Several bisimidazoacridones (BIA) are potent, selective antineoplastic agents, whereas others have potent anti–human immunodeficiency virus activity. BIA are bifunctional agents that consist of two imidazoacridone (IA) chromophores held together by various linkers. Interaction of BIA with DNA has been postulated to be required for their biological activity. Fluorescence data on free and bound BIA suggest that the binding of BIA and similar drugs to DNA is driven by a transfer of hydrophobic molecules from aqueous media to the more amphiphilic DNA environment. Binding to DNA was sensitive to sequence and depended on the length and rigidity of the linker. Time-resolved fluorescence measurements showed that BIA adopt an extended conformation upon binding and that all of the molecules are tightly associated with DNA. Gel-shift and melting assays indicated that one of the aromatic residues of BIA is intercalated, whereas the other, together with a linker, resides in a groove, probably the minor groove. A continuum of structures may be possible where intercalation and classical minor groove binding are limiting structures. In general, the hypothesis for the mechanism of action of BIA wherein the unintercalated residue, accessible for additional interactions, captures a critical protein involved in repair or transcription, is consistent with this model.

Oxidative damage to plasmenyl-type lipids contributes to decreased membrane barrier function, loss of membrane structure and formation of nonlamellar defects in membrane bilayers. Previous results from this laboratory have shown that membrane-soluble sensitizers (e.g. zinc phthalocyanine and bacteriochlorophyll a) mediate the photooxidation of palmitoyl plasmenylcholine (1-O-alk-1′-Z-enyl-2-palmitoyl-sn-glycero-3-phosphocholine; PPlsC) vesicles with the subsequent creation of lamellar defect structures, vesicle contents leakage and membrane–membrane fusion. Because plasmalogen lipids are significant components of sarcoplasma and myelin membranes, we sought to characterize the products of their photooxidation. This study focuses on the photooxidation of PPlsC vesicles in the presence of the water-soluble sensitizer, aluminum phthalocyanine tetrasulfonate (AlPcS₄⁴⁻). Attack of photogenerated singlet oxygen on the 1-O-alkenyl ether linkage of PPlsC lipids was expected to generate dioxetane- and ene-type photoproducts. The products formed during continuous aerobic irradiation (28 mW/cm², (610 nm) of PPlsC vesicles in the presence of AlPcS₄⁴⁻ were separated via reverse-phase high-performance liquid chromatography (HPLC) with electrochemical detection (ECD) or evaporative light-escattering detection (ELSD). Photooxidized dipalmitoylphosphatidylcholine–cholesterol vesicles (control) were used to optimize the HPLC-ECD conditions, using 7α-hydroperoxycholesterol as standard. HPLC-ECD was found to be most sensitive for PPlsC hydroperoxides, whereas HPLC-ELSD was more sensitive for nonhydroperoxide photoproducts. The three major photoproducts formed during vesicle irradiation were isolated via preparative HPLC and then characterized by ¹H–nuclear magnetic resonance and mass spectrometry. 1-Formyl-2-palmitoyl-sn-glycero-3-phosphocholine and 1-hydroxy-2-palmitoyl-sn-glycero-3-phosphocholine were identified as dioxetane cleavage products that coeluted at ∼3 min. The second fraction (retention time [R_T] = 48 min) was identified as a PPlsC allylic hydroperoxide. The third photoproduct, eluting at R_T = 64 min, is tentatively identified as an oxidation product arising from allylic hydroperoxide degradation via Hock rearrangement or free radical decomposition.

Very little information exists on the amount of natural and artificial UV light required to cause sunburn and tanning in individuals with very pale skin who are at the greatest risk of developing skin cancer. We have investigated minimal erythema dose (MED) and minimal melanogenic dose (MMD) in a group of 31 volunteers with Fitzpatrick skin types I and II using an Oriel 1000 W xenon arc solar simulator and natural sunlight in Sydney, Australia. We measured the erythemal and melanogenic responses using conventional visual scoring, a chromameter and an erythema meter. We found that the average MED measured visually using the artificial UV source was 68.7 ± 3.3 mJ/cm² (3.4 ± 0.2 standard erythema doses [SED]), which was significantly different from the MED of sunlight, which was 93.6 ± 5.6 mJ/cm² (P < 0.001) (11.7 ± 0.7 SED). We also found significant correlations between the solar-simulated MED values, the melanin index (erythema meter) and the L* function (chromameter). The average MMD (obtained in 16 volunteers only) using solar-simulated light was 85.6 ± 4.9 mJ/cm², which was significantly less than that measured with natural sunlight (118.3 ± 8.6 mJ/cm²; P < 0.05). We mathematically modeled the data for both the chromameter and the erythema meter to see if we were able to obtain a more objective measure of MED and differentiation between skin types. Using this model, we were able to detect erythemal responses using the erythema index function of the erythema meter and the a* function of the chromameter at lower UV doses than either the standard visual or COLIPA methods.

Using normal, untransformed, human fibroblasts, the effectiveness of aminolevulinic (ALA)-mediated photodynamic therapy (PDT) was investigated in terms of both clonogenic survival and DNA damage. The response of normal fibroblasts was then compared with Gorlin syndrome–derived fibroblasts (basal cell nevus syndrome [BCNS]). In terms of clonogenic survival, no significant differences were observed between the two groups of cells. Using the alkaline comet assay, initial DNA damage after PDT was measured. Some DNA damage was detected at higher doses, but this was fully repaired within 24 h of treatment. The BCNS-derived cells showed levels of initial damage that did not differ significantly from normal lines.

The risk to outdoor workers of exposure to solar ultraviolet radiation (UVR) has been known for some time, particularly in the building and construction industry, where workers often use little in the way of protection against solar UVR. In recent years there have been attempts by authorities in Australia and in Queensland in particular, where UVR levels in spring and summer are very high to extreme, to instigate and to encourage the use of personal UVR protection by outdoor workers. To quantify UVR exposure of building and construction industry workers involved in typical outdoor work, a study was conducted using UVR-sensitive polysulphone film badges. The results indicated that the doses were significant, often well in excess of recommended exposure limits. The measured exposures varied between trades. Data on the use of personal UVR-protective equipment and the skin type of workers were also collected. Many of the workers had skin types that were sensitive to UVR and showed signs of sunburn. In summary, the study found that at-risk individuals were exposed to extreme levels of UVR, in most cases without adequate and appropriate sun protection.

The mechanism by which UV-C irradiation inactivates M13 bacteriophage was studied by analyzing the M13 genome using agarose gel electrophoresis and South-Western blotting for pyrimidine dimers. The involvement of singlet oxygen (¹O₂) was also investigated using azide and deuterium oxide and under deoxygenated conditions. With a decrease in M13 infectivity on irradiation, single-stranded circular genomic DNA (sc-DNA) was converted to Form I and Form II, which had an electrophoretic mobility between that of sc-DNA and linear-form DNA. However, the amount of sc-DNA remaining was not correlated with the survival of M13. The formation of cyclobutane pyrimidine dimers (CPD) and pyrimidine (6–4) pyrimidone photoproducts ((6–4)PP) increased as a function of irradiation dose. The decrease in M13 infectivity was highly correlated with the increase in CPD and (6–4)PP, whereas no change was seen in M13 coat protein on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. 8-Oxo-7,8-dihydro-2′-deoxyguanosine did not form in the M13 genome after UV-C irradiation. Inactivation of M13 was neither enhanced by deuterium oxide nor inhibited by azide. Deoxygenation of the M13 suspension did not affect the inactivation, indicating that ¹O₂ did not participate in the inactivation of M13 by UV-C irradiation under these conditions. These results indicated that UV-C irradiation induced not only CPD and (6–4)PP formation but also additional tertiary structural change in DNA inside the M13 virions, resulting in primary damage and a loss of infectivity. The indirect effect of UV-C irradiation such as ¹O₂ production followed by oxidative damage to nucleic acids and proteins might have contributed less, if at all, to the inactivation of M13 than the direct effect of UV-C.

Chronic ultraviolet irradiation leads to photoaging in human skin, which is associated with degradation of connective tissue. This is partly due to the fibroblast collagenase (matrix metalloproteinase 1 [MMP-1]). Using complementary DNA array technique we demonstrate that after UV irradiation, MMP-1, MMP-3 and the tissue inhibitor of matrix metalloproteinase 1 (TIMP-1) are time-dependently induced on the messenger RNA level in dermal fibroblasts in vitro and in vivo in human buttock skin. This increase in gene expression is paralleled by an increase of latent and active MMP-1 protein after low-dose UV-A exposure in vitro. In vivo the concentration of latent MMP-1 in suction blister fluids peaks 24 h after irradiation with 2 minimal erythema doses of solar simulated radiation. However, only a small proportion of MMP-1 in vitro (5.5 ± 1.5%) and in vivo is active, whereas the majority of MMP-1 remains in its inactive proform. Interestingly, in suction blister fluid the concentration and duration of TIMP-1 expression exceeds that of MMP-1. Taken together, these data indicate that MMP-1 activity is tightly regulated transcriptionally and posttranscriptionally. Furthermore, the pronounced individual differences in all targets investigated provide a possible explanation for the different susceptibility of individuals to UV exposure and, thus, to the clinical features of photodamage.

Singlet oxygen (¹O₂)–induced cytotoxicity is believed to be responsible for responses to photodynamic therapy and for apoptosis of T helper cells after UV-A treatment. Other cytotoxic oxidants, such as hydrogen peroxide and peroxynitrite have been shown to stimulate cell survival signaling pathways in addition to causing cell death. Both these oxidants stimulate the Akt/protein kinase B survival signaling pathway through activation of membrane tyrosine kinase growth factor receptors. We evaluated the ability of ¹O₂ to activate the Akt/protein kinase B pathway in NIH 3T3 cells and examined potential activation pathways. Exposure of fibroblasts to ¹O₂ elicited a strong and sustained phosphorylation of Akt, which occurred concurrently with phosphorylation of p38 kinase, a proapoptotic signal. Inhibition of phosphatidylinositol-3-OH kinase (PI3-K) completely blocked Akt phosphorylation. Significantly, cell death induced by ¹O₂ was enhanced by inhibition of PI3-K, suggesting that activation of Akt by ¹O₂ may contribute to fibroblast survival under this form of oxidative stress. ¹O₂ treatment did not induce phosphorylation of platelet-derived growth factor receptor (PDGFR) or activate SH-PTP2, a substrate of growth factor receptors, suggesting that PDGFR was not activated. In addition, specific inhibition of PDGFR did not affect Akt phosphorylation elicited by ¹O₂. Activation of neither focal adhesion kinase (FAK) nor Ras protein, both of which mediate responses to reactive oxygen species, appeared to be pathways for the ¹O₂-induced activation of the PI3-K–Akt survival pathway. Thus, activation of Akt by ¹O₂ is mediated by PI3-K and contributes to a survival response that counteracts cell death after ¹O₂-induced injury. However, unlike the response to other oxidants, activation of the PI3-K–Akt by ¹O₂ does not involve activation of growth factor receptors, FAK or Ras protein.

Several studies have shown that UV irradiation may be effective for preventing vascular restenosis or vasospasm. However, the long-term effects of UV light on the physiological properties of vessels such as arterial tension have not been elucidated. We therefore studied the long-term effects of UV using rat carotid arteries treated with UV-B light (wavelength = 313 nm, total energy = 14 mJ/mm²). The animals were sacrificed at 1, 7 and 14 days after UV light exposure, and the carotid arteries were studied by light microscopy and the contractile responses of isolated arterial rings were recorded under isometric tension. UV treatment had induced a substantial loss of smooth muscle cells (SMC) along the entire circumference of the media on days 7 and 14, whereas loss of SMC on day 1 was negligible. Contractile responses of arteries that had been exposed to UV light were significantly reduced on days 1, 7 and 14. The susceptibility of UV-treated arteries to phenylephrine and prostaglandin F_2α was significantly decreased on days 1 and 7, but decreased susceptibility was not seen on day 14. Acetylcholine-induced relaxations were not altered by UV treatment. These results suggest that the long-term effect of UV light is an attenuation of smooth muscle contractility without impairment of endothelial function.

We have evaluated the efficacy of the new photosensitizer (PS) Tookad® in photodynamic therapy (PDT) in vivo. This PS is a palladium-bacteriopheophorbide presenting absorption peaks at 762 and 538 nm. The light dose, drug dose and drug injection–light irradiation interval (DLI), ranging between 100 and 300 J/cm², 1 and 5 mg/kg and from 10 to 240 min, respectively, were varied, and the response to PDT was analyzed by staging the macroscopic response and by the histological examination of the sections of the irradiated cheek pouch. The level of PDT response, macroscopically and histologically, shows a strong dependence on the DLI, light dose and drug dose at the applied conditions in the normal hamster cheek pouch. A decay of the tissular response with increasing DLI is observed corresponding to a time of half-maximum response ranging from 10 to 120 min, depending on drug dose and light dose. The tissues affected at the lowest doses are predominantly the vascularized diffuse connective tissue situated between the inner and outer striated muscle (SM) layers as well as these muscle layers themselves. The highest response at the shortest DLI and the absence of a measurable response at DLI longer than 240 min at 300 J/cm² and drug dose of 5 mg/kg are characteristics of a predominantly vascular effect of this PS. This observation suggests that Tookad® could be effective in PDT of vascularized lesions or pathologies associated with the proliferation of neovessels.

In vivo and ex vivo studies of fluorescence from endogenous and exogenous molecules in tissues and cells are common for applications such as detection or characterization of early disease. A systematic determination of the excitation–emission matrices (EEM) of known and putative endogenous fluorophores and a number of exogenous fluorescent photodynamic therapy drugs has been performed in solution. The excitation wavelength range was 250–520 nm, with fluorescence emission spectra collected in the range 260–750 nm. In addition, EEM of intact normal and adenomatous human colon tissues are presented as an example of the relationship to the EEM of constituent fluorophores and illustrating the effects of tissue chromophore absorption. As a means to make this large quantity of spectral data generally available, an interactive database has been developed. This currently includes EEM and also absorption spectra of 35 different endogenous and exogenous fluorophores and chromophores and six photosensitizing agents. It is intended to maintain and extend this database in the public domain, accessible through the Photochemistry and Photobiology website ( http://www.aspjournal.com/).

Photodynamic therapy (PDT) is a new treatment modality for solid tumors as well as for flat lesions of the gastrointestinal tract. Although the use of 5-aminolevulinic acid–induced protoporphyrin IX (PPIX) shows important advantages over other photosensitizers, the main mechanisms of phototoxicity induced are still poorly understood. Three human colon carcinoma cell lines with variable degrees of differentiation and a normal colon fibroblast cell line were used to generate a suitable in vitro model for investigation of photosensitizer concentration as well as the applied light dose. Also, the effects of intracellular photosensitizer localization on efficiency of PDT were examined, and cellular parameters after PDT (morphology, mitochondrial transmembrane potential, membrane integrity and DNA fragmentation) were analyzed to distinguish between PDT-induced apoptosis from necrosis. The fibroblast cell line was less affected by phototoxicity than the tumor cells to a variable degree. Well-differentiated tumor cells showed higher toxicity than less-differentiated cells. After irradiation, cell lines with cytosolic or mitochondrial PPIX localization indicate a loss of mitochondrial transmembrane potential resulting in growth arrest, whereas membrane-bound PPIX induces a loss of membrane integrity and consequent necrosis. Although the absolute amount of intracellular photosensitizer concentration plays the main determining role for PDT efficiency, data indicate that intracellular localization has additional effects on the mode of cell damage.

Poly(adenosine diphosphate–ribose) polymerase (PARP) has recently been characterized as a key regulator of cell death–survival transcriptional programs associated with stress and inflammation. Possible participation of this enzyme in the response of tumors to photodynamic therapy (PDT) was investigated in this study. Immunohistochemical analysis of mouse FsaR tumors treated by PDT based on photosensitizers Photofrin or 5,10,15,20-tetra-(m-hydroxyphenyl)chlorine (mTHPC) revealed a strong positive staining for PARP product poly(ADP-ribose) at 30 min and 1 h after PDT, respectively, and even more intense positivity at 2 h after PDT with both photosensitizers. Flow cytometry–based examination showed the induction of poly-ADP-ribosylation in FsaR tumors at 30 min after PDT, with a trend for a further increase in the intensity by 2 h after PDT in both cancer cells and tumor-associated leukocytes. In FsaR cells treated in vitro by mTHPC-based PDT, flow cytometric analysis indicated that the activation of PARP concentrated in cells undergoing apoptosis and reached a maximum by 30 min after PDT. The administration of PARP inhibitors, 3-aminobenzamide or 1,5-isoquinolinediol, to FsaR tumor–bearing mice before PDT light treatment increased the resistance of these tumors to PDT. PARP appears to control the balance between apoptotic and necrotic cell death in PDT-treated tumors and regulate the progression of PDT-induced inflammatory or innate immune response.

We previously reported that the efficacy of photodynamic therapy (PDT) in cell culture was enhanced by ursodeoxycholic acid (UDCA), a nontoxic bile acid. In this study, we examined the ability of UDCA to promote tumor control by PDT in the mouse, using the radiation-induced fibrosarcoma tumor and the photosensitizing agent tin etiopurpurin (SnET2). These experiments revealed that the addition of UDCA to a PDT protocol promoted inhibition of tumor growth, a phenomenon unrelated to either altered SnET2 biodistribution or the level of vascular shutdown during irradiation. These results indicate that UDCA acts solely by promoting direct tumor cell kill by PDT.

To improve the amphiphilicities and red absorption of the hypocrellins, three novel 2-amino-2-demethoxy-hypocrellins were synthesized by the mild reactions of hypocrellin B with 4-(2-amino-ethyl)morpholine, N,N-dimethylethylenediamine and 1-(2-amino-ethyl)piperazine, respectively. The structures of these derivatives were characterized with proton nuclear magnetic resonance, infrared and mass spectra (MS). The ultraviolet–visible absorption and fluorescence spectra of the derivatives were measured and the new amino-substituted hypocrellins showed strong absorption in the domain of the phototherapeutic window (600–900 nm). Their amphiphilicities evaluated via the partition coefficients between n-octanol and phosphate-buffered saline buffer improved remarkably. Electron paramagnetic resonance spin-trapping measurements were used to investigate the photodynamic action of the three compounds in the presence of oxygen. Singlet oxygen (¹O₂) and superoxide anion radical (O₂^•−) generated by illuminating the hypocrellin derivatives in aerobic solution were observed.

In this communication we propose a novel application for prefluorescent probes in the detection of free carbon-centered radicals in enzymatic processes. Prefluorescent probes combine a fluorescent moiety tethered to a paramagnetic nitroxide that acts as a fluorescence quencher. Trapping of a radical by the nitroxide group restores the fluorescence properties. The increase in fluorescence intensity with time reflects the formation and quenching of carbon-centered radicals and can be used for the quantitative evaluation of yields and kinetics. As a test system we used horseradish peroxidase, an oxidoreductase that is widely accepted to operate by a radical-mediated mechanism. We used the prefluorescent probe (quinoline-TEMPO), where a quinoline moiety has been tethered to 2,2,6,6-tetramethylpiperidin-1-oxyl.

Effects of methanol on growth and photosynthetic activity of Lemna gibba exposed under continuous illumination were examined. As a higher plant, L. gibba appeared to be much more sensitive to methanol inhibitory effect compared with some algae (Theodoridou et al. [2002] Biochim. Biophys. Acta, 1573, 189–198). We found that stimulatory or inhibitory effects were strongly dependent on the methanol concentration and the time of exposure. When the exposure was up to 0.2% methanol, the growth rate of biomass was improved by 50%. However, stimulatory effect of methanol appeared to be smaller when plants were exposed for 48 h compared with 24 h. Increase in biomass induced by methanol was not based on the increase in primary photosynthetic process but rather on accommodation of energy dissipation during photosynthesis. Inhibitory effect on the growth of L. gibba already observed for 0.5% methanol was strongly associated with the increase in the nonphotochemical energy dissipation. The ratio between biomass and methanol concentration appeared to determine the stimulatory or the inhibitory effect. Suggested explanations for the stimulatory and the inhibitory effects are presented.