Phage T7 can be used as a biological dosimeter; its reading, the biologically effective dose (BED), is proportional to the inactivation rate |ln (n/n₀)|. For the measurement of DNA damage in phage T7 dosimeter, a quantitative polymerase chain reaction (QPCR) methodology has been developed using 555 and 3826 bp fragments of phage T7 DNA. Both optimized reactions are so robust that an equally good amplification was obtained when intact phage T7 was used in the reaction mixture. In the biologically relevant dose range a good correlation was obtained between the BED of the phage T7 dosimeter and the amount of ultraviolet (UV) photoproducts determined by QPCR with both fragments under the effect of five various UV sources. A significant decrease in the yield of photoproducts was detected by QPCR in isolated T7 DNA and in heated phage compared with intraphage DNA with all irradiation sources. Because the yield of photoproducts was the same in B, C and A conformational states of T7 DNA, a possible explanation for modulation of photoproduct frequency in intraphage T7 DNA is that the presence of bound phage proteins induces an alteration in DNA structure that can result in increased induction of photoproducts.

Sheets of polysulphone film have been extensively used as detectors to monitor solar UVB radiation. The advantages of polysulphone detectors are that they are small in size, they have good thermal stability and they are sensitive to UVB radiation. The principal disadvantage of polysulphone detectors is that their spectral sensitivity includes part of the short-wavelength UVA. In this study, we investigate the spectral sensitivity of the polysulphone detector with a series of monochromatic (±2 nm) excitations. We then compare the polysulphone-effective solar radiation with the erythemally effective solar radiation by comparing solar UVB data obtained with polysulphone films with those obtained with a spectroradiometer. From polysulphone data on the seasonal variation of solar UVB radiation, we estimate the corresponding fluctuations of the absorption of the ozone layer. We show that the spectral sensitivity of the polysulphone film is closer to the erythema action spectrum than that indicated by earlier data and that polysulphone detectors can be used to predict the erythema risk of solar UVB. Measurements on solar UVB with polysulphone films and with a spectroradiometer were found to be strongly correlated (R² > 0.95). Finally, polysulphone-based measurements provide a good measure of the fluctuations of the stratospheric ozone layer.

Aquatic organisms, ranging from bacteria to fish, living in clear lakes are presently receiving damaging levels of UV radiation. Photoreactivation is a light-dependent mechanism by which some organisms deal with DNA damage caused by UV radiation. Yet, photoreactivation is a mechanism that confounds long-term predictive modeling of UV effects on the survival of these organisms. Here we show that a short-lived rotifer species, Asplanchna girodi, previously thought to have little to no photoreactivation, does indeed have a significant amount of it. The ability to undergo photoreactivation in A. girodi is dependent on age and becomes apparent only after several days of observation after UV exposure.

UV radiation suppresses the immune response, and UV-induced immune suppression contributes to UV-induced photocarcinogenesis. For UV-induced immune suppression to occur, electromagnetic energy (i.e. UV radiation) must be converted to a biological signal. Two photoreceptors have been identified in the skin that serves this purpose, epidermal DNA and trans-urocanic acid (UCA). Although compelling evidence exists to support a role for each pathway (UV-induced DNA damage or photoisomerization of UCA) in UV-induced immune suppression, it is not clear what determines which photoreceptor pathway is activated. To address this question, we injected UV-irradiated mice with a monoclonal antibody with specificity for cis-UCA or applied liposomes containing DNA repair enzymes to the skin of UV-irradiated mice. The effect that each had on UV-induced suppression of delayed-type hypersensitivity was measured. We asked whether the light source used (FS-40 sunlamps vs solar-simulated UV radiation) altered whichever pathway of immune suppression was activated. Different doses of UV radiation and the viability of the antigen were also considered. Neither the dose of UV nor the light source had any influence on determining which pathway was activated. Rather, we found that the viability of the antigen was the critical determinant. When live antigens were used, UV-induced immune suppression was blocked with monoclonal anti–cis-UCA but not with T4 endonuclease V–containing liposomes. The reverse was observed when formalin-fixed or killed antigens were used. Our findings indicate that antigen viability dictates which photoreceptor pathway predominates after UV exposure.

We have investigated tumor immunological effects of photodynamic therapy (PDT) of liver metastases. Livers of Wag/Rij rats were inoculated with three tumors of a syngeneic rat colon carcinoma cell line, CC531. One tumor in each rat was illuminated, with or without previous administration of the photosensitizer metatetrahydroxyphenylchlorin (mTHPC). PDT was effective in causing necrosis of tumors, but it did not affect the growth rate of nearby, nonilluminated tumors in the liver. Immunological staining of tumors showed natural killer (NK) cells to be significantly lower in PDT-treated tumors than in control tumors (P < 0.05). T cells in PDT-treated tumors and in their margins were lower than in tumors that received only sensitizer or only illumination (P = 0.015) at day 2 after treatment but reappeared at the tumor margins from day 7 after treatment. For macrophages, a similar pattern was found. NK cells, T cells or macrophages in nonilluminated tumors in mTHPC-treated rats did not increase significantly when compared with tumors in rats without mTHPC treatment. These findings indicated that no antitumor effect of a systemic immune response was present, as measured by the effect of PDT on growth of distant tumors and the number of T lymphocytes, NK cells and macrophages in these tumors.

To clarify the role of the Golgi apparatus in photodynamic therapy–induced apoptosis, its signaling pathway was studied after photodynamic treatment of human cervix carcinoma cell line HeLa, in which a photosensitizer, 2,4,5,7-tetrabromorhodamine 123 bromide (TBR), was incorporated into the Golgi apparatus. Laser scanning microscopic analysis of TBR-loaded HeLa cells confirmed that TBR was exclusively located in the Golgi apparatus. HeLa cells incubated with TBR for 1 h were then exposed to visible light using an Xe lamp. Light of wavelength below 670 nm was eliminated with a filter. Morphological observation of nuclei stained with Hoechst 33342 revealed that apoptosis of cells was induced by exposure to light. Electron spin resonance spectrometry showed that light-exposed TBR produced both singlet oxygen (¹O₂) and superoxide anion (O₂^–). Apoptosis induction by TBR was inhibited by pyrrolidine dithiocarbamate, an O₂^– scavenger, but not by NaN₃, a quencher of ¹O₂. Furthermore, TBR-induced apoptosis was inhibited by aurintricarboxylic acid and ZnCl₂, which are known as inhibitors of deoxyribonuclease (DNase) γ, and (acetoxymethyl)-1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, a chelator of Ca² , but not by acetyl Asp-Glu-Val-Asp-aldehyde, an inhibitor of caspase-3. These results suggested that O₂^– was responsible for TBR-induced apoptosis, and Ca² -dependent and caspase-3–independent nuclease such as DNase γ played an important role in apoptotic signaling triggered by Golgi dysfunction.

Biomolecules common in blood plasma, including 2-methyl-1,4-naphthoquinone (vitamin K-0, 2), 2,3-dimethoxy-5-methyl-1,4-benzoquinone (ubiquinone-0, 3), bilirubin, 4, and urocanic acid, 5, were used as photoactivators for the photooxidation of methyl linoleate (ML) in 0.50 M sodium dodecyl sulfate micelles to mimic a bioenvironment. UV irradiation of 2 in this system initiated H-atom abstraction from ML (Type-I mechanism). The evidence includes kinetics of oxygen uptake, inhibition of oxidation by an antioxidant ((R)-( )-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid [Trolox], 7) and the analysis of four geometric hydroperoxides formed (cis, trans to trans, trans ratio of 0.5). In contrast, irradiation with a singlet-oxygen sensitizer, 3,5-di-t-butyl-1,2-benzoquinone, 1, formed six isomers by a Type-II mechanism, yielding a cis, trans to trans, trans isomer ratio of 6. Peroxidation activated by 3 or 4 with visible light occurred by a singlet-oxygen pathway (Type-II mechanism), as shown by kinetics of oxygen uptake and the effect of quenchers. In contrast, peroxidation in the presence of 5 in this system initiated H-atom abstraction from ML as shown by oxygen uptake and inhibition by Trolox. A comparison of thermal free-radical peroxidation with direct photooxidation of human blood plasma samples showed important differences. Blood plasma resisted thermal peroxidation because of natural antioxidants or on the addition of Trolox. In contrast, direct photooxidation involved singlet oxygen, according to the effect of quenchers and the lack of inhibition by antioxidants.

There is considerable interest in the biological properties of flavonoids in terms of their antioxidant and cytoprotective actions. The interaction of the flavanone hesperetin with human skin fibroblasts (FEK4) has revealed the potential for metabolism to hesperetin glucuronide and its subsequent extrusion. As a consequence of this observation, the effectiveness of hesperetin glucuronides, in comparison with that of the aglycone form, in protecting against UV-A radiation has been investigated. The results indicate that hesperetin glucuronides, but not hesperetin, protect against UV-A-induced necrotic cell death.

Nitric oxide (^•NO) has a multitude of physiological roles, including the ability to protect cells against oxidant-induced killing, e.g. by inhibiting caspase-mediated apoptosis or by intercepting damaging free radicals derived from membrane lipids. The purpose of this study was to test the hypothesis that low flux ^•NO acting in the latter fashion can enhance tumor-cell resistance to photodynamic killing, specifically that sensitized by 5-aminolevulinic acid (ALA)–derived protoporphyrin IX (PpIX). Preliminary model experiments with iron-ascorbate–treated, PpIX-sensitized liposomes showed that spermine NONOate (SPER/NO)–derived ^•NO had no effect on photoinduced accumulation of primary singlet oxygen adducts, e.g. the cholesterol hydroperoxide 5α-OOH, but dose-dependently inhibited the buildup of free radical–generated oxidation products arising from one-electron turnover of primary peroxides. In subsequent studies, breast tumor COH-BR1 cells in serum-free medium were treated with 1 mM ALA for 15 min and then without ALA for 3.75 h, allowing biogenerated PpIX to diffuse to extramitochondrial sites, including plasma membrane. Cells were irradiated in the absence or presence of SPER/NO and compared for peroxidative damage and Hoechst-assessed viability after 5 h in the dark. Iron-stimulated necrotic photokilling and accumulation of chain lipid peroxidation products were observed, and this was inhibited strongly by SPER/NO, but not by decomposed SPER/NO, confirming that ^•NO was the active agent. When introduced after irradiation, ^•NO became progressively less inhibitory, consistent with ongoing but waning free-radical activity. These findings provide new insights into the possible role of ^•NO in tumor resistance to ALA–photodynamic therapy and other photodynamic treatments.

Experimental therapies for Barrett's esophagus, such as 5-aminolevulinic acid (ALA)–based photodynamic therapy (PDT), aim to ablate the premalignant Barrett's epithelium. However, the reproducibility of the effects should be improved to optimize treatment. Accurate irradiation with light of a proper wavelength (633 nm), fluence and fluence rate has shown to be critical for successful ALA-PDT. Here, we have used in situ light dosimetry to adjust the fluence rate measured within the esophagus for individual animals and monitored protoporphyrin IX (PpIX) fluorescence photobleaching simultaneously. Rats were administered 200 mg kg^–1 ALA (n = 14) or served as control (n = 7). Animals were irradiated with an in situ measured fluence rate of 75 mW cm^–2 and a fluence of 54 J cm^–2. However, this more accurate method of light dosimetry did not decrease the variation in tissue response. Large differences were also observed in the dynamics of PpIX fluorescence photobleaching in animals that received the same measured illumination parameters. We found that higher PpIX fluorescence photobleaching rates corresponded with more epithelial damage, whereas lower rates corresponded with no response. A two-phased decay in PpIX fluorescence could be identified in the response group, with a rapid initial phase followed by a slower rate of photobleaching. Nonresponders did not show the rapid initial decay and had a significantly lower rate of photobleaching during the second phase of the decay (P = 0.012).

Hypericin, a polycyclic quinone obtained from plants of the genus Hypericum, has been shown to be a promising photosensitizer. We investigated the combination of hypericin–photodynamic therapy (PDT) and a bioreductive drug mitomycin C (MMC) in the present study. The radiation-induced fibrosarcoma–1 tumors were exposed to laser light (120 J/cm² at 595 nm) 24 h after an intravenous injection of hypericin (1 mg/kg). Hypericin-PDT alone significantly decreased tumor perfusion and oxygen tension as demonstrated by India ink staining technique and OxyLite pO₂ measurement, respectively. The in vivo–in vitro cell-survival assay revealed about 60% direct tumor cell killing immediately after PDT. No significant delayed tumor cell death was observed after PDT, which suggests that vascular damage does not contribute significantly to the overall tumor cell death. Injection of a 2.5 mg/kg dose of MMC 20 min before light application significantly decreased tumor cell survival and delayed tumor growth compared with PDT or MMC alone. No greater skin reaction was observed after the combination of MMC and PDT than after PDT alone. Our study demonstrates that combining hypericin-PDT with MMC can be effective in enhancing tumor response with little side effect.

Further advantages in the treatment of soft-tissue sarcomas will only be achieved by tailoring the adjuvant therapy after surgery. The photochemically directed release of macromolecules from endosomes and lysosomes into the cytosol is a novel technology, named photochemical internalization (PCI), that has been evaluated for treatment of sarcoma cells in vitro. Two human synovial sarcoma cell lines (SW 982 and CME-1) were treated with the photosensitizer meso-tetraphenylporphine with two sulfonate groups on adjacent phenyl rings (TPPS_2a) and a plasmid encoding enhanced green fluorescent protein (EGFP) complexed to poly-l-lysine to investigate the influence of PCI on gene transfer and with 5 µg/mL gelonin to investigate PCI of a Type-I ribosome-inactivating protein toxin. In addition, both cell lines were transduced with an Adenovirus serotype 5 encoding the Escherichia coli lacZ gene (AdHCMV-lacZ, expressing β-galactosidase) and treated with TPPS_2a and light to evaluate the effect of PCI on the transduction rate. Photochemically induced transfection with the reporter gene EGFP in CME-1 cells increased from 0% of cells at no light to 40% of the cells after 60 s of light exposure. In contrast, the SW 982 cells showed no enhanced expression of the gene. The fraction of virally transduced cells was about doubled in both cell lines by means of PCI, although the transduction was more efficient in the CME-1 cells. Both cell lines became up to four-fold more sensitive to light when combining photochemical treatment with gelonin incubation. Our experiments showed that PCI induced the endocytic escape of therapeutic substances in cells derived from human soft-tissue sarcomas.

The flavin adenine dinucleotide (FAD)–containing photoreceptor protein AppA (in which the FAD is bound to a novel so-called BLUF domain) from the purple nonsulfur bacterium Rhodobacter sphaeroides was previously shown to be photoactive by the formation of a slightly redshifted long-lived intermediate that is thought to be the signaling state. In this study, we provide further characterization of the primary photochemistry of this photoreceptor protein using UV–Vis and Fourier-transform infrared spectroscopy, pH measurements and site-directed mutagenesis. Available evidence indicates that the FAD chromophore of AppA may be protonated in the receptor state, and that it becomes exposed to solvent in the signaling state. Furthermore, experimental data lead to the suggestion that intramolecular proton transfer (that may involve [anionic] Tyr-17) forms the basis for the stabilization of the signaling state.

A derivative of all-trans-retinal (RAL) and ethanolamine, A2-E, is the main fluorescent component of human retinal lipofuscin. The accumulation of lipofuscin has been correlated with exposure to ambient radiation and loss of photoreceptors. A possible precursor to A2-E is the imine formed from RAL and ethanolamine. This compound, (E,E,E,E)-2-[9-(2-hydroxyethyl)imino-3,7-dimethyl-1,3,5,7-decatrien-1-yl]-1,3,3-trimethylcyclohexene (HIDD), has been synthesized and structurally characterized. The photophysical and photochemical properties of HIDD and its protonated form, HIDD-H , have been investigated using steady-state and time-resolved methods. Both HIDD and HIDD-H are weakly fluorescent, and the fluorescence lifetime and quantum yield for HIDD are ca 0.6 ns and 4.0 ± 0.5 × 10^–4, respectively. HIDD forms a triplet excited state on direct excitation that decays with k_d = 3.4 × 10⁴ s^–1. The extinction coefficient and quantum yield of intersystem crossing for the HIDD triplet are measured as 7.6 ± 1.3 × 10⁴ M^–1cm^–1 and 0.055 ± 0.006, respectively. The triplet excited state of HIDD-H can be sensitized by triplet energy transfer and has a decay rate constant of 1.6 × 10⁴ s^–1. The lifetime of the HIDD triplet excited state is observed to decrease with increasing concentration of the well-known electron or hydrogen atom donors, 2,3,5,6-tetramethyl-1,4-phenylenediamine and 2,3,5-trimethylhydroquinone, and the bimolecular rate constants for these reactions are approximately 5.4 × 10⁶ M^–1s^–1 and 1.7 × 10⁸ M^–1s^–1, respectively. These types of reactions may model photooxidative mechanisms of damage in the retina.

In vivo progressive effects of UV irradiation on the lens epithelium were studied using various histomorphological and biochemical parameters. Fifteen day old rat pups were exposed to 600 mW/m² of radiation, including UV-A and UV-B, 12 h daily for 90, 120, 150 and 180 days. Biochemical parameters such as protein-bound and non–protein-bound sulfhydryl groups in both soluble and insoluble fractions and enzymes, which play an important role in combating the oxidative stress, were studied. Decreased cell density of lens epithelial cells (LEC) was observed in all three zones along with the decrease in the levels of soluble sulfhydryls (S-SH), glutathione reductase (GR), superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT). On the other hand, an increase in insoluble sulfhydryls was observed. Because of the decrease in S-SH and GR activities, the LEC became vulnerable to oxidative stress. Decreased activities of SOD, GPx and CAT suggest elevated oxidative stress. This effect of UV radiation may lead to cell death that may be responsible for the observed decrease in the cell density in all three zones of the lens epithelium.