It was demonstrated that the interaction of the aminoacridizinium salts 2a–2d with DNA depends on the substitution pattern of the chromophore. Spectrophotometric and fluorometric titrations of the acridizinium salts 2a–2d with natural and synthetic polynucleotides reveal that the degree of interaction of the acridizinium salts 2a–2d with the nucleic acid differs significantly. The binding mode of the dyes with DNA was evaluated by circular dichroism and linear dichroism spectroscopy and compared with the parent system 2c. Whereas the 9-aminoacridizinium (2a) mainly intercalates into DNA, the salts 2b–c show a higher degree of association to the DNA backbone. The intercalated aminoacridizinium 2a caused few strand breaks upon UVA exposure, whereas the salts 2b–2d exhibit relatively efficient DNA-damaging properties. All acridizinium salts showed a sequence-selective strand cleavage for guanine-rich DNA regions.

Under physiological conditions B-form DNA is an exceedingly stable structure. However, experimental evidences obtained through nuclear magnetic resonance and fluorescence anisotropy suggest that the structure of the double helix fluctuates substantially. We describe photoacoustic phase modulation frequency measurements of ethidium bromide (Eb) with calf thymus DNA. As in fluorescence phase modulation measurements, we used an intercalating dye as a probe; however, we monitored the triplet excited state lifetime at different ionic strengths. The triplet lifetime of Eb varied from about 0.30 ms, with no DNA present, to 20 ms (at a DNA:Eb molar ratio of 5). With salt titration, this value falls to about 2.0 ms. This result suggests a strong coupling between the phenantridinium ring of the ethidium and the base pairs because of the stacking movement of the DNA molecule under salt effect. This effect may be understood considering DNA as a polyelectrolyte. The counterions in the solution shield the phosphate groups, reducing the electrostatic repulsion force between them, hence compacting the DNA molecule. The results from Fourier transform infrared demonstrated two important bands: 3187 cm⁻¹ corresponding to the symmetric stretching of the NH group of the bases and 1225 cm⁻¹ corresponding to the asymmetric stretching of phosphate groups shifted toward higher wavenumbers, suggesting a proximity between the intercalant and base pairs and a modification of the DNA backbone state, both induced by salt accretion.

Using water-soluble 1,8-naphthalimide derivatives, the mechanisms of photosensitized DNA damage have been elucidated. Specifically, a comparison of rate constants for the photoinduced relaxation of supercoiled to circular DNA, as a function of dissolved halide, oxygen and naphthalimide concentration, has been carried out. The singlet excited states of the naphthalimide derivatives were quenched by chloride, bromide and iodide. In all cases the quenching products were naphthalimide triplet states, produced by induced intersystem crossing within the collision complex. Similarly, the halides were found to quench the triplet excited state of the 1,8-naphthalimide derivatives by an electron transfer mechanism. Bimolecular rate constants were <10⁵ M⁻¹ s⁻¹ for quenching by bromide and chloride. As expected from thermodynamic considerations quenching by iodide was 6.7 × 10⁹ and 8.8 × 10⁹ M⁻¹ s⁻¹ for the two 1,8-naphthalimide derivatives employed. At sufficiently high ground-state concentration self-quenching of the naphthalimide triplet excited state also occurs. The photosensitized conversion of supercoiled to circular DNA is fastest when self-quenching reactions are favored. The results suggest that, in the case of 1,8-naphthalimide derivatives, radicals derived from quenching of the triplet state by ground-state chromophores are more effective in cleaving DNA than reactive oxygen species or radicals derived from halogen atoms.

“Light-up” probes, icosa-α-thymidylate-thiazole orange conjugates, for the in situ time-resolved detection of messenger ribonucleic acid (mRNA) in living cells are evaluated. Upon annealing with polyA in aqueous solutions, the icosa-α-thymidylate-thiazole orange conjugates were shown to be up to 15 times more fluorescent. Microinjection of these probes into adherent fibroblasts resulted in high yields of hybridization and fluorescent signals. Incubation of cells in the presence of these probes resulted in facile internalization of the probe and similar painting of the messenger RNA in the nuclear and cytosolic regions.

The photoreactivity of methoxy-p-benzoquinone (MQ) and methoxyhydroquinone (MHQ) in dilute solution (10⁻⁴–10⁻³ M) was studied using continuous irradiation and laser flash photolysis (LFP). The quinone irradiated in degassed tetrahydrofuran (THF) gives MHQ and an adduct with the solvent. Only the formation of hydroquinone is observed in ethanol, and hydroxylation is evidenced in water, whereas the compound is stable in CCl₄. The bis-quinone, 4,4′-dimethoxybiphenyl-2,5,2′,5′-bisquinone, and the dibenzofurane-quinone, 8-hydroxy-3,7-dimethoxydibenzofuran-1,4-quinone, are formed in the presence of MHQ, whereas the reactivity is low with ethylconiferyl alcohol. When MHQ is irradiated selectively in degassed THF, the formation of MQ and of the bis-hydroquinone, 4,4′-dimethoxy-2,5,2′,5′-tetrahydroxybiphenyl, are observed. The dimer is oxidized photochemically or thermally into the mono- or bis-quinones, the process being accelerated in alkaline medium. The formation of the dimers is strongly favored by the contiguous presence of quinone and hydroquinone. When MHQ is selectively irradiated in the presence of trans-ethylconiferaldehyde (EtC), quinone formation and isomerization of EtC are observed. LFP experiments, performed with a selective excitation of MQ, indicate that the triplet state of the quinone is strongly quenched by MHQ to conduce to a semiquinone radical. The interaction between ³MQ* and MQ is mainly driven by an electron transfer process according to the similar value of the quenching rate constant found with another electron donor compound such as 1,4-dimethoxybenzene. By contrast, no strong quenching of ³EtC* by MHQ was observed. It is proposed that the photochemistry of the couple MQ/MHQ is governed by the formation of encounter complex between either ³MQ* and MHQ or ³MHQ* and MQ. Consequently, the fast part of the photoyellowing of lignocellulosics does not appear to involve the couple MHQ/MQ or MHQ/etherified coniferaldehyde, but more likely a combination of oxidation of the hydroquinone by ground-state oxygen and photohydration of the formed quinone from its triplet state, giving inter-alia more colored o-quinonoid type molecules.

By means of steady-state fluorescence spectroscopy we explore the photophysics of two lowest lying singlet excited states in two natural 15-cis-carotenoids, namely phytoene and phytofluene, possessing three and five conjugated double bonds (N), respectively. The results are interpreted in relation to the photophysics of all-trans-carotenoids with varying N. The fluorescence of phytofluene is more Stokes-shifted relative to that of phytoene, and is ascribed to the forbidden S₁ → S₀ transition, with its first excited singlet state (S₁) lying 3340 cm⁻¹ below the dipole allowed second excited singlet state (S₂), at 77 K. For phytoene the S₂ and S₁ potential surfaces are closer in energy, probably giving rise to the mixed S₂ and S₁ fluorescence characteristics. The origin of phytoene fluorescence is discussed and is suggested to be due to the S₁ → S₀ transition; with the S₁ state located 1100 cm⁻¹ below S₂ at 77 K. The dependence of the fluorescence quantum yield on temperature and viscosity shows that large amplitude molecular motions are involved in the radiationless relaxation process of phytoene. The transition dipole moment of absorption and emission are parallel in phytoene and nonparallel in phytofluene.

We report the formation of host–guest complexes between water-soluble calix[n]arene-p-tetrasulfonates (n = 4, 6, 8) or 2-hydroxypropyl-cyclodextrins (α-, β-, γ-) and the tetratosylate salt of 5,10,15,20-tetrakis(4-N-methylpyridyl)porphyrin (TMPyP). The binding constants ranging between 10² and 10⁵ M⁻¹ were calculated from the absorption and fluorescence changes. Calix[4]arene-p-tetrasulfonate has a high binding affinity and forms with TMPyP a 1:1 complex, whereas other calixarenes bind two molecules of TMPyP. Electrostatic attraction is the dominating binding mode. Binding to calixarenes leads to a considerable decrease of the quantum yields of the triplet and excited singlet states and to shortening of the singlet and triplet lifetimes of TMPyP. The quenching mechanism is attributed to electron transfer between calixarene phenolates and excited TMPyP. Photoinduced electron transfer within a novel supramolecular complex calixarene/TMPyP (electron donor)/methyl viologen (electron acceptor) has been proven by absorption and fluorescence measurements. Electrostatic attraction between the cationic donor and cationic acceptor, on the one hand, and the anionic host, on the other, overcomes the electrostatic repulsion forces. In contrast, the interaction of cyclodextrin with TMPyP is hydrophobic in nature and only slightly influences the photophysical properties of TMPyP. The different behavior of TMPyP bound to either of the hosts has been assigned to the specific effects of the dominant binding modes, viz. the electrostatic attraction for calixarenes and the hydrophobic interactions for inclusion complexes with cyclodextrins.

The phenomenon of protonation of phthalocyanines (Pc) and its effect upon their photophysical properties has seen considerable neglect in the literature. The work reported here clearly shows that tetrasulfonated zinc Pc, a known photodynamic therapy (PDT) agent, is strongly susceptible to protonation at the azomethine bridges. Absorption and fluorescence spectra demonstrate the absolute dependence of the redshifted peak on the pH of the solution. The fluorescence spectra and lifetimes of the protonated Pc are reported, and the potential application of this phenomenon to the development of a PDT agent with increased selectivity is discussed.

During July 2000 we used an electronic personal dosimeter (X-2000) and a biological dosimeter (Deutsches Zentrum für Luft- und Raumfahrt: Biofilm) to characterize the UV radiation exposure of arctic field scientists involved in biological and geological fieldwork. These personnel were working at the Haughton impact structure on Devon Island (75°N) in the Canadian High Arctic under a 24 h photoperiod. During a typical day of field activities under a clear sky, the total daily erythemally weighted exposure, as measured by electronic dosimetry, was up to 5.8 standard erythemal dose (SED). Overcast skies (typically 7–8 okta of stratus) reduced exposures by a mean of 54%. We estimate that during a month of field activity in July a typical field scientist at this latitude could potentially receive ∼80 SED to the face. Because of body movements the upper body was exposed to a UV regimen that often changed on second-to-second timescales as assessed by electronic dosimetry. Over a typical 10 min period on vehicle traverse, we found that erythemal exposure could vary to up to 87% of the mean exposure. Time-integrated exposures showed that the type of outdoor field activities in the treeless expanse of the polar desert had little effect on the exposure received. Although absolute exposure changed in accordance with the time of day, the exposure ratio (dose received over horizontal dose) did not vary much over the day. Under clear skies the mean exposure ratio was 0.35 ± 0.12 for individual activities at different times of the day assessed using electronic dosimetry. Biological dosimetry showed that the occupation was important in determining daily exposures. In our study, scientists in the field received an approximately two-fold higher dose than individuals, such as medics and computer scientists, who spent the majority of their time in tents.

Bcl-2 is a member of the large Bcl-2 family and protects cells from apoptosis. Ultraviolet B (UVB) irradiation induces apoptosis of keratinocytes that is known as “sunburn cells.” Previously we reported that UVB irradiation induces apoptosis accompanied by sequential activation of caspase 8, 3 and 1 in keratinocytes, and that the process is inhibited by various caspase inhibitors. Using bcl-2–expressing adenovirus vector we investigated the effect of Bcl-2 on UVB-induced apoptosis. Adenovirus vector efficiently introduced bcl-2 gene in cultured normal mouse keratinocytes (NMK cells); almost all NMK cells (1 × 10⁶) were transfected at 1 × 10⁸ plaque-forming unit (PFU)/mL. Bcl-2–transfected NMK cells were significantly resistant to UVB-induced apoptosis with the suppressive effect dependent on the Bcl-2 expression level. Following UVB irradiation caspase 8, 3 and 9 activities were stimulated in NMK cells, whereas in bcl-2–transfected cells only caspase 8, but not caspase 3 or 9, activity was stimulated. In order to investigate the effect of Bcl-2 in vivo topical application of Ad-bcl-2 on tape-stripped mouse skin was performed. Following the application Bcl-2 was efficiently overexpressed in almost all viable keratinocytes. The expression was transient with the maximal expression of Bcl-2 on the first day following the application of 1 × 10⁹ PFU in 200 μL. The introduced Bcl-2 remained at least for 6 days. UVB irradiation (1250 J/m²) induced apoptosis within 12 h and the maximal effect was observed at 24 h in control mouse skin. Both bcl-2–transfected and topical caspase 3 inhibitor-treated mice skin were resistant to UVB-induced apoptosis. The suppressive effect of Bcl-2 was more potent than that of caspase 3 inhibitor application. Topical application of empty adenovirus vector alone had no effect on Bcl-2 expression or UVB-induced apoptosis. These results indicate that adenovirus vector is an efficient gene delivery system into keratinocytes and that Bcl-2 is a potent inhibitor of UVB-induced apoptosis both in vitro and in vivo.

UV light exerts hazardous effects such as induction of skin cancer and premature skin aging. In this study we evaluated an assumptive anti-inflammatory effect of the nonsedative histamine H1-receptor antagonist, mizolastine, on UV-induced acute sunburn reaction. Therefore, a clinical, randomized, double-blind, four-arm, crossover study was conducted in healthy young female volunteers (skin type II) comparing the UV sensitivity under mizolastine, acetyl-salicylic acid (ASA), indomethacin or a mizolastine/ASA combination. Moreover, HaCaT keratinocytes were incubated with mizolastine under various UV treatment modalities in vitro to study its effect on the release of inflammatory cytokines, i.e. interleukin (IL)-1α, IL-6 and tumor necrosis factor α (TNF-α). All three drugs were effective in suppressing the UVB-, UVA- and combined UVA/UVB-erythema. However, the strongest effects were observed using the combined treatment with both 250 mg ASA and 10 mg mizolastine. An inhibitory effect in vitro of 10 nM mizolastine upon UV-induced cytokine release from HaCaT keratinocytes was observed for IL-1α at 24 h after 10 J/cm² UVA1, for IL-6 at 48 h after 10 J/cm² UVA1 and 30 mJ/cm² UVB, and also for TNF-α at 4 h after 10 J/cm² UVA, 10 J/cm² UVA1 and 30 mJ/cm² UVB, respectively. The combination of mizolastine and ASA can be strongly recommended as a protective measure against UV erythema development with a lower unwanted side effect profile than that of the hitherto treatment modality, i.e. indomethacin.

Leaf discs, enclosed in a photoacoustic (PA) chamber, generate two types of PA gas-uptake signals under certain conditions. Type I is manifested by a severe signal decrease that develops slowly under very low-light intensity and often reaches negative values. It is partially reversed by low-intensity far-red light. Type II occurs transiently in modulated far-red light. It is manifested by a rapid and dramatic decrease of the PA signal, upon the addition of short-wave background light, which is subsequently reversed. It differs from type-I uptake in that it occurs at much higher total light intensities. A thorough study, including modulation frequency and atmospheric composition dependencies, indicates different mechanisms for the two types of uptakes. Type-I uptake results from CO₂ accumulation in the PA cell by leaf respiration and reflects modulations in CO₂ solubilization. Type-II uptake likely reflects oxygen photoreduction in photosystem I, occurring prior to the activation of photosynthesis (i.e. during photosynthesis induction). This is supported by the complete suppression of type-II uptake when O₂ was removed. Also, type-II uptake was only mildly sensitive to CO₂ elimination, whereas type-I uptake was totally dependent on the presence of CO₂. Type-II uptake consists usually of two uptake waves. Fluorescence transients measured in parallel give further support to the reality and interpretation of these two uptake waves. PA could thus provide a unique opportunity to monitor oxygen photoreduction in vivo with high sensitivity and time resolution.

Illumination of H9c2 rat heart cells in the presence of Rose Bengal resulted in dose-dependent cell killing (assessed by trypan blue staining) and modification of ionic currents flowing through the heart cell membrane. Inhibitors of voltage-gated ionic currents were shown to have little effect on cell killing. Ionic current measurements were used to assess the increase in leak conductance of these cells, which has been suggested to be a causal factor in killing of other cell types (1). Inhibitors of voltage-gated ionic currents, including the sodium channel blocker tetrodotoxin (100 μM) and the calcium channel blocker lanthanum (10 μM) were shown to have little effect on cell killing. The potassium channel inhibitor tetraethylammonium (20 mM) inhibited cell killing, but the effect is viewed as being caused by an inhibition of leak current. The time course of block of voltage-activated ionic currents during illumination, in the presence of Rose Bengal, was rapid compared with that for induction of leak current and for cell killing. These observations are consistent with a role for leak current in photosensitized killing of cardiac cells. They are interpreted with respect to calcium influx through the leak current pathway as a trigger for the cellular response.

Radical tumor resection is the basis for the prolonged survival of patients suffering from malignant brain tumors such as glioblastoma multiforme. We have carried out a phase-II study involving 22 patients with malignant brain tumors to assess the feasibility and the effectiveness of the combination of intraoperative photodynamic diagnosis and fluorescence-guided resection (FGR) mediated by the second-generation photosensitizer meta-tetrahydroxyphenylchlorin (mTHPC). In addition, intraoperative photodynamic therapy (PDT) was performed. Several commercially available fluorescence diagnostic systems were investigated for their applicability in clinical practice. We have adapted and optimized a diagnostic system that includes a surgical microscope, an excitation light source (filtered to 370–440 nm), a video camera detection system and a spectrometer for clear identification of the mTHPC fluorescence emission at 652 nm. Especially in regions of faint fluorescence, it turned out to be essential to maximize the spectral information by optimizing and matching the spectral properties of all components, such as excitation source, camera and color filters. To sum up, on the basis of 138 tissue samples derived from 22 tumor specimens, we have been able to achieve a sensitivity of 87.9% and a specificity of 95.7%. This study demonstrates that mTHPC-mediated intraoperative FGR followed by PDT is a highly promising concept in improving the radicality of tumor resection combined with a therapeutic approach.

Fluorescence diagnosis and photodynamic therapy using 5-aminolevulinic acid (ALA) provide new methods for the detection and treatment of cervical cancer and especially its precursors. However, these techniques are restricted by the rate of uptake of the hydrophilic ALA, its poor diffusion through the bilayer of biological membranes or both. In this study we evaluated the effect of some esterified ALA derivatives on the induction of the endogenous photosensitizer, protoporphyrin IX (PpIX), and the photodamage in cultured human cervical cells (C33-A and CaSki). The kinetics of PpIX accumulation showed that ALA esters, especially the ALA-hexylester (h-ALA), induced significantly faster PpIX formation than ALA at the same concentration (0.5 mM). The PpIX induction showed a dose-dependent characteristic. The highest PpIX values could be achieved by an up to 1.3–13-fold lower concentration of ALA esters than with ALA. Using the Annexin V assay, apoptosis was found to be induced rapidly after irradiation in both ALA- and ALA esters–treated cells. On measuring mitochondrial activity, the incubation with h-ALA induced a more pronounced photodamage. The results indicate that improved or at least comparable photodynamic effects can be achieved by using remarkably lower doses of ALA esters.

The time-resolved enthalpy and the structural volume changes after excitation of native oat phytochrome A were studied in the micro- to milliseconds range by photothermal beam deflection (PBD), a technique that follows the time-resolved refractive index changes upon decay of the excited species. The first set of intermediates, I₇₀₀¹ and I₇₀₀², stores ca 83% of the energy of the first excited state, in agreement with previous optoacoustic data, whereas the second set stores only ca 18%. The temperature dependence of the amplitudes ratio for the optical absorbances of the (I₇₀₀¹ I₇₀₀²) intermediates set is explained on the basis of the thermochromic equilibrium between P_r,657 and P_r,672, which also is in line with the present PBD data. These data were best fitted with a parallel mechanism (with equal yield in each branch) for the production of the first set of intermediates, I₇₀₀¹ and I₇₀₀², as well as the second set of intermediates, I_bl¹ and I_bl². Thus, the final steps toward P_fr should be largely driven by positive entropic changes brought about by protein movements, in line with previous resonance Raman data. For the production of the first set of intermediates (I₇₀₀¹ and I₇₀₀²) an expansion of 18 ± 13 mL mol⁻¹ was determined, and a further expansion ≥7 mL mol⁻¹ was estimated for the decay from I₇₀₀ⁱ to the set of I_bl intermediates, indicating that the far red–absorbing form of phytochrome (P_fr) has a larger volume than the red-absorbing form of phytochrome. This is in agreement with previous chromatographic and circular dichroism data according to which P_fr shows a larger volume and the chromophore shows a higher accessibility, respectively, in the P_fr state.