This review of our work, presented at the Photocarcinogenesis Symposium of the 14th International Congress on Photobiology, shows that UV-A causes a similar number of gene mutations as UV-B in human skin cancer. Areas of about 20 keratinocytes from solar keratoses and squamous cell carcinomas, which are benign and malignant skin cancers, respectively, were sampled by laser capture microdissection. Automated sequencing of the p53 gene was used to detect mutations in these tumor areas, and the cause of the mutations was attributed on the basis of previously published studies. UV-A and UV-B caused similar numbers of p53 gene mutations in both benign and malignant human skin tumors, with UV-B–induced mutations being restricted to the upper areas of the tumors and UV-A–induced mutations predominating at the basal layer. Furthermore, each microdissected region within a tumor had distinct mutations showing that the skin tumors consisted of different clones of cells. This is not consistent with how human skin carcinogenesis is currently understood, and hypotheses to explain our data are presented. We propose that the UV-A waveband of sunlight is as important as UV-B in causing skin cancer in humans.

Protein kinase C (PKC), a family of phospholipid-dependent serine/threonine kinases, is not only the major intracellular receptor for the mouse skin tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) but also is activated by a variety of stress factors including ultraviolet radiation (UVR). PKCϵ is among six isoforms (α, δ, ϵ, η;, μ and ζ) expressed in the mouse skin. To determine the in vivo functional specificity of PKCϵ in mouse skin carcinogenesis, we generated PKCϵ transgenic mouse (FVB/N) lines 224 and 215 that overexpress PKCϵ protein approximately 8- and 18-fold, respectively, over endogenous levels in the basal epidermal cells and cells of the hair follicle. PKCϵ transgenic mice were observed to be highly sensitive to the development of papilloma-independent metastatic squamous cell carcinoma (mSCC) elicited either by repeated exposure to UVR or by the 7,12-Dimethylbenzanthracene–TPA tumor promotion protocol. The development of squamous cell carcinoma (SCC) appears to be linked to the PKCϵ-mediated induction of cytokine tumor necrosis factor–α(TNFα). Immunohistochemical analysis for the expression of PKCϵ in the SCC of PKCϵ transgenic mice revealed that PKCϵ was not expressed in the tumor itself; however, the uninvolved tissue surrounding the SCC exhibited intense PKCϵ expression. Also, human SCC, similar to mouse SCC, did not express PKCϵ in the tumor, whereas the surrounding uninvolved epidermis revealed strong PKCϵ expression. These findings in both the PKCϵ mouse model and human SCC indicate that overexpression of PKCϵ in epidermis may lead to a microenvironment, which is suitable for enhancing the development of mSCC by a paracrine mechanism involving specific cytokines including TNFα.

Xeroderma pigmentosum (XP) is a rare, recessive, photosensitive and cancer-prone syndrome, the biochemical hallmark of which is a defect in nucleotide excision repair of ultraviolet (UV)–induced mutagenic lesions. After isolation and amplification of several strains of XP-C keratinocytes and fibroblasts, a three-dimensional skin model in vitro comprising both epidermis and a dermal equivalent could be obtained. XP dermal tissues and XP epidermis displayed specific morphological and biochemical characteristics compared with tissues obtained with normal cells. One of the major features was the formation of epidermal invaginations into the dermal equivalent. After UV-B exposure, and contrary to repair of DNA lesions in normal cells, the XP model displayed repair deficiency with long-lasting persistence of UV-induced DNA damage and p53 positive nuclei. Recent data obtained after genetic correction leading to functional XPC gene in keratinocytes and fibroblasts revealed that several abnormal features could be normalized. In conclusion, reconstruction of XP skin in vitro provides a very promising system to study genetic hyperphotosensitivity and opens a rational perspective to XP tissue therapy.

Nonmelanoma skin cancer is the most frequently diagnosed malignancy in the United States, and multiple exposures to solar ultraviolet (UV) radiation (particularly its UV-B component, 290–320 nm) is its major cause. “Chemoprevention” by naturally occurring agents is being appreciated as a newer dimension in the management of neoplasia including skin cancer. We recently demonstrated that resveratrol (trans-3,5,4-trihydroxystilbene), an antioxidant found in grapes, red wines and a variety of nuts and berries, imparts protection from acute UV-B–mediated cutaneous damages in SKH-1 hairless mice. Understanding the mechanism of resveratrol-mediated protection of UV responses is important. We earlier demonstrated that resveratrol imparts chemopreventive effects against multiple UV-exposure–mediated modulations in (1) cki-cyclin-cdk network and (2) mitogen activated protein kinase (MAPK)-pathway. This study was conducted to assess the involvement of inhibitor of apoptosis protein family protein Survivin during resveratrol-mediated protection from multiple exposures of UV-B (180 mJ/cm²; on alternate days; for a total of seven exposures) radiations in the SKH-1 hairless mouse skin. Our data demonstrated that topical pretreatment of resveratrol (10 μmol in 200 μL acetone/mouse) resulted in significant inhibition of UV-B exposure–mediated increases in (1) cellular proliferations (Ki-67 immunostaining), (2) protein levels of epidermal cyclooxygenase-2 and ornithine decarboxylase, established markers of tumor promotion, (3) protein and messenger RNA levels of Survivin, and (4) phosphorylation of survivin in the skin of SKH-1 hairless mouse. Resveratrol pretreatment also resulted in (1) reversal of UV-B–mediated decrease of Smac/DIABLO and (2) enhancement of UV-B–mediated induction of apoptosis, in mouse skin. Taken together, our study suggested that resveratrol imparts chemopreventive effects against UV-B exposure–mediated damages in SKH-1 hairless mouse skin via inhibiting Survivin and the associated events.

Isoflavones derived from many edible plants, such as genistein from the soybean, have well-documented antioxidant and estrogenic activity but may also be anticarcinogenic. In this study, we examined the potential of the isoflavone equol [(S)-4′,7-dihydroxyisoflavane] to protect from skin carcinogenesis in the hairless mouse. Daily topical applications of equol lotions significantly protected against skin carcinogenesis induced by chronic exposure to solar-simulated UV radiation (SSUV) or by topical treatment with the chemical carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) or by the combined cocarcinogenic treatment of DMBA followed by chronic SSUV. Monitoring of tumor development for 40 weeks showed significantly delayed tumor appearance and reduced tumor multiplicity in all equol-treated groups. In mice treated with either SSUV or DMBA SSUV, equol significantly reduced the proportion of tumors progressing from benign papillomas to malignant squamous cell carcinoma (SCC) by 33–58% and reduced the average diameter of SCC by 71–82%. In a short-term study, equol dose dependently inhibited the SSUV induction of the tumor promotion biomarker enzyme, ornithine decarboxylase, in the skin, suggesting the anticarcinogenic activity of equol may be attributed to its inhibition of the tumor promotion phase of carcinogenesis.

Excessive exposure of solar ultraviolet (UV) radiation, particularly its UV-B component, to humans causes many adverse effects that include erythema, hyperplasia, hyperpigmentation, immunosuppression, photoaging and skin cancer. In recent years, there is increasing use of botanical agents in skin care products. Pomegranate derived from the tree Punica granatum contains anthocyanins (such as delphinidin, cyanidin and pelargonidin) and hydrolyzable tannins (such as punicalin, pedunculagin, punicalagin, gallagic and ellagic acid esters of glucose) and possesses strong antioxidant and anti-inflammatory properties. Recently, we have shown that pomegranate fruit extract (PFE) possesses antitumor promoting effects in a mouse model of chemical carcinogenesis. To begin to establish the effect of PFE for humans in this study, we determined its effect on UV-B–induced adverse effects in normal human epidermal keratinocytes (NHEK). We first assessed the effect of PFE on UV-B–mediated phosphorylation of mitogen-activated protein kinases (MAPK) pathway in NHEK. Immunoblot analysis demonstrated that the treatment of NHEK with PFE (10–40 μg/mL) for 24 h before UV-B (40 mJ/cm²) exposure dose dependently inhibited UV-B–mediated phosphorylation of ERKl/2, JNK1/2 and p38 protein. We also observed that PFE (20 μg/mL) inhibited UV-B–mediated phosphorylation of MAPK in a time-dependent manner. Furthermore, in dose- and time-dependent studies, we evaluated the effect of PFE on UV-B–mediated activation of nuclear factor kappa B (NF-κB) pathway. Using Western blot analysis, we found that PFE treatment of NHEK resulted in a dose- and time-dependent inhibition of UV-B–mediated degradation and phosphorylation of IκBα and activation of IKKα. Using immunoblot analysis, enzyme-linked immunosorbent assay and electrophoretic mobility shift assay, we found that PFE treatment to NHEK resulted in a dose- and time-dependent inhibition of UV-B–mediated nuclear translocation and phosphorylation of NF-κB/p65 at Ser⁵³⁶. Taken together, our data shows that PFE protects against the adverse effects of UV-B radiation by inhibiting UV-B–induced modulations of NF-κB and MAPK pathways and provides a molecular basis for the photochemopreventive effects of PFE.

We previously demonstrated that CD1d knockout mice were resistant to ultraviolet (UV)–induced immunosuppression. Because immune suppression is a critical factor in the development of UV-induced skin cancers, we investigated the response of wild type (WT) and CD1d−/− mice to UV carcinogenesis. We found that although 100% of WT mice developed skin tumors after 45 weeks of UV irradiation, only 60% of CD1d−/− mice developed skin tumors. To investigate the mechanisms involved in the resistance of CD1d−/− mice to UV-induced carcinogenesis, we determined the time course and kinetics of keratinocyte cell death after UV irradiation. After acute UV exposure, the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL)-positive keratinocytes were eliminated from the skin of WT mice by 72 h post-UV, but they still persisted until 96 h in CD1d−/− mice. The kinetics of p53 protein expression closely followed the kinetics of apoptotic cell death. Chronic UV irradiation resulted in induction of a significantly higher number of apoptotic keratinocytes in CD1d−/− than WT mice. In addition, epidermis and dermis from chronically UV-irradiated CD1d−/− mice harbored significantly fewer p53 mutations than WT mice. These results indicate that the resistance of CD1d−/− mice to UV carcinogenesis may be due to increased cell death and elimination of keratinocytes and fibroblasts containing DNA damage and p53 mutations.

The genetic changes and corruption of kinase activity in melanomas appear to revolve around a central axis: mitogenic signaling along the RAS pathway down to transcription regulation by pRB. Epidemiological studies point to the importance of ultraviolet (UV) radiation in the etiology of melanoma, but where and how UV radiation is targeted to contribute to the oncogenic signaling remains obscure. Animal models of melanoma genesis could serve to clarify this issue, but many of these models are not responsive to UV exposure. Most interesting advances have been made by using transgenic mice that carry genetic defects that are known to be relevant to human melanoma: specifically, dysfunction in the tumor suppressive action of p16INK4a or a receptor tyrosine kinase/RAS pathway, that is constitutively activated in melanocytes. The latter types of mice appear to be most responsive to (neonatal) UV exposure. Whether this is due to a general increase in target cells by melanocytosis and a paucity or complete lack of pigment, or a possible UV-induced response of the promoter–enhancer of the transgene or a genuinely independent and additional genetic alteration caused by UV exposure needs to be established. Importantly, the full effect of UV radiation needs to be ascertained in mice with different pigmentation by varying the wavelengths, UV-B versus UV-A1, and the exposure schedules, i.e. neonatal versus adult and chronic versus intermittent overexposure. Intermittent UV-B overexposure deserves special attention because it most strongly evokes proliferative responses in melanocytes.

Synthetic DNA conjugates in which one or both ends of a short duplex is capped by a stilbene chromophore have been prepared and characterized crystallographically. Selective excitation of the chromophore can be used to initiate electron transfer processes in which a nucleobase serves as either an electron donor or an electron acceptor. These processes include hole- and electron injection and hole migration. The dynamics of these processes and its dependence on distance, driving force, and base sequence have been investigated by means of femtosecond time-resolved spectroscopy. Duplexes with identical chromophores at both ends have been used to study both the dynamics of electron transfer processes and exciton coupling between the two chromophores by means of circular dichroism spectroscopy. Duplexes with different chromophores can also be used to study distance dependence of both electron transfer and exciton coupling.

Recent developments in phototropin biology have provided exciting new findings on the roles of these photoreceptor proteins in plants. Much of the recent work has focused on phototropin photochemistry and the structural alterations in both the chromophoric and peptide components of the molecule associated with light perception. In this review, specific aspects of phototropin action in higher plants will be discussed in the context of these new findings. Although, as their name suggests, phototropins play a key role in phototropic responses in plants, increasing evidence shows they have many other functions in plants. In this review, the roles of phototropins in additional plant “movement” responses will be addressed; in particular their roles in stomatal aperture control and chloroplast movements. In discussing these various movement responses special attention will be given to identified and hypothesized downstream signaling partners or events that enable the phototropins to selectively participate in any one or more of these responses in a given light condition.

Phloxine B (PhB) (2′,4′,5′,7′-tetrabromo-4,5,6,7-tetrachlorofluorescein; D&C Red No. 28) is a red dye found in drugs, cosmetics and foods; it is also currently being evaluated as a phototoxin for the potential control of fruit flies. Previous studies have shown that PhB is an efficient photosensitizer of damage to cellular membranes; thus, exposure of the skin to the dye and sunlight or artificial light may result in phototoxicity. Therefore, we have studied the phototoxicity of PhB and its structural analogue 2′,7′-dichlorofluorescein (DCF) to HaCaT keratinocytes. Anaerobic visible irradiation (>400 nm) of PhB generated a semiquinone type radical, as detected by direct electron paramagnetic resonance. Aerobic visible irradiation of a reaction mixture containing PhB, the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and nicotinamide adenine dinucleotide (reduced) generated a superoxide dismutase–sensitive DMPO/O₂^·− adduct. Irradiation of PhB and DCF in D₂O generated singlet oxygen with quantum yields of 0.59 and 0.06, respectively. PhB was much more phototoxic than DCF when cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. Visible irradiation of HaCaT keratinocytes in the presence of PhB (5 μM) resulted in a 90% decrease in cell viability. 3β-Hydroxy-5α-cholest-6-ene-5-hydroperoxide, a singlet oxygen photoproduct of cholesterol, was isolated from HaCaT keratinocytes irradiated in the presence of PhB. Furthermore, PhB phototoxicity was inhibited by histidine and cysteine, quenchers of singlet oxygen. PhB (0.5 μM) and light irradiation also resulted in DNA damage, as measured by the Comet assay. The phototoxicity mechanism of PhB most probably initially involves a Type-II reaction with free radicals playing a minor role. However, secondary oxidative species such as radicals generated as a result of lipid peroxidation may serve to further promote oxidative damage. Our findings suggest that concern is warranted about the use of this dye in cosmetic products, as a food additive and in insecticidal sprays.

Aromatic amines, such as o-phenylenediamine (OPD), have been used extensively in commercial hair dyes and in the synthesis of agricultural pesticides. Air oxidation of OPD results in the formation of 2,3-diaminophenazine (DAP). Although the mutagenic toxicity of DAP has been shown in both prokaryotic and eukaryotic systems, its phototoxicity remains largely unexplored. This study focuses on the pH-dependent photophysical properties of DAP and demonstrates its ability to photoinduce DNA damage to pUC19 plasmid in vitro. The photocytotoxicity of DAP toward human skin fibroblasts was also measured. DAP exhibits weak intercalative binding to double-stranded DNA with a binding constant K_b = 3.5 × 10³ M⁻¹. Furthermore, upon irradiation with visible light, DAP is able to nick plasmid DNA in the presence of oxygen. The concentration of DAP that resulted in 50% cell death was 172 ± 9 μM in the dark and 13 ± 1 μM after irradiation of the DAP-treated cell cultures with visible light (400–700 nm, 30 min, 5 J/cm²). The 13-fold increase in toxicity upon exposure to visible light shows the need for further study of the photocytotoxicity of contaminants such as DAP.

Characterization of the tissue light penetration in prostate photodynamic therapy (PDT) is important to plan the arrangement and weighting of light sources so that sufficient light fluence is delivered to the treatment volume. The optical properties (absorption [μ_a], transport scattering [μ_s^′] and effective attenuation [μ_eff] coefficients) of 13 patients with locally recurrent prostate cancer were measured in situ using interstitial isotropic detectors. Measurements were made at 732 nm before and after motexafin lutetium (MLu)–mediated PDT in four quadrants. Optical properties were derived by applying the diffusion theory to the fluence rates measured at several distances (0.5–5 cm) from a point source. μ_a and μ_s^′ varied between 0.07 and 1.62 cm⁻¹ (mean 0.37 ± 0.24 cm⁻¹) and 1.1 and 44 cm⁻¹ (mean 14 ± 11 cm⁻¹), respectively. μ_a was proportional to the concentration of MLu measured by an ex vivo fluorescence assay. We have observed, on average, a reduction of the MLu concentration after PDT, presumably due to the PDT consumption of MLu. μ_eff varied between 0.91 and 6.7 cm⁻¹ (mean 2.9 ± 0.7 cm⁻¹), corresponding to an optical penetration depth (δ = 1/μ_eff) of 0.1–1.1 cm (mean 0.4 ± 0.1 cm). The mean penetration depth at 732 nm in human prostate is at least two times smaller than that found in normal canine prostates, which can be explained by a four times increase of the mean value of μ_s^′ in human prostates. The mean light fluence rate per unit source strength at 0.5 cm from a point source was 1.5 ± 1.1 cm⁻², excluding situations when bleeding occurs. The total number of measurements was N = 121 for all mean quantities listed above. This study showed significant inter- and intraprostatic differences in the optical properties, suggesting that a real-time dosimetry measurement and feedback system for monitoring light fluences during treatment should be considered for future PDT studies.

TOOKAD (WST09) is a new, long-wavelength palladium bacteriopheophorbide photosensitizer that targets tissue vasculature. The cutaneous phototoxicity of TOOKAD was assessed in normal rat and pig animal models and in patients in a Phase-I trial of TOOKAD-mediated photodynamic therapy (PDT) for recurrent prostate cancer. Controlled skin exposures were administered using solar-simulated light at various times after drug administration. Two different spectral ranges were used. In the first, the UV portion of the spectrum was removed (UV⁻) because UV irradiation in nondrugged control animals produced an erythema response at incident energy densities (J/cm²) lower than those required to induce a PDT response. In the second, the full solar spectrum (UV ) was used, and the potentiation by the photosensitizer of the UV-mediated minimum erythema dose was assessed. Results showed that the PDT skin response was negligible at clinical drug doses of 2 mg/kg for any period after administration at light doses of 128 J/cm² in the animal models. In patients, there was no observed UV⁻ skin response at doses of up to 2 mg/kg, drug–light intervals of 1–3 h or greater and light exposures up to 128 J/cm². At higher drug doses in the rat and pig models, the duration of skin phototoxicity was found to be ∼3 h and less than 1 h, respectively. Using the full spectrum of solar-simulated light, the presence of TOOKAD did not measurably enhance the UV -induced erythema in the rats, pigs or patients.

We previously reported that approximately 10% of V79 Chinese hamster fibroblast populations clonally derived from single cells immediately after irradiation with either ultraviolet B (UV-B, 290–320 nm, mainly 311 nm) or ultraviolet A (UV-A, 320–400 nm, mainly 350–390 nm) radiation exhibit genomic instability. The instability is revealed by relatively high mutation frequencies in the hypoxanthine phosphoribosyl transferase (hprt) gene up to 23 cell generations after irradiation. These delayed mutant clones exhibited higher levels of oxidative stress than normal cells. Therefore, persistently increased oxidative stress has been proposed as a mechanism for UV-induced genomic instability. This study investigates whether this mechanism is reflected in the deletion spectrum of delayed mutant clones. Eighty-eight percent of the delayed mutant clones derived from UV-A–irradiated populations were found to have total deletion of the hprt gene. Correspondingly, 81% of UV-A–induced early mutations (i.e. detected shortly after irradiation) also had total deletions. Among delayed UV-B–induced mutant clones, 23% had total deletions and 8% had deletion of one exon, whereas all early UV-B events were either point mutations or small deletions or insertions. In conclusion, the multiplex polymerase chain reaction deletion screen showed that there were explicit differences in the occurrence of large gene alterations between early and delayed mutations induced by UV-B radiation. For UV-A radiation the deletion spectra were similar for delayed and early mutations. UV-A radiation is, in contrast to UV-B radiation, only weakly absorbed by DNA and probably induces mutation almost solely via production of reactive oxygen species. Therefore, the present results support the hypothesis that persistent increase in oxidative stress is involved in the mechanism of UV-induced genomic instability.

Oxoglaucine (OG) is an oxoaporphine alkaloid, which has been linked to plant defense mechanisms. It contains a phenalenone (PN)–like chromophore, which suggests a role as singlet oxygen (¹O₂) photosensitizer. Indeed, OG is able to photosensitize ¹O₂ with 100% efficiency in nonpolar environments, similar to PN. However, this efficiency decreases in polar and protic media, although ¹O₂ is formed in all environments ranging from benzene to water. OG is a rather inefficient ¹O₂ quencher (k_q = 8 × 10⁵ M⁻¹ s⁻¹) unlike the related alkaloids boldine and glaucine, for which an antioxidant role has been suggested. The results of this study contribute to the view that plant defense mediated by PN-like secondary metabolites may have a photochemical component.

Cancer chemopreventive agents such as N-4-(hydroxyphenyl)retinamide (4HPR) are thought to prevent cancers by suppressing growth or inducing apoptosis in precancerous cells. Mechanisms by which these drugs affect cells are often not known, and the means to monitor their effects is not available. In this study endogenous fluorescence spectroscopy was used to measure metabolic changes in response to treatment with 4HPR in ovarian and bladder cancer cell lines. Fluorescence signals consistent with nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD) and tryptophan were measured to monitor cellular activity through redox status and protein content. Cells were treated with varying concentrations of 4HPR and measured in a stable environment with a sensitive fluorescence spectrometer. Results suggest that redox signal of all cells changed in a similar dose-dependant manner but started at different baseline levels. Redox signal changes depended primarily on changes consistent with NADH fluorescence, whereas the FAD fluorescence remained relatively constant. Similarly, tryptophan fluorescence decreased with increased drug treatment, suggesting a decrease in protein production. Given that each cell line has been shown to have a different apoptotic response to 4HPR, fluorescence redox values along with changes in tryptophan fluorescence may be a response as well as an endpoint marker for chemopreventive drugs.

Melanosomes in black and red human hair are isolated and characterized by various chemical and physical techniques. Different yields of 4-amino-hydroxyphenolanaline by HI hydrolysis (a marker for pheomelanin) and pyrrole-2,3,5-tricarboxylic acid by KMnO₄/H oxidation (a marker for eumelanin) indicate that the melanosomes in black hair are eumelanosomes, whereas those in red hair are mainly pheomelanosomes. Atomic force microscopy reveals that eumelanosomes and pheomelanosomes have ellipsoidal and spherical shapes, respectively. Eumelanosomes maintain structural integrity upon extraction from the keratin matrix, whereas pheomelanosomes tend to fall apart. The black-hair eumelanosomes have an average of 14.6 ± 0.5% amino acids content, which is attributed to the internal proteins entrapped in the melanosomes granules. The red-hair melanosomes contain more than 44% of amino acid content even after extensive proteolytic digestion. This high content of amino acids and the poorly reserved integrity of red-hair melanosomes suggest that some proteins are possibly covalently bonded with the melanin constituents in addition to those that are entrapped inside the melanin species. Soluene solubilization assay indicates the absorbance of melanin per gram of sample, adjusted for the amino acid content, is a factor of 2.9 greater for the black-hair melanosomes than the red-hair melanosomes. Metal analysis reveals significant amounts of diverse heavy metal ions bound to the two types of melanosomes. The amount of Cu(II) and Zn(II) are similar but Fe(III) content is four times higher in the red-hair melanosomes. ¹³C solid-state nuclear magnetic resonance spectra and infrared spectra are presented and are shown to be powerful techniques for discerning differences in the amino acid contents, the 5,6-dihydroxyindole-2-carboxylic acid:5,6-dihydroxyindole ratio, and the degree of cross-linking in the pigment. Excellent agreement is observed between these spectral results and the chemical degradation data.

Eumelanosomes and pheomelanosomes isolated from black and red human hair, respectively, were studied by photoelectron emission microscopy (PEEM). PEEM images were collected at various wavelengths between 207 and 344 nm, using the spontaneous emission output of the Duke OK-4 free electron laser (FEL). Analysis of the FEL-PEEM data revealed ionization thresholds of 4.6 and 3.9 eV corresponding to oxidation potentials of −0.2 and 0.5 V vs normal hydrogen electrode for eumelanosomes and pheomelanosomes, respectively. The difference in oxidation potential is attributed to the pigment content of the melanosome, namely whether it contains primarily eumelanin and pheomelanin. The effect of added melanosomes on the reduction of Fe(III)-cytochrome showed pheomelanosomes are stronger reducing agents than eumelanosomes, consistent with the measured oxidation potentials. The FEL-PEEM experiment offers to be an important new approach for quantifying the effects of age, oxidation and metal accumulation on the oxidation potentials of intact melanosomes.

Formulations of the tin etiopurpurin (SnET2) have been observed to undergo a spectral change on storage in dimethylformamide solution. This results in an alteration in the action spectrum, with enhanced photodynamic activity at lower wavelengths and decreased activity at 660 nm. On the basis of structural considerations, a reduced analog of SnET2 was prepared with an absorbance maximum at 640 nm. Formation of this product, termed SnET2H₂ relieves steric strain inherent in the parent molecule. Nuclear magnetic resonance, spectral and photodynamic data are consistent with the formation of SnET2H₂ during SnET2 storage. Slight modifications in the original synthesis are also reported, resulting in improved yields of intermediate products.

A worldwide forecast of the erythemally effective ultraviolet (UV) radiation is presented. The forecast was established to inform the public about the expected amount of erythemally effective UV radiation for the next day. Besides the irradiance, the daily dose is forecasted to enable people to choose the appropriate sun protection tools. Following the UV Index as the measure of global erythemally effective irradiance, the daily dose is expressed in units of UV Index hours. In this study, we have validated the model and the forecast against measurements from broadband UV radiometers of the Robertson–Berger type. The measurements were made at four continents ranging from the northern polar circle (67.4°N) to the Antarctic coast (61.1°S). As additional quality criteria the frequency of underestimation was taken into account because the forecast is a tool of radiation protection and made to avoid overexposure. A value closer than one minimal erythemal dose for the most sensitive skin type 1 to the observed value was counted as hit and greater deviations as underestimation or overestimation. The Austrian forecast model underestimates the daily dose in 3.7% of all cases, whereas 1.7% results from the model and 2.0% from the assumed total ozone content. The hit rate could be found in the order of 40%.

The main lesion produced in DNA by UV-C irradiation of spores of Bacillus subtilis is 5-thyminyl-5,6-dihydrothymine (spore photoproduct [SP]). In contrast, cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproducts (6-4PP) are the main photolesions in other cell types. The novel photochemistry of spore DNA is accounted for in part by its reduced hydration, but largely by the saturation of spore DNA with α/β-type small, acid-soluble spore proteins (SASP). Using high-performance liquid chromatography–mass spectrometry analysis of the photoproducts, we showed that in wild-type B. subtilis spores (1) UV-C irradiation generates almost exclusively SP with little if any CPD and 6-4PP; (2) the SP generated is ∼99% of the intrastrand derivative, but ∼1% is in the interstrand form; and (3) there is no detectable formation of the SP analog between adjacent C and T residues. UV-C irradiation of spores lacking the majority of their α/β-type SASP gave less SP than with wild-type spores and significant levels of CPD and 6-4PP. The binding of an α/β-type SASP to isolated DNA either in dry films or in aqueous solution led to a large decrease in the yield of CPD and 6-4PP, and a concomitant increase in the yield of SP, although levels of interstrand photoproducts were extremely low.

3¹-Racemically pure zinc 3¹-hydroxy-13¹-oxo-porphyrins (zinc methyl 17,18-dehydro-bacteriopheophorbides-d) as well as their 3¹-demethyl form were prepared by modifying chlorophyll-a through oxidation by 2,3-dichloro-5,6-dicyano-benzoquinone. From visible, circular dichroism and infrared spectral analyses, these synthetic pigments self-aggregated in 1%(vol/vol) tetrahydrofuran and cyclohexane to give large oligomers by an intermolecular bonding of 13-C=O⋯H–O(3¹)⋯Zn(central) and π–π interaction of the porphyrin chromophores. The supramolecular structures are similar to those of the corresponding chlorins and a core part of extramembranous light-harvesting antennas of photosynthetic green bacteria. The 17,18-dehydrogenation of a chlorin to porphyrin moiety did not disturb its self-aggregation, and the synthetic zinc porphyrins are good models for naturally occurring, self-aggregative bacteriochlorophylls.

Phototropins are UV-A/blue light photoreceptors containing two flavin mononucleotide (FMN)–binding domains, light, oxygen and voltage (LOV)1 and LOV2, of which LOV2 is more sensitive toward light and more important for the physiological response compared with LOV1. Some physiological responses are plant phototropism, chloroplast migration and stomatal opening. Oat phototropin 1 together with light-dependent autophosphorylation shows a reduced electrophoretic mobility and reduced immunoreaction against a heterologous antiserum; both effects were suggested to be caused by phosphorylation at the same sites (M. Salomon, E. Knieb, T. von Zeppelin and W. Rüdiger [2003] Biochemistry 42, 4217–4225). In this study, we show that both effects can be separated from each other: at low temperature, reduced immunoreaction preceded the mobility shift, and irradiation with UV-C light led to the mobility shift without the loss of immunoreactivity. We demonstrated that UV-C light at 280 nm, which does not match any absorption maximum of FMN, leads to autophosphorylation of phototropin. It is hypothesized that UV-C light causes differential activation of the LOV domains via energy transfer from aromatic amino acids.

We report the first separation of the enantiomers of hypericin. Their steady-state optical spectra and ultrafast primary photoprocesses are investigated in chiral environments. Within experimental error, there is no difference between the two enantiomers in any of the systems considered. This is consistent with the emerging picture that the rich and extended absorption spectrum of hypericin is not a result of ground-state heterogeneity. It is also consistent with the observation that the spectra and photophysics of hypericin are generally insensitive to environments in which it does not aggregate.

Understanding macroscale processes controlling solar radiation in marine systems will be important in interpreting the potential effects of global change from increasing ultraviolet radiation (UV) and glacial retreat. This study provides the first quantitative assessment of UV in the water column of Prince William Sound, a subarctic, semienclosed sea surrounded by mountains, glaciers, rivers, bays and fjords in south central Alaska. Glacial influences on diffuse attenuation coefficients (Kd) were determined along an approximate 120 km transect running NE (61°07′43″N, 146°17′1″W) to SW (60°27′25″N, 148°05′27″ W). Glacial meltwater and flour caused a 10-fold increase in Kd for visible light, UV-A and UV-B, whereas high optical clarity was present in a diversity of areas away from glacial influences. Transition areas and locations affected by calving of a tidewater glacier had intermediate Kd values. Depths at 99% attenuation ranged from less than 0.2 m near glacial streams to greater than 5 m in bays and open ocean distant from sources of glacial sediments. These results suggest that potential global change from increasing UV and glacial retreat may have heterogeneous effects on subarctic marine systems.

A chitosan derivative, glycated chitosan (GC), has been used as an immunostimulant for cancer treatment in laser immunotherapy. The function of GC is to enhance the host immune response after direct cancer cell destruction by a selective laser photothermal interaction. To further test its effects, laser immunotherapy was extended to include several different adjuvants for immunological stimulation and to include photodynamic therapy (PDT) as a different tumor-destruction mechanism. Complete Freund (CF) adjuvant, incomplete Freund (IF) adjuvant and Corynebacterium parvum (CP) were selected for treatment of metastatic mammary tumors in rats, in combination with a selective photothermal interaction. The solution of the immunoadjuvants admixed with indocyanine green (ICG), a light-absorbing dye, was injected directly into the tumors, followed by noninvasive irradiation of an 805 nm laser. Combined with PDT, in the treatment of tumors in mice, GC was administered peritumorally immediately after laser irradiation. The survivals of treated animals were compared with untreated control animals. In the treatment of rat tumors, CF, IF and CP raised the cure rates from 0% to 18%, 7% and 9%, respectively. In comparison, GC resulted in a 29% long-term survival. In the treatment of EMT6 mammary sarcoma in mice, GC of 0.5% and 1.5% concentrations increased the cure rates of Photofrin-based PDT treatment from 38% to 63% and 75%, respectively. In the treatment of Line 1 lung adenocarcinoma in mice, a 1.67% GC solution enabled a noncurative meso-substituted tetra(meta-hydroxy-phenyl)chlorin–based PDT to cure 37% of the tumor-bearing mice. The experimental results of this study confirmed our previous studies, showing that immunoadjuvants played an active role in laser-related cancer treatment and that GC significantly enhanced the efficacy of laser cancer treatment.

Predicting the therapeutic outcome of photodynamic therapy (PDT) requires knowledge of the amount of cytoxic species generated. An implicit approach to assessing PDT efficacy has been proposed where changes in photosensitizer (PS) fluorescence during treatment are used to predict treatment outcome. To investigate this, in vitro experiments were performed in which Mat-LyLu cells were incubated in meta-tetra(hydroxyphenyl)chlorin (mTHPC) and then irradiated with 652 nm light. PS concentration, fluence rate and oxygenation were independently controlled and monitored during the treatment. Fluorescence of mTHPC was monitored during treatment and, at selected fluence levels, cell viability was determined using a colony-formation assay. Singlet oxygen dose was calculated using four different models and was compared with cell survival. For the dose metric based on singlet oxygen–mediated PS photobleaching, a universal relationship between cell survival and singlet oxygen dose was found for all treatment parameters. Analysis of the concentration dependence of bleaching suggests that the lifetime of singlet oxygen within the cell is 0.05–0.25 μs. Generation of about 9 × 10⁸ molecules of singlet oxygen per cell reduces the surviving fraction by 1/e.

We have investigated the riboflavin (RF)-sensitized inactivation of glucose 6-phosphate dehydrogenase (G6PD) in the presence and absence of trans-urocanic acid (UCA). The inactivation of the enzyme results from its direct oxidation by the excited triplet RF in a Type-I–photosensitized reaction whose efficiency increases at low oxygen concentration. The addition of histidine to the system produced no change in the inactivation rate, discarding the participation of singlet oxygen in the reaction. On the other hand, the presence of UCA results in its bleaching, with a significant enhancement of RF-mediated inactivation of G6PD. Both the consumption of UCA and G6PD are faster at low oxygen concentrations. UCA also produced a decrease in the sensitizer photodecomposition yield. These results indicate that the enhancement of the RF-mediated G6PD inactivation observed in the presence of UCA is not a singlet oxygen–mediated process. It is proposed that UCA consumption and its effect on G6PD inactivation are due to a complex reaction sequence initiated by a direct oxidation of UCA by the excited sensitizer triplet. The oxidation of the semireduced flavin gives rise to reactive oxygen species (ROS) responsible for the increased rate of the process. This is supported by the protection afforded by several additives with ROS removal capacity: benzoate, superoxide dismutase and catalase.

The PhotochemCAD program has been revised extensively. Calculations can be performed using eight modules (oscillator strength, transition dipole moment and natural radiative lifetime, Förster energy transfer, multicomponent analysis, blackbody radiator, artificial spectrum creation, transmission calculation, and analysis of energy transfer among linear multichromophore arrays). The user interface has been streamlined to facilitate visual display, operation of the various modules, input of user data via a wizard and output of spectra and calculations. The database of absorption and fluorescence spectra has been expanded to 150 photochemically relevant compounds. A database of solar spectra has been added. The program runs under Windows and is equipped with extensive literature references and help features, including a tutorial section with video files.