During the past several years, phototoxicity has been studied at the molecular level, and these studies have provided new insights in the field of DNA lesion characterization, DNA repair and cell response to ultraviolet (UV)-induced stress. The development of new antibiotics and antiinflammatory drugs has highlighted the necessity to develop the assessment of phototoxicity in the safety evaluation of new chemical compounds. This paper aims at reviewing the known molecular mechanisms of the cellular response to UV-induced stress, the in vitro methods that can be proposed and used to screen for toxicity of sunlight and the photosensitization process resulting from the activation of drugs by light. UV sources, biological systems and endpoints of interest in that particular objective are listed. Phototoxic effects span from the cytotoxic–apoptotic effect to the induction of primary DNA damage, DNA repair and a variety of stress genes acting on the cell cycle and the fate of the cell. Ultimately, it can lead to the induction of hereditary DNA modification. A variety of assays are proposed to specifically address all these particular consequences of UV-induced toxicity.

Intramolecular processes of deactivation of 1,3-dimethyl-4-thiouracil (DMTU) from the second excited singlet (S₂) (π, π*) and the lowest excited triplet (T₁) (π, π*) states have been studied using perfluoro-1,3-dimethylcyclohexane (PFDMCH) as a solvent. The spectral and photophysical (PP) properties of DMTU in CCl₄, hexane and water have also been described. For the first time, the fluorescence from S₂ state DMTU has been observed. The picosecond lifetime of DMTU in the S₂ state (τ_S2) in PFDMCH has been proposed to be determined by a very fast intramolecular reversible process of hydrogen abstraction from the ortho methyl group by the thiocarbonyl group. The shortening of τ_S2 in CCl₄ is interpreted to be caused by the intermolecular interactions between DMTU (S₂) and the solvent. Results of the phosphorescence decay as a function of DMTU concentration were analyzed using the Stern–Volmer formalism, which enabled determination of the intrinsic lifetime of the T₁ state ( ) and rate constants of self-quenching (k_sq). The lifetimes, , of DMTU in PFDMCH and CCl₄ are much longer than the values hitherto obtained in more reactive solvents. The PP properties of DMTU both in the S₂ and T₁ states have been shown to be determined by the thiocarbonyl group.

1-Hydroxybenzotriazole and 1-hydroxypyridine-2-thione were incorporated as ligands with the cluster Ru₃(CO)₁₀(NCMe)₂ to give [(μ-H)Ru₃(CO)₁₀(μ₂-2,3-η;²-NNN(O)C₆H₄)] and [(μ-H)Ru₃(CO)₉(μ₂-η;¹ : η;²-C₅H₄N(O)S)], respectively. Irradiation of these two new triruthenium metal clusters individually with 350 nm UV light in a phosphate buffer (pH 6.0) containing form I DNA resulted in single-strand cleavage. Cluster [(μ-H)Ru₃(CO)₁₀(μ₂-2,3-η;²-NNN(O)C₆H₄)] was also found to bind to calf thymus DNA upon UV irradiation.

A series of derivatives of 5,10,15,20-tetrakis-(4-N-methylpyridyl)-porphine, where one N-methyl group was replaced by a hydrocarbon chain ranging from C6 to C22, were characterized for their photophysical and photosensitizing properties. The absorption and fluorescence features of the various compounds in neutral aqueous solutions were typical of largely monomeric porphyrins, with the exception of the C22 derivative, which appeared to be extensively aggregated. This was confirmed by the very low triplet quantum yield and lifetime of the C22 derivative as compared with 0.2–0.7 quantum yields and 88–167 μs lifetimes for the other porphyrins. The photophysical properties and photosensitizing activity toward N-acetyl-l-tryptophanamide of the C22 porphyrin became comparable to those typical of the other derivatives in 2% aqueous sodium dodecyl sulfate, where the C22 compound is fully monomerized. All the porphyrin derivatives exhibited at micromolar concentrations photoinactivation activity against both Staphylococcus aureus and Escherichia coli, even though the gram-negative bacteria were markedly less photosensitive. The photosensitizing efficiency was influenced by (1) the amount of cell-bound porphyrin, which increased with increasing length of the hydrocarbon chain; and (2) the tendency to undergo partial aggregation in the cell, which seems to be especially important for the C22 derivative.

The biological activities of a congeneric series of pyropheophorbides used as sensitizers in photodynamic therapy have been predicted on the basis of their molecular structures, using multiple linear regression and artificial neural network (ANN) computations. Theoretical descriptors (a total of 81) were calculated by the 3DNET program based on the three-dimensional structure (3D) of the geometry-optimized molecules. These input descriptors were tested as independent variables and used for model building. Systematic descriptor selections yielded models with one, two or three descriptors with good cross-validation results. The predictive abilities of the best fitting models were checked by shuffling and cross-validation procedures. ANN was suitable for building models for both linear and nonlinear relationships. Lipophilicity was sufficient to predict the accumulation of the sensitizers in the target tissue. Weighted holistic invariant molecular descriptors weighted by atomic mass, Van der Waals volume or electronegativity were also needed to predict photodynamic activity properly. Our models were able to predict the biological activities of 13 pyropheophorbide derivatives solely on the basis of their 3D molecular structures. Moreover, linear and nonlinear variable selection methods were compared in models built linearly and nonlinearly. It is expedient to use the same method (linear or nonlinear) for variable selection as for parameter estimation.

The absorption and emission behavior of flavin mononucleotide (FMN) in the light-, oxygen- and voltage-sensitive (LOV) domain LOV1 of the photoreceptor Phot1 from the green alga Chlamydomonas reinhardtii was studied. The results from the wild-type (LOV1-WT) were compared with those from a mutant in which cysteine 57 was replaced by serine (LOV1-C57S), and with free FMN in aqueous solution. A fluorescence quantum yield of ϕ_F = 0.30 and a fluorescence lifetime of τ_F = 4.6 ns were determined for FMN in the mutant LOV1-C57S, whereas these quantities are reduced to about ϕ_F = 0.17 and τ_F = 2.9 ns for LOV1-WT, indicating an enhanced intersystem crossing in LOV1-WT because of the adjacent sulfur of C57. A single-exponential fluorescence decay was observed in picosecond laser time-resolved fluorescence measurements for both LOV1-WT and LOV1-C57S as expected for excited singlet state relaxation by intersystem crossing and internal conversion. An excitation intensity dependent fluorescence signal saturation was observed in steady-state fluorescence measurements for LOV1-WT, which is thought to be because of the formation of a long-lived intermediate flavin-C(4a)–cysteinyl adduct in the triplet state (few microseconds triplet lifetime, adduct lifetime around 150 s). No photobleaching was observed for LOV1-C57S, because no thiol group is present in the vicinity of FMN for an adduct formation.

Steady state and time-resolved fluorescence properties of chlorin p₆, a potential drug for photodynamic therapy, have been investigated as functions of pH. A decrease in pH of the medium has been shown to cause protonation of the ionizable carboxylic acid side chain, leading to an increase in hydrophobicity and consequent aggregation. The aggregates dissociate on further protonation. The dissociation is explained in terms of formation of cations and their mutual repulsion. A synchronous fluorescence spectroscopic study revealed the presence of two anionic forms in equilibrium at physiological pH, with a shift in the equilibrium on slight decrease in the pH. The anionic nature of chlorin p₆ in aqueous solutions at physiological pH has been confirmed by complexation with surfactants. The nature of the charge on the headgroups of the surfactants has been found to govern the formation of chlorin–surfactant complexes.

Cells receive signals for survival as well as for death, and the balance between the two ultimately determines the fate of the cells. UV-triggered apoptotic signaling has been well documented, whereas UV-induced survival effects have received little attention. We have reported previously that UVB irradiation prevented apoptosis, which is partly dependent on activation of the phosphatidylinositol 3-kinase (PI3-kinase)–Akt pathway (Ibuki Y. and Goto, R. [2000] Biochem. Biophys. Res. Commun. 279, 872–878). In this study, antiapoptotic effects and survival signals of UV with different wavelength ranges, UVA, UVB and UVC, were examined. NIH3T3 cells showed apoptotic cell death by detachment from the extracellular matrix under serum-free conditions, which was prevented by all wavelengths. However, the effect of UVA was less than those of UVB and UVC, as determined by metabolism of fluoresceine diacetate and the appearance of chromatin-condensed cells. Furthermore, the effects of three wavelengths of UV on the apoptotic pathway upstream of the nuclear signals were examined. Reduction of mitochondrial transmembrane potential (ΔΨ) and activation of caspase-9 and -3 were suppressed by all three wavelengths of UV, showing wavelength-dependent effects as mentioned previously. Shorter wavelengths showed stronger inhibitory effects on caspase-8 activity. The PI3-kinase inhibitor wortmannin partially inhibited the UVB- and UVC-induced suppression of apoptosis but not the inhibitory effect of UVA. Furthermore, normal ΔΨ maintained by UVA was not changed in the presence of wortmannin, but those by UVB and UVC were reduced. Akt was clearly phosphorylated by all three wavelengths. The phosphorylation by UVB and UVC was completely inhibited by addition of wortmannin, but that by UVA was not, in agreement with the results of survival and of ΔΨ. These results suggested the existence of two different survival pathways leading to suppression of apoptosis, one for UVA that is independent of the PI3-kinase–Akt pathway and the other for UVB and UVC that is dependent on this pathway.

Exposure of living organisms to UV light leads to photooxidative reactions. Peroxyl radicals are involved in the propagation of lipid peroxidation. Carotenoids are dietary antioxidants and show photoprotective effects in human skin, efficiently scavenging peroxyl radicals and inhibiting lipid peroxidation. Cultured human skin fibroblasts were used to examine the protective effects of the carotenoids, lycopene, β-carotene and lutein on UVB-induced lipid peroxidation. The carotenoids were delivered to the cells using liposomes as the vehicle. The cells were exposed to UVB light for 20 min. Lycopene, β-carotene and lutein were capable of decreasing UV-induced formation of thiobarbituric acid-reactive substances at 1 h to levels 40–50% of controls free of carotenoids. The amounts of carotenoid needed for optimal protection were divergent at 0.05, 0.40 and 0.30 nmol/mg protein for lycopene, β-carotene and lutein, respectively. Beyond the optimum levels, further increases of carotenoid levels in cells led to prooxidant effects.

Oxidative DNA damage has been implicated in some of the biological properties of UVA but so far not in the acute photosensitivity or cellular sensitivity. In contrast to pyrimidine dimers, oxidative DNA damage is predominantly processed by base excision repair (BER). In order to further clarify the role of oxidative DNA damage and its repair in the acute cellular response to UV light, we studied UVA1 and UVB sensitivities in three different cell model systems with modified BER. 8-Oxoguanine-DNA-glycosylase 1^–/– (OGG1^–/–) mouse embryonal fibroblasts and human fibroblasts in which BER was inhibited by incubation with methoxyamine were hypersensitive to UVA1, in particular to low doses. This hypersensitivity could be partially corrected by reexpression of OGG1 in OGG1^–/– cells. The Chinese hamster ovary (CHO) cells with upregulated AP-endonuclease 1 exhibited reduced UVA1 sensitivity. UVB sensitivity was not altered in any of the cell models. These results indicate that DNA damage, in particular oxidative DNA damage, contributes to cellular UVA1 sensitivity and underline a pivotal role of its repair in the cellular responses to UVA1.

An earlier mechanistic phase of iron toxicity in photosynthetic cells was interpreted in terms of enhanced photodynamic action by the cytochrome b₆/f complex (Cyt b₆/f) via singlet oxygen (¹O₂) on the photosystem II complex (PS II). Iron excess was induced in hydroponically cultured pea (Pisum sativum L.) plants, and its effect on the function of PS II in vivo as well as in vitro was studied under high-irradiance conditions. Iron excess in plants gave rise to a significant increase in Cyt b₆/f content of thylakoids. It appeared that the larger the content of Cyt b₆/f, the more susceptible PS II was to photoinhibition, and the higher the rate of ¹O₂ photoproduction in thylakoids was. The action spectrum for degradation of the D1 protein in thylakoids revealed that photosensitization by nonporphyrin chromophore(s) was apparently associated with near UV to blue light–induced deterioration of PS II. The results are pertinent to the concept that photooxidative damage to PS II, exacerbated by iron accumulation in thylakoid membranes in the form of Cyt b₆/f, is involved in the mechanism of iron toxicity in leaf cells.

A method for monitoring respiratory chain (RC) activity in single live cells is described. It is based on the registration of the photothermal (PT) response after a laser pulse of a live single cell. The dependence of the PT-response amplitude and shape upon redox state of RC components was studied with a PT microscope for two in vitro models: (1) solutions of the RC component cytochrome c and (2) mice hepatocytes. The parameters of the PT responses differed for oxidized and reduced forms of cytochrome c solutions and for inhibited RC and intact RC. The latter difference may be caused by alteration of the quantum yields of thermal (nonradiative) relaxation for light-absorbing molecules, i.e. RC components, as they undergo reduction during RC inhibition.

It is generally assumed that a central metal is essential for the efficiency of phthalocyanines in photodynamic therapy (PDT) of cancer. Contrary to the set opinion, the results of the present study indicate that the metal-free sulfonated phthalocyanines (H₂PcS_n, where n is the number of sulfonate groups per molecule) possess a considerable photoactivity. The relative phototoxicities of H₂PcS_1.5, H₂PcS_2.4, H₂PcS_3.1 and H₂PcS_3.8 on HEp2 human epidermoid carcinoma cells were 3.3, 20, 3.3 and 1, respectively, thus demonstrating dependence of the activity on the sulfonation degree, known for metallo-PcS_n. A significant delay in tumor growth and a decrease in tumor regrowth rate were observed in mice after PDT with H₂PcS_2.4. The antitumor effect declined in the order H₂PcS_2.4 > H₂PcS_3.1 > H₂PcS_1.5 and vanished for H₂PcS_3.8. We demonstrate here that the high photodynamic activity of H₂PcS_2.4 can be explained by its physicochemical properties in living cells and tissues. Thus, H₂PcS_n (n is about 2) can be considered as a new alternative in PDT of light-accessible neoplasms and further clinic-oriented studies are warranted.

Selectivity of photodynamic therapy can be improved with localized photosensitizer delivery, but topical administration is restricted by poor diffusion across the stratum corneum. We used electric pulses to increase transdermal transport of δ-aminolevulinic acid (ALA), a precursor to the photosensitizer protoporphyrin IX (PpIX). ALA-filled electrodes were attached to the surface of excised porcine skin or the dorsal surface of mice. Pulses were administered and, in some in vivo cases, a continuous DC potential (6 V) was concomitantly applied. For in vitro ¹⁴C ALA penetration, 10 μm layers parallel to the stratum corneum were assayed by liquid scintillation analysis, and 10 μm cross sections were examined autoradiographically. As the electrical dose (voltage × frequency × pulse width × treatment duration) increased, there was an increase in penetration depth. In vivo delivery was assayed by measuring the fluorescence of PpIX in skin samples. A greater than two-fold enhancement of PpIX production with electroporative delivery was seen versus that obtained with passive delivery. Superimposition of a DC potential resulted in a nearly three-fold enhancement of PpIX production versus passive delivery. Levels were higher than the sum of PpIX detected after pulse-alone and DC-alone delivery. Electroporation and electrophoresis are likely factors in electrically enhanced delivery.

Ultraviolet light exposure can impair immune responses that are not restricted to the exposed skin but is also found at other sites, i.e. systemic immunosuppression. Therefore, we investigated the UV-induced modulating effects on vaccination against hepatitis B in a mouse model. Two different mouse strains, BALB/c and C57Bl/6, were vaccinated intramuscularly against hepatitis B. Mice were exposed to different doses of ultraviolet B (UVB) for five consecutive days on shaved back skin before the vaccination. Vaccination against hepatitis B induced cellular (delayed-type hypersensitivity [DTH] and lymphocyte stimulation test) as well as humoral immune responses in both mouse strains. The DTH responses in C57Bl/6 mice were statistically significantly higher compared with BALB/c mice. UVB exposure induced a dose-dependent suppression of cellular immunity in both strains of mice. C57Bl/6 mice seemed to be more susceptible to this suppression. Anti-hepatitis B surface antibodies (total-Ig) were only marginally suppressed after UVB exposure. IgG2a and interferon-γ levels, both indicators for Th1 immune response, were suppressed in both mouse strains after UVB exposure. In summary, UVB exposure induced a dose-dependent suppression of both cellular and humoral immune responses after hepatitis B vaccination, although the suppressive effects on humoral immunity were limited to IgG2a production. Susceptibility to UVB-induced immunomodulation depended on the strain of mice and their predilection for developing different T cell responses.

This study investigated a possible circadian rhythm of light damage susceptibility in photoreceptors of both cyclic light-reared and dark-reared rats. A single exposure to intense green light was administered, beginning either in the early light period, the late light period or the dark period. In some animals exposed in the dark period, the synthetic antioxidant dimethylthiourea was administered before or after the onset of intense light exposure. Retinas were examined either immediately after exposure or after 2 weeks of recovery in darkness. Rod outer segment length and outer nuclear layer thickness measurements were used to assess light damage, along with qualitative analysis of swelling and disruption of the outer retinal layers. In all animals, retinal light damage was the most severe when intense light exposure began during the dark period. However, this severe damage was significantly reduced by pretreatment with the antioxidant. In a separate set of unexposed animals, fluctuations in plasma adrenocorticotropic hormone (ACTH) and corticosterone concentrations followed the same time course, regardless of the light regime during rearing. Our data support the notion of a circadian rhythm of light damage susceptibility that peaks in the dark period and yet can be modulated by the exogenous administration of an antioxidant.

Chromophore–apoprotein interactions were studied with recombinant apoproteins, oat phytochrome (phyA) and CphB of the cyanobacterium Calothrix PCC7601, which were both incubated with the bilin compounds biliverdin (BV) IXα, phycocyanobilin (PCB) and the 3′-methoxy derivative of PCB. Previously it was shown that CphB and its homolog in Calothrix, CphA, show strong sequence similarities with each other and with the phytochromes of higher and lower plants, despite the fact that CphB carries a leucine instead of a cysteine at the chromophore attachment position and thus holds the chromophore only noncovalently. CphA binds tetrapyrrole chromophores in a covalent, phytochrome-like manner. For both cyanobacterial phytochromes, red and far-red light–induced photochemistry has been reported. Thus, the role of the binding site of CphB in directing the photochemistry of noncovalently bound tetrapyrroles was analyzed in comparison with the apoprotein from phyA phytochrome. Both the aforementioned compounds, which were used as chromophores, are not able to form covalent bonds with a phytochrome-type apoprotein because of their chemical structure (vinyl group at position 3 or methoxy group at position 3′). The BV adducts of both apoproteins showed phytochrome-like photochemistry (formation of red and far-red–absorbing forms of phytochrome [P_r and P_fr forms]). However, incubation of the oat apophytochrome with BV primarily yields a 700 nm form from which the P_r–P_fr photochemistry can be initiated and to which the system relaxes in the dark after illumination. The results for CphB were compared with a CphB mutant where the chromophore-binding cysteine had been introduced, which, upon incubation with PCB, shows spectral properties nearly identical with its (covalently binding) CphA homolog. A comparison of the spectral properties (P_r and P_fr forms) of all the PCB- and BV-containing chromoproteins reveals that the binding site of the cyanobacterial apoprotein is better suited than the plant (oat) phytochrome to noncovalently incorporate the chromophore and to regulate its photochemistry. Our findings support the proposal that the recently identified phytochrome-like prokaryotic photoreceptors, which do not contain a covalently bound chromophore, may trigger a light-induced physiological response.