The relationship between purified transcription factor p50 binding and ultraviolet light–induced DNA damage formation in the NF-κB promoter element was investigated. The effect of bound transcription factor on cyclobutane dimer formation was quantified using Maxam–Gilbert analysis of irradiated substrate digested with T4 phage endonuclease V. Two methods were employed for cleaving (6-4) photoproducts. Sites of (6-4) photoproducts cleaved by piperidine showed a general suppression in the presence of bound p50 protein similar to that observed for cyclobutane dimers. In contrast to piperidine, digestion with ultraviolet damage endonuclease (UVDE) from Saccharomyces pombe subsequent to cyclobutane dimer reversal by photolyase displayed a broader spectrum of damaged sites. Whereas some of these sites were suppressed by bound p50 protein, some remained unaffected and one site showed increased (6-4) photoproduct induction. These data illustrate the advantage of UVDE over piperidine for studying (6-4) photoproducts at the sequence level and suggest that this approach may be useful for footprinting transcription factor binding in other promoters.

The photochemistry of the anticancer drug flutamide (FM), 2-methyl-N-[4-nitro-3-(trifluoromethyl)phenyl]propan-amide, in homogeneous media and in the β-cyclodextrin (β-CD) cavity has been investigated. The photoreactivity of the free molecule has been rationalized on the basis of an intramolecular nitro to nitrite rearrangement followed by cleavage of the nitrite intermediate. The twisted geometry of the nitro group with respect to the aromatic plane plays a key role in triggering such a photoprocess. Incorporation of FM in the β-CD cavity leads to dramatic effects on both the efficiency and the nature of the photochemical deactivation pathways of the guest molecule. A 20-fold increase in the FM photodecomposition quantum yield and the formation of photoproducts originated by both reduction of the nitro group and cleavage of the amide bond were observed in the presence of the macrocycle. Such a behavior cannot be attributed exclusively to the micropolarity of β-CD and/or to its role as a reactant. The induced circular dichroism spectra and the nature of the photoproducts formed in these experimental conditions provide indications that the photoreactivity in the β-CD microenvironment could likely be mediated by structural changes of FM upon complexation.

A fluorescence titration method was applied for the determination of pK_a of ciprofloxacin (CPX) in solution. Values of 6.18 ± 0.05 and 8.76 ± 0.03 were obtained for pK_a1 and pK_a2, respectively. The method was used to determine the ionization constants in the presence of liposomes of dipalmitoylphosphatidylcholine (DPPC) and DPPC with 10 mol% of dipalmitoylphosphatidylglycerol. A dependence on the surface charge of liposomes was found which supported the existence of a basic electrostatic interaction between CPX and the phospholipid bilayer. Both pK values for the N-4 butyl-piperazinyl derivative (BCPX) of the parent compound were also determined in solution and in the presence of liposomes. The competition of both drugs for the same binding site as 1-anilino-8-naphtalene sulfonate demonstrate that the interaction is governed by electrostatic forces.

Four groups of 30 dorsally shaved opossums (Monodelphis domestica) were exposed to graded doses of ultraviolet radiation A (UVA) (320–400 nm) three times per week for 90 weeks. Animals were monitored for the appearance of focal melanocytic hyperplasia (FMH) and nonmelanoma skin tumors (NMST) during the course of the exposures and for an additional 20 weeks following termination of exposures. FMH is the putative precursor for melanoma in the opossum. The lowest dose of UVA (2.5 × 10³ J/m²) used in this study was selected based on the action spectrum for the induction of melanoma in a fish model. The prediction was that 2.5 × 10³ J/m² would induce FMH in the opossum if the action spectra for the induction of FMH in the opossum and melanoma in the fish were the same. The highest UVA dose was 2.5 × 10⁵ J/m². Only the highest dose of UVA gave a statistically significant induction of FMH and NMST in the opossum. As in previous studies, the FMH appeared earlier than the NMST during the course of exposures and the final prevalence of FMH was lower than the final prevalence of NMST. Overall, the results of this study indicate that the efficacy of UVA to induce FMH in the opossum is not as great as would be predicted from the action spectrum for melanoma induction in a fish model.

Irradiance measurements of short wave (SW), photosynthetically active (PAR), ultraviolet-A (UVA) and ultraviolet-B (UVB) solar radiations were made on horizontal and vertical surfaces in the shade of trees under cloud-free and partly cloudy skies. All measurements were referenced to the irradiance of a horizontal surface above the canopy. For horizontal shaded surfaces under cloud-free skies, the values of the ratio (R_h) of below- to above-canopy horizontal irradiance were similar for the UVA and UVB wavebands and for the SW and PAR wavebands. However, R_h for the UV wavebands differed from that for the PAR and SW wavebands. Overall, values of R_h in the shade typically varied as PAR < SW ≪ UVA < UVB. The irradiance ratios for vertical surface in the shade typically varied as UVB > UVA = SW > PAR. In absolute terms, UVB irradiance (I_h) on tree-shaded horizontal surfaces increased relative to a cloud-free sky when a translucent cirroform cloud was in front of the sun, but decreased when the cloud was in a region of sky away from the sun. Translucent cirroform cloud cover also tended to decrease the UVB irradiance (I_v) for a shaded vertical surface (either facing the sun or south) relative to that under cloud-free skies, regardless of where the clouds were in the sky. In all other wavebands the shaded I_h and I_v increased under translucent cirroform cloud cover relative to cloud-free skies, regardless of where the clouds were in the sky.

Discrimination between normal and premalignant tissues by fluorescence imaging and/or spectroscopy may be enhanced by a tumor-localizing fluorescent drug. Ethyl Nile Blue A (EtNBA), a dye with no phototoxic activity, was investigated for this purpose. The pharmacokinetics and tissue-localizing properties were investigated in a rat palate model with chemically induced premalignant mucosal lesions (0.5 mg/kg EtNBA intravenous [i.v.]), a hairless mouse model with UVB-induced premalignant skin lesions (1 mg/kg EtNBA intraperitoneal) and in a rat skin-fold observation chamber model on the back of a rat with a transplanted solid tumor (2.5 mg/kg EtNBA i.v.). Fluorescence images and spectra were recorded in vivo (600 nm excitation, 665–900 nm detection) and in frozen tissue sections at several time points after EtNBA administration. In the rat palate the EtNBA fluorescence was maximum almost immediately after injection, whereas in the mouse skin and the observation chamber the fluorescence maximum was reached between 2 and 3 h after injection. EtNBA cleared from tissues after 8–24 h. EtNBA localizes in the transplantable solid tumor, but is not targeted specifically to the dysplastic location in the rat palate and mouse skin. However, in the rat palate the EtNBA fluorescence increased significantly with increasing dysplasia, apparently due to the increasing thickness of the upper keratinized layer of the epithelium where the dye was found to localize. Localization in this layer occurred both in the rat palate and in hairless mouse skin.

Two distinct photodynamic therapy–resistant variants of the murine radiation-induced fibrosarcoma (RIF) cell line have been isolated. One strain displayed relative resistance over the parental RIF-1 strain to treatment with the porphyrin-based compound, polyhaematoporphyrin (PHP), whereas the other strain displayed relative resistance over the RIF-1 strain to treatment using the cationic zinc (II) pyridinium-substituted phthalocyanine (PPC). The PHP-resistant strain did not display crossresistance to PPC-mediated treatment, and vice versa. In both PDT-resistant strains, the increased resistance could not be attributed to altered cellular growth rate, antioxidant capacity or intracellular sensitizer localization. The PHP-resistant strain displayed resistance to treatment with both short (1 h) and extended (16 h) sensitizer incubation periods, which may indicate that in this strain, the resistance has arisen through an alteration in a membrane component. Conversely, the PPC-resistant strain only displayed increased resistance over the parental cells to treatment involving the short drug incubation, which is likely to reflect the existence of a threshold effect caused by the alteration of an individual cellular target. Each resistant strain has been compared to the parental strain in terms of cellular sensitivity to treatment with a range of other photosensitizers, hyperthermia, UV light and the anticancer agent cis-diamminedichloroplatinum. The PHP-resistant strain exhibited crossresistance to photosensitization treatment using exogenously added protoporphyrin IX, and also to treatment with the anionic phthalocyanine sensitizers, zinc (II) tetrasulfonated phthalocyanine and zinc (II) tetra-glycine-substituted phthalocyanine. The PPC-resistant strain did not display cross-resistance to any of the treatment strategies employed in this investigation. The results of this investigation indicate that there are at least two distinct mechanisms of PDT resistance in RIF cells, and that the mechanism of PHP resistance may, to some extent depend, upon the physical nature of the sensitizer molecule.

Identifying the cellular responses to photodynamic therapy (PDT) is important if the mechanisms of cellular damage are to be fully understood. The relationship between sensitizer, fluence rate and the removal of cells by trypsinization was studied using the RIF-1 cell line. Following treatment of RIF-1 cells with pyridinium zinc (II) phthalocyanine (PPC), or polyhaematoporphyrin at 10 mW cm⁻² (3 J cm⁻²), there was a significant number of cells that were not removed by trypsin incubation compared to controls. Decreasing the fluence rate from 10 to 2.5 mW cm⁻² resulted in a two-fold increase in the number of cells attached to the substratum when PPC used as sensitizer; however, with 5,10,15,20 meso-tetra(hydroxyphenyl) chlorin (m-THPC) there was no resistance to trypsinization following treatment at either fluence rate. The results indicate that resistance of cells to trypsinization following PDT is likely to be both sensitizer and fluence rate dependent. Increased activity of the enzyme tissue-transglutaminase (tTGase) was observed following PPC-PDT, but not following m-THPC-PDT. Similar results were obtained using HT29 human colonic carcinoma and ECV304 human umbilical vein endothelial cell lines. Hamster fibrosarcoma cell (Met B) clones transfected with human tTGase also exhibited resistance to trypsinization following PPC-mediated photosensitization; however, a similar degree of resistance was observed in PDT-treated control Met B cells suggesting that tTGase activity alone was not involved in this process.

Photobleaching kinetics of aminolevulinic acid–induced protoporphyrin IX (PpIX) were measured in the normal skin of rats in vivo using a technique in which fluorescence spectra were corrected for the effects of tissue optical properties in the emission spectral window through division by reflectance spectra acquired in the same geometry and wavelength interval and for changes in excitation wavelength optical properties using diffuse reflectance measured at the excitation wavelength. Loss of PpIX fluorescence was monitored during photodynamic therapy (PDT) performed using 514 nm irradiation. Bleaching in response to irradiances of 1, 5 and 100 mW cm⁻² was evaluated. The results demonstrate an irradiance dependence to the rate of photobleaching vs irradiation fluence, with the lowest irradiance leading to the most efficient loss of fluorescence. The kinetics for the accumulation of the primary fluorescent photoproduct of PpIX also exhibit an irradiance dependence, with greater peak accumulation at higher irradiance. These findings are consistent with a predominantly oxygen-dependent photobleaching reaction mechanism in vivo, and they provide spectroscopic evidence that PDT delivered at low irradiance deposits greater photodynamic dose for a given irradiation fluence. We also observed an irradiance dependence to the appearance of a fluorescence emission peak near 620 nm, consistent with accumulation of uroporphyrin/coproporphyrin in response to mitochondrial damage.

UVB-induced immunosuppression, a promoter of photocarcinogenesis, involves the formation of pyrimidine dimers and cis-urocanic acid (cis-UCA), but reactive oxygen species (ROS) also plays an important role. Eicosapentaenoic acid (EPA) can inhibit photocarcinogenesis, but due to its polyunsaturated nature it is susceptible to oxidative damage by ROS. The antioxidant defense system may therefore be challenged upon ultraviolet-B (UVB) irradiation in the presence of EPA. We investigated whether topically applied EPA in mice could protect against local immunosuppression (contact hypersensitivity response to dinitrofluorobenzene) induced by UVB radiation (1.5 J/cm²), or topically applied cis-UCA (150 nmol/cm²) or thymidine dinucleotides (pTpT) (5 nmol/cm²). The influence of EPA on epidermal lipid peroxidation and antioxidant status was also measured. UVB irradiation, cis-UCA and pTpT all caused 70% immunosuppression. Topical pretreatment of mice with EPA partially protected against immunosuppression; the EPA dose needed to accomplish this was 10 nmol/cm² for UVB irradiation, 100 nmol/cm² for cis-UCA and 1000 nmol/cm² for pTpT. Higher EPA doses caused higher UVB-induced lipid peroxidation and lower vitamin C levels. Glutathione only decreased with the highest EPA dose whereas vitamin E was not decreased after UVB irradiation. In conclusion, topically applied EPA protects against UVB-, cis-UCA- and pTpT-induced immunosuppression and maintenance of an adequate antioxidant defense seems to be an important prerequisite for the protective action by EPA.

The photophysical properties of all-trans-retinal (RAL) have been extensively studied because of the importance of the retinoids in the visual process. However, little information is available regarding the participation of RAL in photochemical transformations such as photooxidation. RAL is one of several native chromophores that have been suggested to act as photosensitizers of damage in the human retina, and this damage would likely occur through oxidative pathways. Time-resolved and steady state techniques have been used to examine the photoreactivity of RAL toward several suitable substrates. The lifetime of the RAL triplet excited state is observed to decrease with increasing concentration of the well-known electron and hydrogen atom donors, 2,3,5,6-tetramethyl-1,4-phenylenediamine (DAD), hydroquinone (HQ), methylhydroquinone (MHQ), 2,3-dimethylhydroquinone (DMHQ) and trimethylhydroquinone (TMHQ), although the bimolecular rate constants for the reaction are much less than that of diffusion controlled (2.9 × 10⁷ M⁻¹ s⁻¹, 1.2 × 10⁵ M⁻¹ s⁻¹, 1.2 × 10⁵ M⁻¹ s⁻¹, 1.5 × 10⁵ M⁻¹ s⁻¹ and 1.6 × 10⁶ M⁻¹ s⁻¹, for DAD, HQ, MHQ, DMHQ and TMHQ, respectively). In the presence of the donors, new absorptions grow concomitant with the decay of the triplet excited state, and for DAD and TMHQ, the observed spectra are similar to the spectra of p-phenylenediamine and TMHQ radicals. Irradiation of RAL in argon-saturated methanol results in fairly efficient photobleaching of RAL and in the formation of two new compounds having absorption spectra that are shifted below 300 nm. Irradiation of RAL in argon-saturated acetonitrile also results in photobleaching of RAL, but the reaction proceeds at a slower rate.

Continuous red light controls starch degradation in turions of Spirodela polyrhiza [Dölger, K., U. K. Tirlapur and K.-J. Appenroth [1997] Photochem. Photobiol. 66, 126–127 (1997)]. This light could be replaced by repeated red light pulses with the reciprocity law fulfilled over a large range of fluence rates. The effect of red light pulses repeated every 24 or 12 h for 6 days was reversible by subsequent far-red light pulses. In contrast, hourly applied red pulses were irreversible by far-red light. This discrepancy was explained by showing the starch degradation activity of far-red pulses themselves. The investigated process was categorized as a phytochrome low fluence response with an unusual property: requirement of light treatment for several days. A partial fulfillment of this requirement was obtained with a red pulse followed by a dark period and a 24 h continuous irradiation. These results suggest the existence of two separate steps in the process of starch degradation in turions: formation of a sprout (=sink) during the pulse-induced germination, and starch degradation in the storage tissue (=source) induced by the second light treatment.

Mammalian skin is vulnerable to the photocarcinogenic and photoaging effects of solar UV radiation and defends itself using a variety of photoprotective responses including epidermal thickening, tanning and the induction of repair and antiradical systems. We treated Skh-1 albino hairless mice for 60 days with ultraviolet-A (UVA) or ultraviolet-B (UVB) radiation and measured the frequency of cyclobutane pyrimidine dimers and pyrimidine(6–4)pyrimidone photoproducts induced by a single acute sunburn dose of UVB at different stages of the chronic treatment. We found that both UVA and UVB exposure produced a photoprotective response in the dermis and epidermis and that the degree of photoproduct attenuation was dependent on dose, wavelength and the type of damage induced. Although epidermal thickening was important, our data suggest that UV protective compounds other than melanin may be involved in mitigating the damaging effects of sunlight in the skin.

Unicellular thermophilic cyanobacterium Synechococcus elongatus displayed phototaxis on agar plate at 55°C. Equal-quantum action spectra for phototactic migration were determined at various fluence rates using the Okazaki Large Spectrograph as the light source. The shapes of the action spectra drastically changed depending on the fluence rate of the unilateral monochromatic irradiation: at a low fluence rate (3 μmol/m²/s), only lights in the red region had significant effect; at a medium fluence rate (10 μmol/m²/s), four major action peaks were observed at 530 nm (green), 570 nm (yellow), 640 nm (red) and 680 nm (red). At high fluence rates (30–90 μmol/m²/s), the former two peaks remained, while red peaks at 640 nm and 680 nm disappeared and, interestingly, an action peak around 700–740 nm (far-red) newly appeared. These results indicate that two or more distinct photoreceptors are involved in the phototaxis and that suitable photoreceptors are selectively active in response to the stimulus of light fluence rates. Far-red or red background lights irradiated vertically from above drastically inhibited phototaxis toward red light or far-red light, respectively. These results indicate involvement of some phytochrome(s).