, the applicability of this drug for photodynamic therapy (PDT) was assessed in terms of fluorescence pharmacokinetics, i.e. leakage from the CAM vessels, and photothrombic activity. The influence of different PDT parameters including drug and light doses on the photodynamic activity of BLC 1010 has been investigated. It was found that, irrespective of drug dose, an identical continuous decrease in fluorescence contrast between the drug inside and outside the blood vessels was observed. The optimal treatment conditions leading to desired vascular damage were obtained by varying drug and light doses. Indeed, observable damage was achieved when irradiation was performed at light doses up to 5 J/cm² 1 min after i.v. injection of drug doses up to 0.5 mg/kg body weight(b.w.). However, when irradiation with light doses of more than 10 J/cm² was performed 1 min after injection of drug doses up to 2 mg/kg body weight, this led to occlusion of large blood vessels. It has been demonstrated that it is possible to obtain the desired vascular occlusion and stasis with BLC 1010 for different combinations of drug and/or light doses.

Water-coupled excimer lamp systems have been developed to inactivate microorganisms within complex, low–optical quality, fluids. Monochromatic lamps were selected to minimize UV-B and UV-C absorption within the carrier fluids while maximizing deposition within specific chemical targets. Fundamentals, system scaling and power supply design are discussed. This work used two large-surface area excimer lamps as intense sources of near monochromatic radiation at 308 and 282 nm. Data are presented for two distinct fluid systems: flow-through processing of large-volume metalworking fluids used in heavy industry and batch irradiation of human blood plasma and platelet suspensions used in transfusion medicine. In the first, a 200–600 L/min reactor is used to control bacterial concentrations within metalworking fluids used in large-scale metal machining processes. Control is defined as the maintenance of 10³ to 10⁴ CFU/mL in fluids that without treatment would have concentrations over 10⁷ CFU/mL. The second is a batch process for viral inactivation in undiluted blood bank products. Samples of fresh frozen plasma and platelet suspensions were spiked with high titers of porcine parvovirus (PPV) and irradiated at 308 and 282 nm. Although both wavelengths were effective at reducing PPV levels, 308 nm light resulted in both higher rates of viral inactivation (greater than 6 log units) and lower rates of fluid degradation.

The rate constants of thermal (irreversible) damage of bacteriochlorin pigments (bacteriochlorophyll monomer [B], bacteriochlorophyll dimer [P] and bacteriopheophytine [H]) in reaction center [RC] protein from the photosynthetic bacterium Rhodobacter sphaeroides were studied in the dark and during intense (400 mW·cm⁻²) laser light excitation (wavelengths 488 and 515 nm) under deoxygenated conditions. While the kinetics of degradation of P and B were monoexponential, the decay kinetics of H were overlapped by an initial lag phase at elevated (>40°C) temperature. This is explained by removal of the central metal ion from the bacteriochlorophylls as part of their degradation processes. At all temperatures, the rates of damage were very similar for all bacteriochlorin pigments and were larger in the light than in the dark. The logarithm of the rate constant of pigment degradation and loss of photochemistry as a function of reciprocal (absolute) temperature (Arrhenius/Eyring plot) showed single phase in the light and double phases in the dark. Below 20°C, the rate of pigment degradation in the RC decreased so dramatically in the dark that it became limited by the natural degradation process of bacteriochlorophyll measured in solution. The function of loss of photochemistry in the dark was also biphasic and had a break point at 40°C. The damage in the dark required high enthalpy change (ΔH^‡ = 64 kcal/mol for P and ΔH^‡ = 60 kcal/mol for B) and entropy increase (T·ΔS^‡ = 38 kcal/mol for P and T·ΔS^‡ = 34 kcal/mol for B at T = 300 K), whereas significantly smaller enthalpy change (ΔH^‡ = 21 kcal/mol for P and B and ΔH^‡ = 13 kcal/mol for H) and practically no (T·ΔS^‡ = −1 kcal/mol for P and B at T = 300 K) or small (T·ΔS^‡ = −9 kcal/mol for H at T = 300 K) entropy change was needed in the light. The thermodynamic parameters of activation reveal major steps common in the degradation of all bacteriochlorin pigments: ring opening reactions at C5 or C20 meso-bridges (or both) and breaking/removal of the phytyl chain. Their contribution in the degradation is probably reflected in the observed enthalpy/entropy compensation at an almost constant (ΔG^‡ = 22–26 kcal/mol at T = 300 K) free energy change of activation.

The values of the rate constants for the reactions of the sulfate (2.5 × 10⁹ M⁻¹ s⁻¹) and hydrogen phosphate (2.2 × 10⁸ M⁻¹ s⁻¹) radicals with silica nanoparticles are obtained by flash photolysis experiments with silica suspensions containing S₂O₈²⁻ or P₂O₈⁴⁻, respectively. The interaction of these radicals with the silica nanoparticles leads to formation of transients, probably adsorbed sulfate and hydrogen phosphate radicals, with absorption maxima at around 320 and 350 nm, respectively. A different mechanism takes place for the interaction of the less oxidizing dithiocyanate radicals with the silica nanoparticles. These radicals selectively react with the dissociated silanol groups of the nanoparticles with a rate constant at 298.2K of 7 × 10⁷ M⁻¹ s⁻¹ (per mol of SiO⁻ groups), and there is no evidence for their adsorption at the surface. All the results are discussed in terms of the Smoluchowski equation and redox potential of the inorganic radicals.

Singlet oxygen (¹O₂) is the most important cytotoxic agent in photodynamic therapy (PDT). The feasibility of using a chemiluminescence (CL) probe, 3,7-dihydro-6-[4-(2-(N′-(5-fluoresceinyl)thioureido)ethoxy)phenyl]-2-methylimidazo{1,2-a}pyrazin-3-one sodium salt (fluoresceinyl Cypridina luciferin analog, FCLA), to monitor ¹O₂ production during PDT is evaluated in vitro. Lymphoma cells were treated with various protocols of PDT. The results show that the FCLA-CL production during PDT is linearly related to the corresponding cytotoxicity, regardless of the treatment protocol. With minimum cytotoxicity and interference to the PDT treatment outcome, the FCLA-CL system is an effective means to quantify PDT ¹O₂ production and may provide an alternative real-time dosimeter.

This work presents the application of an on-line photoreactor to the detection of 3,5-diamino-trifluoromethyl-benzene (3,5-DABTF) in aqueous solutions. When irradiated at 310 nm, this compound is defluorinated to 3,5-diaminobenzoic acid by a nucleophilic substitution of the fluoride by water. Concomitantly, defluorination intermediates polymerize through amide bonds to give dark-colored compounds. We take advantage of the photocatalyzed defluorination and the subsequent decrease in pH to develop an original and specific photoreactor. Continuous recording of pH and temperature in the outlet of the reactor by a dual electrode gives us an opportunity to optimize the system. In the photoreactor, 3,5-DABTF is immediately and totally transformed as attested by the rapid drop of the flowing solution pH from 6.2 to 3.2 and the chromatographic analysis of the outgoing solutions. The detection remains effective from 1 to 1000 parts per million.

We have measured the intrinsic fluorescence anisotropies of six photosensitizers in homogeneous solution, and we have imaged the anisotropies of these sensitizers in tumor cell monolayers using polarization-sensitive laser-scanning confocal microscopy. The intrinsic anisotropies are unremarkable and are within the approximate range of 0.2–0.27. In cells, however, very interesting behavior is exhibited by meso-tetrahydroxyphenyl chlorin (mTHPC). Polarization-sensitive images of mTHPC's fluorescence show a pronounced banding of alternating high and low anisotropy consistent with an ordering of the sensitizer in the nuclear envelope, indicating that this structure is a target of photodynamic damage with this sensitizer. None of the other sensitizers exhibits localization to the nuclear envelope. The frequency distributions of the intracellular anisotropies of the sensitizers exhibit variable peaks and widths. An unusual case is that of Photofrin, with a peak in its anisotropy frequency distribution of −0.12. The change from a positive intrinsic anisotropy in homogeneous solution to a negative value in cells suggests an environmentally induced change in the relative orientations of the absorption and emission dipole moments.

Topical application of the isoflavone equol immediately following solar-simulated UV (SSUV) radiation exposure has previously been demonstrated to have significant photoprotective effects. Equol reduced both the inflammatory edema and the systemic suppression of the contact hypersensitivity reaction in hairless mice. Furthermore, daily topical equol application immediately following irradiation during a 10-week chronic SSUV exposure regime also reduced photocarcinogenesis severity in the mouse. This study examines the potential for topical equol to prevent photoaging in response to chronic SSUV irradiation for up to 30 weeks. We did not find consistent expression of the characteristic markers of photoaging until 30 weeks, although moderate epidermal hyperplasia and a transient increase in dermal mast cell numbers were evident after 1 week. Daily application of 10 μM equol lotion significantly reduced these early changes. However after 30 weeks of SSUV exposure, photoaging was well developed, as shown histologically by markedly increased epidermal hyperplasia, increased dermal mast cell number, pronounced focal elastotic deposits, degraded dermal collagen and deposition of glycosaminoglycans in the lower dermis. Topical equol treatment protected significantly from each of these impairments, as demonstrated histologically and quantitatively. Additionally, equol was found to have strong antioxidant action against acute UVA (320–400 nm)–induced lipid peroxidation of mouse skin, this property accounting for its antiphotoaging mechanism. The evidence for equol's antiphotoaging activity, taken together with its anti-inflammatory, immunoprotective and anticarcinogenic efficacy against SSUV irradiation in the mouse, suggests that equol could be developed as a helpful topical photoprotective agent for daily use by humans.

Specimens of malignant and normal female human breast tissues were analyzed after surgery by means of synchronous luminescence spectroscopy. Measurements were performed in the ranges of excitation wavelengths from 330 to 650 nm and synchronous wavelengths from 30 to 120 nm to obtain ordinary and first derivative three-dimensional total synchronous luminescence spectra (3d-TSLS) of each specimen. Arithmetic mean of these spectra has been calculated for normal and malignant specimens and analyzed to establish criteria for tissue differentiation. Spectral domain volumes (volumes below luminescence intensity surface) and mean spectral slopes have been calculated and also analyzed as tissue discrimination criteria. The obtained results are discussed in view of the possible relevance of synchronous luminescence spectroscopy in discrimination between normal and malignant breast tissue.

We previously reported that Shewanella oneidensis MR-1 is extremely sensitive to natural solar radiation (NSR). Here we analyzed the global transcriptional profile of MR-1 during a 1-h recovering period after exposure to ambient solar light at a dose that yields about 20% survival rate on a Luria-Bertani (LB) plate. We observed the induction of DNA damage-repair genes, the SOS response as well as detoxification strategies that we previously observed in MR-1 following artificial UV-A irradiation. Few prophage-related genes were induced by natural solar UV radiation, however, in contrast to what was observed following artificial UV-B irradiation. Overall, the cellular response to NSR in MR-1 was more similar to that of UV-A than that of UV-B, but additional genes involved in detoxification were induced compared with induction by either UV-B or UV-A or their sum. Thus, oxidative stress appeared to contribute greatly to the NSR-induced cytotoxic effects in MR-1. A total of 29.1% of genome showed differential expression following NSR exposure, which is much greater than following exposure by UV-B (4.0%), UV-A (8.2%) or their sum (10.7%). Our data suggest that NSR may impact biological processes in a much more complex manner than previously thought.

The observed distribution of mitochondria in a cell can vary with environmental influence, degree of differentiation and disease. Differences in the distribution of mitochondrial autofluorescence may be used to distinguish these different cellular states.