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Trisbipyrazyl ruthenium(II) (Ru[bpz]32 ) was examined as DNA photosensitizer. Damage resulting from the photolysis of synthetic oligonucleotides has been monitored by polyacrylamide gel electrophoresis. Photoadduct formation is found on both single- and double-stranded oligonucleotides. On oligonucleotide duplex, oxidative damage occurs selectively at the 5′G of the 5′GG3′ site and to a lesser extent at the 5′G of a GA sequence. These findings suggest the involvement of electron transfer and show that this mechanism is the main DNA damaging process involved in Ru(bpz)32 photosensitization. In addition, photoadducts and oxidative damage are both highly affected by an increase of salt concentration in the reaction medium, stressing the importance of direct interactions between nucleic acid bases and the excited ruthenium complex for efficient electron transfer. On single-stranded oligonucleotides, all the guanines are oxidized to the same extent. In this case, oxidative damage, which is not affected by an increase of salt in the solution, has been attributed, in part, to singlet oxygen. More importantly, Cu/Zn superoxide dismutase (SOD) strongly enhances the yield of all damage, correlated to an increase of both electron transfer and singlet oxygen production. This original activity of SOD is the first example of bioactivation of a polyazaaromatic ruthenium complex.
Structural volume changes upon excitation of isomerization-blocked 5,12-trans-locked bacteriorhodopsin (bR) (bacterio-opsin 5-12-trans-locked retinal) were studied using photothermal methods. The very small prompt expansion detected using laser-induced optoacoustics (0.3 mL/mol of absorbed photons) is assigned to a charge reorganization in the chromophore protein pocket concomitant with the formation of the intermediate T5.12. The subsequent contraction associated with a 300 ns lifetime is assigned to protein movements required to reach the entire chromoprotein free energy minimum, after the 17 ps optical decay of T5.12. The volume changes comprise the entropy of medium rearrangement during T5.12 formation and decay. The slow changes detected in previous studies by atomic force microscopy might be explained by the slowing down of movements in films containing 5,12-trans-locked bR. Photothermal beam deflection data with the 5,12-trans-locked bR suspensions indicate no further changes in microseconds to hundreds of milliseconds. Thus, all the absorbed energy is either released to the solution as heat or used for entropy changes within the first 300 ns after the pulse, supporting the paradigm that isomerization is required for signal transduction in retinal proteins. Bacterio-opsin assembled with all-trans-retinal afforded (similar to data reported with wild-type bR) an expansion of 2.6 mL/mol (assigned to the production of KE) followed by a further expansion of 0.8 mL/mol (KE → KL; KE, KL, early and late K's) involving no heat loss. For KL decay to L, a contraction of 6 mL/mol of phototransformed reconstituted all-trans bR was determined.
Tuning thermodynamic driving force and electronic coupling through structural modifications of a carotene (C) porphyrin (P) fullerene (C60) molecular triad has permitted control of five electron and energy transfer rate constants and two excited state lifetimes in order to prepare a high-energy charge-separated state by photoinduced electron transfer with a quantum yield of essentially unity (≥96%). Excitation of the porphyrin moiety of C–P–C60 is followed by a combination of photoinduced electron transfer to give C–P· –C60·− and singlet–singlet energy transfer to yield C–P–1C60. The fullerene excited state accepts an electron from the porphyrin to also generate C–P· –C60·−. Overall, this initial state is formed with a quantum yield of 0.97. Charge shift from the carotenoid to yield C· –P–C60·− is at least 60 times faster than recombination of C–P· –C60·−, leading to the overall quantum yield near unity for the final state. Formation of a similar charge-separated species from the zinc analog of the triad with a yield of 40% is also observed. Charge recombination of C· –P–C60·− in 2-methyltetrahydrofuran yields the carotenoid triplet state, rather than the ground state. Comparison of the results for this triad with those for related triads with different structural features provides information concerning the effects of driving force and electronic coupling on each of the electron transfer steps.
The well-characterized, monodispersed nature of reverse micelles formed by sodium bis(2-ethylhexyl)sulfosuccinate/heptane and their usefulness in approximating a membrane-like environment have been exploited to investigate the effect of pH and water pool size on the photophysical properties of hypericin (Hyp). Our measurements reveal two titratable groups of pKa ∼1.5 and ∼12.5. These are assigned to the HypH/Hyp equilibrium (the deprotonation of a carbonyl group) and the Hyp−/Hyp2− equilibrium (the deprotonation of a peri hydroxyl group). The low-energy absorbance maxima of HypH, of Hyp and Hyp− and of Hyp2− are 583, 594 and 613 nm, respectively. Neither at pH 13 nor at 1 M HCl is the system entirely in the Hyp2− or the HypH forms. Ours is the first study of Hyp in reverse micelles as well as the first time-resolved study of Hyp as a function of pH.
On irradiation of N-hydroxythiazole-2(3H)-thione 3 at 300 nm, the photoproducts disulfide 4, bisthiazole 5 and thiazole 6 are formed. During this photolysis, hydroxyl radicals are released, which have been detected by spin trapping with 5,5-dimethyl-1-pyrroline N-oxide (DMPO), coupled with electron paramagnetic resonance spectroscopy. In the presence of supercoiled pBR322 DNA, irradiation of thiazolethione 3 induces strand breaks through the photogenerated hydroxyl-radicals, as confirmed by control experiment with the hydroxyl-radical scavenger isopropanol. Singlet oxygen appears not to be involved, as attested by the lack of a D2O isotope effect. During the photoreaction of thiazolethione 3 in the presence of 2′-deoxyguanosine (dG), the latter is photooxidized (ca 10% conversion after 2 h of irradiation) to the 7,8-dihydro-8-oxo-2′-deoxyguanosine as the main oxidation product. The dG conversion levels off after complete consumption of thiazolethione 3 and is suppressed by the addition of the hydroxyl-radical scavenger 2,6-di-tert-butylcresol or DMPO. Since the photoproducts 4–6 are ineffective as sensitizers for the photooxidation of dG and DNA, the hydroxyl radicals released in the photolysis of thiazolethione 3 are the oxidizing species of DNA and dG. These results suggest that the thiazolethione 3 may serve as a novel and effective photochemical hydroxyl-radical source for photobiological studies.
Photophysical properties of curcumin, 1,7-bis-(4-hydroxy-3-methoxy phenyl)-1,6-heptadiene-2,5-dione, a pigment found in the rhizomes of Curcuma longa (turmeric) have been studied in different kinds of organic solvent and also in Triton X-100 aqueous micellar media using time-resolved fluorescence and transient absorption techniques having pico and nanosecond time resolution, in addition to steady-state absorption and fluorescence spectroscopic techniques. Steady-state absorption and fluorescence characteristics of curcumin have been found to be sensitive to the solvent characteristics. Large change (Δμ = 6.1 Debye) in dipole moments due to photoexcitation to the excited singlet state (S1) indicates strong intramolecular charge transfer character of the latter. Curcumin is a weakly fluorescent molecule and the fluorescence decay properties in most of the solvents could be fitted well to a double-exponential decay function. The shorter component having lifetime in the range 50–350 ps and percent contribution of amplitude more than 90% in different solvents may be assigned to the enol form, whereas the longer component, having lifetime in the range 500–1180 ps with less than 10% contribution may be assigned to the di-keto form of curcumin. Our nuclear magnetic resonance study in CDCl3 and dimethyl sulfoxide-D6 also supports the fact that the enol form is present in the solution by more than about 95% in these solvents. Excited singlet (S1) and triplet (T1) absorption spectrum and decay kinetics have been characterized by pico and nanosecond laser flash photolysis. Quantum yield of the triplet is low (ϕT ≤ 0.12). Both the fluorescence and triplet quantum yields being low (ϕf ϕT < 0.18), the photophysics of curcumin is dominated by the energy relaxation mechanism via the internal conversion process.
The photokinetic behavior of a photochromic compound, 2,2-spiro-adamantylidene-2H-naphtho[1,2-b]pyran (Py), has been investigated by using monochromatic irradiation in the UV and visible ranges. An unusually complex photochemistry occurs whereby two colored forms (o-quinone–allides) are produced, one of which is thermoreversible whereas the other is not thermoreversible but photoreversible. These are the result of ring opening of the pyran C–O bond to a short lived open-form intermediate which converts to the two different colored forms by twisting around different C–C bonds. Their spectra and molar absorption coefficients were obtained in acetonitrile solution. The kinetic parameters of the thermal bleaching (rate constant and activation energy) and photobleaching (quantum yield) were measured. A plausible reaction mechanism is proposed. Based on this mechanism, mathematical methods were devised which were capable of analyzing the system during its dynamic evolution and evaluating the quantum yields of the color-forming photoreactions.
Photochemical hole-burning spectroscopy was used to study the excited-state electronic structure of the 4-hydroxycinnamyl chromophore in photoactive yellow protein (PYP). This system is known to undergo a trans-to-cis isomerization process on a femtosecond-to-picosecond time scale, similar to membrane-bound rhodopsins, and is characterized by a broad featureless absorbance at 446 nm. Resolved vibronic structure was observed for the hole-burned spectra obtained when PYP in phosphate buffer at pH 7 was frozen at low temperature and irradiated with narrow bandwidth laser light at 431 nm. The approximate homogeneous width of 752 cm−1 could be calculated from the deconvolution of the hole-burned spectra leading to an estimated dephasing time of ∼14 fs for the PYP excited-state structure. The resolved vibronic structure also enabled us to obtain an estimated change in the C=C stretching frequency, from 1663 cm−1 in the ground state to ∼1429 cm−1 upon photoexcitation. The results obtained allowed us to speculate about the excited-state structure of PYP. We discuss the data for PYP in relation to the excited-state model proposed for the photosynthetic membrane protein bacteriorhodopsin, and use it to explain the primary event in the function of photoactive biological protein systems. Photoexcitation was also carried out at 475 nm. The vibronic structure obtained was quite different both in terms of the frequencies and Franck–Condon envelope. The origin of this spectrum was tentatively assigned.
Herpes simplex virus (HSV) normally causes vescular lesions on mucocutaneous surfaces but can also cause encephalitis. The virus can reactivate from the latent state in neurons to form recrudescent lesions. One common stimulus for reactivation is exposure to sunlight. In the present study, the effects of irradiating rats with suberythemal ultraviolet (UV) before or after infecting them epidermally with HSV was investigated. Preexposure to UV impaired HSV-specific cellular immune responses, as indicated by delayed type hypersensitivity (DTH) and in vitro lymphoproliferation assays. However, the number and severity of the skin lesions were not altered. In contrast, exposure after infection did not affect cellular immunity but resulted in a large increase in the severity and number of lesions. In a second series of experiments, the effects of preirradiating with UV on HSV infection was examined using a route of inoculation which was not skin-associated, namely intranasal, allowing direct noninvasive access to the nervous system. It was found that suppressed DTH resulted, together with an increase in the incidence and severity of neurological symptoms and an increased viral load in the brain. Therefore, unlike the situation in the skin, irradiation of rats before intranasal inoculation led to a suppressed immune response to HSV which correlated with increased viral load and symptoms. These results indicate that the effects of UV may be dependent on whether the animal is exposed before or after the infection, and whether the infection is skin-associated or systemic.
Laboratory tests confirmed a negative and variable response of the following four species to artificial UV radiation: Cypridopsis vidua, an ostracode; Chironomus riparius, a midge larvae; Hyalella azteca, an amphipod; and Daphnia magna, a daphnid. Severe damage occurred at UV-B irradiance ranging from 50 to 80% of incident summer values. Under constant exposure to UV and photosynthetically active radiation (PAR) the acute lethal response was recorded at 0.3, 0.8, 0.8 and 4.9 W m−2 UV-B for D. magna, H. azteca, C. riparius and C. vidua, respectively. Sublethal UV-B damage to invertebrates included impaired movement, partial paralysis, changes in pigmentation and altered water balance (bloating). A series of UV-B, UV-A and PAR treatments, applied separately and in combination, revealed a positive role for both UV-A and PAR in slowing down UV-B damage. Mean lethal concentration values of the species typically more tolerant to UV and PAR (Cypridopsis, Chironomus) decreased conspicuously when both UV-A and PAR were eliminated. For UV-B–sensitive species (Hyalella, Daphnia) these differences were notably smaller. We suggest that this gradation of sensitivity among the tested species demonstrates potential differences in repairing mechanisms which seem to work more efficiently for ostracodes and chironomids than for amphipods and daphnids. Manipulations with a cellulose acetate filter showed that lower range UV-B (280–290 nm), produced by FS-40 lamps, may cause excessive UV damage to invertebrates.
Chlorophyll accumulation during greening implies the continuous transformation of photoactive protochlorophyllide (Pchlide) to chlorophyllide. Since this reaction is a light-dependent step, the study of regeneration of photoactive Pchlide under a continuous illumination is difficult. Therefore this process is best studied on etiolated plants during a period of darkness following the initial photoreduction of photoactive Pchlide. In this study, the regeneration process has been studied using spinach cotyledons, as well as barley and bean leaves, illuminated by a single saturating flash. The regeneration was characterized using 77 K fluorescence emission and excitation spectra and high-performance liquid chromatography. The fluorescence data indicated that the same spectral forms of photoactive Pchlide are regenerated by different pathways: (1) photoactive Pchlide regeneration starts immediately after the photoreduction through the formation of a nonphotoactive Pchlide form, emitting fluorescence at approximately 651 nm. This form is similar to the large aggregate of photoactive Pchlide present before the illumination, but it contains oxidized form of nicotinamide adenine dinucleotide phosphate, instead of the reduced form (NADPH), in the ternary complexes; and (2) after the dislocation of the large aggregates of chlorophyllide–light-dependent NADPH:Pchlide a photooxidoreductase–NADPH ternary complexes, the regeneration occurs at the expense of the several nonphotoactive Pchlide spectral forms present before the illumination.
Time-resolved, laser-induced changes in absorbance, ΔA(λ; t), have been recorded with a view to probing pigment–pigment interactions in chlorosomes (control as well as carotenoid-depleted) and artificial aggregates of bacteriochlorophyll e (BChle). Control chlorosomes were isolated from Chlorobium phaeobacteroides strain CL1401, whose chromophores comprise BChle, bacteriochlorophyll a (BChla) and several carotenoid (Car) pigments; Car-depleted chlorosomes, from cells grown in cultures containing 2-hydroxybiphenyl. Artificial aggregates were prepared by dispersing BChle in aqueous phase in the presence of monogalactosyl diglyceride. In chlorosomes ΔA(λ; t) shows, besides a signal attributable to triplet Car (with a half-life of about 4 μs), signals in the Qy regions of both BChl. The BChla signal decays at the same rate as the Car signal, which is explained by postulating that some Car are in intimate contact with some baseplate BChla pigments, and that when a ground-state Car changes into a triplet Car, the absorption spectrum of its BChla neighbors undergoes a concomitant change (termed transient environment-induced perturbation). The signal in the Qy-region of BChle behaves differently: its amplitude falls, under reducing conditions, by more than a factor of two during the first 0.5 μs (a period during which the Car signal suffers negligible diminution), and is much smaller under nonreducing conditions. The BChle signal is also attributed to transient environment-induced perturbation, but in this case the perturber is a BChle photoproduct (probably a triplet or a radical ion). The absence of long-lived BChle triplets in all three systems, and of long-lived BChla triplets in chlorosomes, indicates that BChle in densely packed assemblies is less vulnerable to photodamage than monomeric BChle and that, in chlorosome, BChla rather than BChle needs, and receives, photoprotection from an adjacent Car.
Photodynamic therapy could provide an alternative to antibiotics for the treatment of local infections since a wide range of microorganisms have been shown to be susceptible to killing by photodynamic action (PDA) in vitro. The purpose of this study was to determine whether PDA was also able to affect the potency of two key bacterial virulence factors—lipopolysaccharide (LPS) and proteases. Suspensions of LPS from Escherichia coli and culture supernatants containing proteases of Pseudomonas aeruginosa were exposed to red light in the presence of toluidine blue O (TBO). The activity of each virulence factor was determined before and after irradiation. The limulus amoebocyte lysate (LAL) assay and the induction of proinflammatory cytokine (interleukin-8 and -6) release from human peripheral blood mononuclear cells (PBMC) were used for assessing the biological activity of LPS. Protease activity was quantified by azocasein hydrolysis. The biological activities of the LPS (both the LAL activity and its ability to induce cytokine release from PBMC) and the proteases were reduced significantly by irradiation with red light in the presence of TBO in a dose-dependent manner with respect to both the light energy dose and the TBO concentration. The ability of TBO-mediated PDA to reduce the activities of key virulence factors may be an additional benefit of using light-activated antimicrobial agents in the treatment of infectious diseases.
An important limitation of topical 5-aminolevulinic acid (ALA)-based photodetection and photodynamic therapy is that the amount of the fluorescing and photosensitizing product protoporphyrin IX (PpIX) formed is limited. The reason for this is probably the limited diffusion of ALA through the stratum corneum. A solution to this problem might be found in the use of ALA derivatives, as these compounds are more lipophilic and therefore might have better penetration properties than ALA itself. Previous studies have shown that ALA hexyl ester (ALAHE) is more successful than ALA for photodetection of early (pre)malignant lesions in the bladder. However, ALA pentyl ester slightly increased the in vivo PpIX fluorescence in early (pre)malignant lesions in hairless mouse skin compared to ALA. The increased PpIX fluorescence is located in the stratum corneum and not in the dysplastic epidermal layer. In the present study, ALA- and ALAHE-induced PpIX fluorescence kinetics are compared in the normal nude mouse skin, of which the permeability properties differ from the bladder. Application times and ALA(HE) concentrations were varied, the effect of a penetration enhancer and the effect of tape stripping the skin before or after application were investigated. Only during application for 24 h, did ALAHE induce slightly more PpIX fluorescence than ALA. After application times ranging from 1 to 60 min, ALA-induced PpIX fluorescence was higher than ALAHE-induced PpIX fluorescence. ALA also induced higher PpIX production than ALAHE after 10 min of application with concentrations ranging from 0.5 to 40%. The results of experiments with the penetration enhancer and tape stripping indicated that the stratum corneum acts a barrier against ALA and ALAHE. Use of penetration enhancer or tape stripping enhanced the PpIX production more in the case of ALAHE application than in the case of ALA application. This, together with the results from the different application times and concentrations indicates that ALAHE diffuses more slowly across the stratum corneum than ALA.
A fluorescence imaging system was used to monitor the emission of disulfonated aluminum phthalocyanine (AlS2Pc) during the photodynamic therapy (PDT) of murine tumors. Cells of the MS-2 fibrosarcoma were injected in mice in two compartments in order to cause the development of tumors in different host tissues. Two drug doses and two uptake times were considered. Moreover, the fluorescence of the AlS2Pc was excited using two wavelengths on the opposite sides of the absorption peak to detect a possible change in the absorption spectrum of the sensitizer induced by the PDT. In the tumors, the treatment induces a variation of the fluorescence intensity: in some mice a mild photobleaching takes place, in others a fluorescence enhancement occurs. Which effect predominates depends on the experimental conditions, even though a large spread of data was found amongst mice of the same group. In all mice, independently of the drug dose, uptake time or tumor compartment, a marked increase in the fluorescence signal takes place at the borders of the irradiated area. To quantify this effect we evaluated the ratio between the fluorescence intensities in the peritumoral area and in the tumor itself. This ratio increases monotonically during the PDT, showing a different behavior with the two excitation wavelengths. This indicates that the AlS2Pc absorption spectrum shifts toward shorter wavelengths as a result of the irradiation.
Photodynamic therapy (PDT) of malignancies uses light to activate a photosensitizer preferentially accumulated in cancer cells. The first pegylated photosensitizer, tetrakis-(m-methoxypolyethylene glycol) derivative of 7,8-dihydro-5,10,15,20-tetrakis(3-hydroxyphenyl)-21-23-[H]-porphyrin (PEG-m-THPC), was evaluated in non–tumor-bearing rats. The aim of this study was to assess the photodynamic threshold for damage and its sequelae in normal rat tissue. Thirty-five Fischer rats were sensitized with 3, 9 or 30 mg/kg body weight PEG-m-THPC. Colon, vagina and perineum were irradiated with laser light of 652 nm wavelength and an optical dose of 50, 150 or 450 J/cm fiber length. Temperature in the pelvis was measured during PDT. Three days following PDT the effect on skin, vagina, colon, striated muscle, connective tissue, nerves and blood vessels was assessed by histology. The healing of the above-mentioned tissues was assessed on two rats 3 and 8 weeks after PDT using 9 mg/kg PEG-m-THPC activated with 450 J/cm laser light. No dark toxicity was observed. PDT using 30 mg/kg PEG-m-THPC induced severe necrosis irrespective of the optical dose. Body weight of 9 or 3 mg/kg activated with less than 450 J/cm induced moderate or no damage. No substantial increase in body temperature was seen during PDT. Tissues with severe PDT-induced damage seem to have a good tendency to regenerate. We conclude that within the dose required for tumor treatment PEG-m-THPC is a safe photosensitizer with promising properties. PDT of the colon mucosa below 9 mg/kg PEG-m-THPC and 150 J/cm seems to be safe. All other tissues can be exposed to 9 mg/kg PEG-m-THPC activated with less than 450 J/cm laser light with little side effects.
UVB and UVA components of the solar spectrum or from artificial UV-sources might be important etiological factors for the induction and development of skin cancer. In particular, deficiencies in the capacity to repair UV-induced DNA-lesions have been linked to this phenomenon. However, until now only limited data are available on the biological and physical parameters governing repair capacity. We have, therefore, developed a flowcytometric assay using fluorescence-labeled monoclonal antibodies to study the dose-dependence of induction and repair of UVB-induced cyclobutane pyrimidine dimers in a spontaneously immortalized keratinocytic cell line (HaCaT). Our results show that the kinetics of recognition and incision of UVB-induced DNA lesions slows down by a factor of about 3 in a dose range of 100–800 J m−2. Furthermore, a thorough analysis of repair kinetics indicates that this reduction in repair capacity might not be dependent on saturation of enzymatic repair capacity (Michalis–Menten) but may be caused by a UV-induced impairment of enzymes involved in DNA repair. Because this effect is evident in vitro at doses comparable to the minimal erythemal dose in vivo, our results might have significant impact on risk assessment for UV-induced carcinogenesis.
In the ciliated protozoan Blepharisma, step-up photophobic response is believed to be mediated by a novel type of photosensory pigment known as “blepharismins“ (BL) that are contained in the pigment granules located just beneath the plasma membrane. We examined the ultrastructure of the pigment granules by freeze-fracture and thin-section electron microscopy and proposed a schematic diagram showing the granules' three-dimensional inner membranous structure. Some of the BL are suggested to be associated with 200 kDa membrane protein. High-pressure liquid chromatography analysis of pigment species associated with 200 kDa protein obtained from blue forms of Blepharisma (oxyblepharisma) revealed that the 200 kDa protein was associated with five types of oxyblepharismin. The fluorescence intensity was increased when the pigments were dissociated from the 200 kDa protein. The result supports the hypothesis that the pigment–200 kDa complex is able to transduce light energy into signals mediating the photobehavior of Blepharisma.
The proton channels of the bacteriorhodopsin (BR) proton pump contain bound water molecules. The channels connect the purple membrane surfaces with the protonated retinal Schiff base at the membrane center. Films of purple membrane equilibrated at low relative humidity display a shift of the 570 nm retinal absorbance maximum to 528 nm, with most of the change occurring below 15% relative humidity. Purple membrane films were dehydrated to defined humidities between about 50 and 4.5% and examined by Fourier transform infrared difference spectroscopy. In spectra of dehydrated-minus-hydrated purple membrane, troughs are observed at 3645 and 3550 cm−1, and peaks are observed at 3665 and 3500 cm−1. We attribute these changes to water dissociation from the proton uptake channel and the resulting changes in hydrogen bonding of water that remains bound. Also, in the carboxylic acid spectral region, a trough was observed at 1742 cm−1 and a peak at 1737 cm−1. The magnitude of the trough to peak difference between 1737 and 1742 cm−1 correlates linearly with the extent of the 528 nm pigment. This suggests that a carboxylic acid group or groups is undergoing a change in environment as a result of dehydration, and that this change is linked to the appearance of the 528 nm pigment. Dehydration difference spectra with BR mutants D96N and D115N show that the 1737–1742 cm−1 change is due to Asp 96 and Asp 115. A possible mechanism is suggested that links dissociation of water in the proton uptake channel to the environmental change at the Schiff base site.
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