Suppression of Delayed-type Hypersensitivity and Hemolysis Induced by Previously Photooxidized Psoralen: Effect of Fluence Rate and Psoralen Concentration

Abstract— The kinetics of the formation of biologically active psoralen photooxidation (POP) products were analyzed by the biological effects produced. Effects of the UV light fluence rate and psoralen concentration during the preir-radiation were investigated to assess the yield of POP products, which were active in vivo (inducing suppression of delayed-type hypersensitivity [DTH] reaction to sheep red blood cells) and in vitro (altering the human erythrocyte membrane permeability). It was shown that the reciprocity law of the irradiation fluence rate and time was not valid in the case of POP-induced hemolysis and DTH suppression. Immunosuppressive POP products were more efficiently formed at low fluence rate (20.8 W/m²), whereas POP hemolysins were more efficiently produced at a high fluence rate (180 W/m²) of UV light. The yield of immunosuppressive POP products was enhanced in dilute psoralen solutions, while the POP hemolysins yield increased with increasing psoralen concentration. A kinetic scheme for psoralen photoproduct formation was proposed. Kinetic analysis showed that a labile intermediate was produced as the result of excitation of psoralen. This intermediate was either converted to a stable immunosuppressive POP product, or two intermediates combined to form a POP hemolysin. It is proposed that PUVA therapy conditions are more favorable for the formation of immunosuppressive rather than membrane-damaging psoralen photooxidation products.     

LIGHT-CONTROLLED ADAPTATION KINETICS IN Phycomyces: EVIDENCE FOR A NOVEL YELLOW-LIGHT ABSORBING PIGMENT

When sporangiophores of the fungus Phycomyces blakesleeanus adapt from high to low fluence rate, dark adaptation (sensitivity recovery) can be accelerated by dim subliminal light [Galland et al. (1989) Photochem. Photobiol. 49, 485-491]. We measured fluence rate-response curves for this acceleration under the following conditions. After sporangiophores were initially adapted symmetrically to a fluence rate of 1 W m-2 (447 nm), they were exposed to unilateral subliminal light (subthreshold for phototropism) of variable wavelength and fluence rate, and then to unilateral test light (447 nm) of fluence rate either 10(-3) or 10(-5) W m-2. The duration of the subliminal light was chosen so that phototropism would not occur during this period. Phototropic latencies could be shortened by subliminal light that was less intense than the test light by several orders of magnitude. In experiments with the final unilateral light of fluence rate 10(-3) W m-2, the 447 nm subliminal light had a threshold (for the acceleration effect) of about 10(-11) W m-2. Yellow light of wavelength 575 nm, which itself is extremely ineffective for phototropism was extremely effective in shortening phototropic latencies in response in response to the test light. At 575 nm, the threshold was about 2 x 10(-12) W m-2. Conversely, near-UV light of wavelength 347 nm, which is highly effective for phototropism, was relatively ineffective (threshold approximately 7 x 10(-8) W m-2) in shortening the phototropic latency. Our results suggest the presence of a novel yellow-light absorbing pigment in Phycomyces that specifically regulates dark adaptation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protection of photosystem II against UV-A and UV-B radiation in the cyanobacterium Plectonema boryanum: the role of growth temperature and growth irradiance

Plectonema boryanum UTEX 485 cells were grown at 29 degrees C and 150 mumol m-2 s-1 photosynthetically active radiation (PAR) and exposed to PAR combined with ultraviolet-A radiation (UV-A) at 15 degrees C. This induced a time-dependent inhibition of photosystem II (PSII) photochemistry measured as a decrease of the chlorophyll a fluorescence ratio, Fv/Fm, to 50% after 2 h of UV-A treatment compared to nontreated control cells. Exposure of the same cells to PAR combined with UV-A + ultraviolet-B radiation (UV-B) caused only a 30% inhibition of PSII photochemistry relative to nontreated cells. In contrast, UV-A and UV-A + UV-B irradiation of cells cultured at 15 degrees C and 150 mumol m-2 s-1 had minimal effects on the Fv/Fm values. However, cells grown at 15 degrees C and lower PAR irradiance (6 mumol m-2 s-1) exhibited similar inhibition patterns of PSII photochemistry as control cells. The decreased sensitivity of PSII photochemistry of P. boryanum grown at 15 degrees C and 150 mumol m-2 s-1 to subsequent exposure to UV radiation relative to either control cells or cells grown at low temperature but low irradiance was correlated with the following: (1) a reduced efficiency of energy transfer to PSII reaction centers; (2) higher levels of a carotenoid tentatively identified as myxoxanthophyll; (3) the accumulation of scytonemin and mycosporine amino acids; and (4) the accumulation of ATP-dependent caseinolytic proteases. Thus, acclimation of P. boryanum at low temperature and moderate irradiance appears to confer significant resistance to UV-induced photoinhibition of PSII. The role of excitation pressure in the induction of this resistance to UV radiation is discussed.     

EFFECTS OF INTENSITY AND FLUENCE UPON DNA SINGLE-STRAND BREAKS INDUCED BY EXCIMER LASER RADIATION

Abstract— A Xenon-chloride excimer laser emitting energy at 308 nm was used to induce single-strand breaks (SSBs, frank breaks plus alkali-labile lesions as assayed by alkaline sucrose sedimentation techniques) in purified DNA from Bacillus subtilis . A fluence response study and a peak pulse intensity study were performed. At a pulse energy of 0.1 mJ/pulse, the radiation induced SSBs in a linear fashion (91 SSB/10⁸ Da per MJ/m²) to a maximum exprimental fluence of 1.28 MJ/m². The pulse intensity study showed that there were no significant changes in DNA breakage (105 SSB/10⁸ Da) between 2.93 times 10⁹ and 5.86 times 10¹¹ W/m² (0.11 and 22.0 mJ/pulse) at a constant total fluence of 1.1 MJ/m² (27000 mJ dose). This study has verified and extended previous work by quantifying the yield of SSBs induced in DNA by this laser radiation.     

PSORALEN-PHOTOSENSITIZED DAMAGE OF RAT PERITONEAL EXUDATE CELLS

Psoralen-sensitized photodamage (PUVA) of rat peritoneal exudate cells was investigated. Quartz-activated luminol-dependent chemiluminescence (ChL) was registered and the amount of trypan-positive cells was determined. Irradiation of peritoneal exudate cells in the presence of psoralen resulted in a dose-dependent monotonous inhibition of ChL. The reciprocity law of irradiation intensity and duration of irradiation was not valid for the observed inhibition of ChL: the inhibition increased with higher intensity. When psoralen previously photooxidized in ethanol (POP) was added to peritoneal exudate cell suspension, a double-phase response depending on psoralen irradiation dose was obtained: ChL activation was observed at low doses of UVA, ChL inhibition at high doses. Chemiluminescence inhibition correlated well with the increase in the number of trypan-positive cells. It may be supposed that the observed effects of PUVA or POP treatment are caused by cell cytoplasmic membrane damage.     

Influence of the laser intensity and spot size on the desorption of molecules and ions in matrix-assisted laser desorption/ionization with a uniform beam profile

The dependence of the number of desorbed particles on laser fluence has been investigated for matrix-assisted laser desorption/ionization (MALDI) of analyte and matrix ions as well as for (photoionized) neutral matrix molecules using a homogeneous “flat-top” laser profile. Laser spot diameters ranging from 10 to 200 μm in size have been used. 2,5-Dihydroxybenzoic acid (DHB) and 3,5-dimethoxy-4-hydroxycinnamic acid (sinapic acid) have been tested as matrices. The threshold (for ion detection) is higher and the dependence of the ion signal upon higher-than-threshold fluences is stronger for directly desorbed ions than for photoionized neutral molecules. Directly desorbed analyte ions exhibit the same dependence on fluence as the matrix ions with only minor differences between the two matrices tested, so both have approximately the same detection threshold. For both ions and photoionized neutral molecules, the fluence threshold increases with decreasing spot size while the slope of the intensity/fluence curves decreases. A quasi-thermal, sublimation/desportion model was found to describe the experimental results with excellent precision. For a complete explanation, non-equilibrium effects had to be taken into account.     

Depth profiling of tungsten coating layer on CuCrZr alloy using LIBS approach

Laser induced breakdown spectroscopy (LIBS) was employed to find the depth profile of W-coated CuCrZr alloy. Cathodic arc deposition method was applied to produce tungsten coating on the CuCrZr alloy. The LIBS measurements were carried out by using a Nd:YAG laser at its fundamental wavelength (1064 nm) with various fluences. The spectral intensity emitted from laser induced plasma of the coating material was investigated for both elemental and depth-profile analyses in terms of linear correlation coefficient. Other surface characterization techniques (scanning electron microscopy and energy dispersive X-ray) were also used to determine morphology and elemental composition. It is observed that nonuniform radial energy distribution of Gaussian laser beam caused gradual decay of spectral lines of the coating and rise of substrate lines. The results indicate that the spectral intensity from W coating was higher than that from the bulk material at same laser fluence. The high-resolution in-depth profile was achieved at low fluence. This study demonstrates that LIBS is a promising technique for the depth-profiling analysis of W coating of EAST-like material and can be used to determine the material erosion and impurity deposition on plasma facing components.     

Repair of furocoumarin-plus-UVA-induced damage and mutagenic consequences in eukaryotic cells

In the presence of near-UV radiation (UVA) furocoumarins (psoralens) photoinduce defined lesions in DNA, i.e. monoadducts and interstrand crosslinks. Their use in photochemotherapy (psoralen plus UVA (PUVA) treatment) and cosmetics raises questions concerning the repairability of these lesions and their genotoxic consequences. We have analysed the repair of psoralen photoadducts in cultured eukaryotic cells, such as yeast and mammalian cells, for furocoumarins of photochemotherapeutic interest. In yeast, the interaction of repair pathways differs in exogenous (plasmid) and endogenous (chromosomal) DNA. The order of mutagenic activity is 4,5',8-trimethylpsoralen greater than 5-methoxypsoralen greater than 8-methoxypsoralen greater than 7-methylpyrido[3,4-c]psoralen greater than 3-carbethoxypsoralen. The mutagenicity is dependent on psoralen functionality, concentration and bioavailability, maximal UVA dose, wavelength, dose (fluence) rate and presence or absence of chemical filters. It probably involves an inducible component. Chromosome breakage occurs during the repair period after PUVA treatment. It appears that the genotoxic effects of psoralens are produced by a specific arrangement of induced photolesions and the interaction of different repair systems.     

AN ULTRAVIOLET RADIATION ACTION SPECTRUM FOR IMMEDIATE PIGMENT DARKENING

The wavelength dependence for immediate pigment darkening (IPD) was investigated by exposing the midback skin of volunteers to a series of incremental fluences of narrow waveband radiation isolated by band-pass filters in the310–400 nm region. The threshold IPD fluence for each waveband was determined by visual assessment of the skin responses immediately after each exposure. The action spectrum, constructed from the mean threshold fluences, was broad and extended from 320 nm to 400 nm with a peak at around 340 nm. No IPD could be evoked at 310 nm, even after erythemogenic fluences. The spectrum was similar in each of the three skin types investigated (III, IV, V). The broad nature of the action spectrum within the UVA region suggests that IPD may serve as an alternative endpoint for measuring photoprotection against these wavelengths.     

Influence of the laser fluence in infrared matrix-assisted laser desorption/ionization with a 2.94 microm Er : YAG laser and a flat-top beam profile

The dependence of the signal intensity of analyte and matrix ions on laser fluence was investigated for infrared matrix-assisted laser desorption/ionization (IR-MALDI) mass spectrometry using a flat-top laser beam profile. The beam of an Er : YAG laser (wavelength, 2.94 microm; pulse width, 90 ns) was coupled into a sapphire fiber and the homogeneously illuminated end surface of the fiber imaged on to the sample by a telescope. Three different laser spot sizes of 175, 350 and 700 microm diameter were realized. Threshold fluences of common IR matrices were determined to range from about 1000 to a few thousand J m(-2), depending on the matrix and the size of the irradiated area. In the MALDI-typical fluence range, above the detection threshold ion signals increase strongly with fluence for all matrices, with a dependence similar to that for UV-MALDI. Despite the strongly different absorption coefficients of the tested matrices, varying by more than an order of magnitude at the excitation laser wavelength, threshold fluences for equal spot sizes were found to be comparable within a factor of two. With the additional dependence of fluence on spot size, the deposited energy per volume of matrix at threshold fluence ranged from about 1 kJ mol(-1) for succinic acid to about 100 kJ mol(-1) for glycerol.     

POTENTIAL ERRORS IN THE USE OF CELLULOSE DIACETATE AND MYLAR FILTERS IN UV-B RADIATION STUDIES

Abstract— The increase in UV-B radiation(290–320 nm) penetrating to the earth's surface as a result of the chemical depletion of the stratospheric ozone layer is an important environmental concern. In most studies using artificial UV-B sources, the determination of enhanced UV-B radiation effects on plants relies on equivalent UV-A radiation(320–400 nm) from the experimental UV-B fluorescent lamp source, filtered with either cellulose diacetate (CA) to create UV-B treatments, or with type S Mylar or polyester (PE) to create controls (no UV-B). The spectral irradiance in the UV-A was measured in the dark below lamps at two daily UV-B irradiance levels (14.1 and 10.7 W m^-2) with CA and PE at two ages. Highly significant differences in UV-A radiation (P 0.01) were measured below the treatment/control pairs at both fluence rates and filter ages. Filter aging was observed, which reduced the UV-A irradiance, especially for PE. The total daily ambient UV-A irradiance was also determined in the glasshouse at three seasons: the fall equinox, summer and winter, from which the total daily UV-A (lamp + ambient) irradiances were calculated. The addition of low to moderate ambient irradiance removed the treatment/control differences in the longwave UV-A(350–400 nm); however, the treatment/contro1 differences remained in the shortwave UV-A(320–350 nm), which was restricted by the glass, and in the total UV-A. The treatment/control differences persisted in the shortwave UV-A for the higher irradiance level, even under high summer ambient light. Also, spectral ratios (UVB:UV-A and shortwave: longwave UV-A) for all treatment groups decreased as the ambient UV-A radiation increased. Therefore, a range of experimental conditions exist where PE-covered lamps do not provide adequate control for UV-A irradiance, relative to the CA treatment, for glasshouse/growth chamber experiments. Potential complications in the interpretation of plant response exist for UV-B experiments conducted under low ambient light conditions (e.g. growth chambers; glasshouse in winter) or high daily UV-B irradiances (e.g. 14 kJ m^-2) for those plant responses that are sensitive to UV-A radiation.     

Antagonistic blue- and red-light regulation of cab-gene expression during photosynthetic adaptation in Scenedesmus obliquus.

During adaptation of the photosynthetic apparatus of the green alga Scenedesmus obliquus to various light qualities, the accumulation of chlorophylls and pigment-protein complexes (with specific consideration of chlorophyll a/b-binding (Cab) proteins) and cab-gene expression were determined. The fluence rate dependences for chlorophyll accumulation and cab-gene expression were very different. Very low fluence rates of violet (404 nm), blue (461 nm) and red (650 nm) light below the photosynthetic threshold, i.e. between 10(-3) and 10(-1) mumol m-2 s-1, inhibited all of these reactions in cells grown under heterotrophic conditions. At elevated fluence rates (above 1 mumol m-2 s-1), red light retained its negative regulation, whereas blue light stimulated pigment accumulation. Under autotrophic conditions the pattern was more complex, because chlorophyll accumulation was unaffected by light below the photosynthetic threshold. However, the expression of cab-genes was inhibited by red light but stimulated by blue light. Cells adapted to fluence rates, which ensured photosynthetic energy supply (above 1 mumol m-2 s-1), showed an increase in chlorophyll accumulation, blue light being more effective than red light. The results confirm and extend our previous discovery of two antagonistically acting photoreceptors in Scenedesmus which mediate and coordinate the complex functional and structural changes associated with photosynthetic adaptation. One of these receptor pigments is a blue-light receptor with positive action; the other is a violet-red-light receptor which can operate far below the photosynthetic threshold and exerts a negative regulation.     

PRIOR EXPOSURE OF HUMAN CELLS TO NEAR UV-RADIATION GIVES A DECREASE IN THE AMOUNT OF THE UNSCHEDULED DNA-SYNTHESIS INDUCED BY FAR UV-RADIATION

Pretreatment of human cells with near UV radiation (UVA) in fluences exceeding 5 × 10⁴ Jm⁻² caused a decrease in the amount of the unscheduled DNA synthesis induced by far UV radiation (UVC). The DNA repair synthesis, as measured by the incorporation of [³H] -thymidine, is reduced by nearly a factor of 2 for a UVA radiation exposure of 1.5 × 10⁵ Jm⁻². Since solar UVA fluence rate is rather independent of latitude, this figure corresponds to a UVA exposure time of 50-60 min from noon sunlight in the summer time.     

Fragmentation of leucine enkephalin as a function of laser fluence in a MALDI TOF-TOF

The effects of laser fluence on ion formation in MALDI were studied using a tandem TOF mass spectrometer with a Nd-YAG laser and alpha-cyano hydrocinnamic acid matrix. Leucine enkephalin ionization and fragmentation were followed as a function of laser fluence ranging from the threshold of ion formation to the maximum available, that is, about 280-930 mJ/mm2. The most notable finding was the appearance of immonium ions at fluence values close to threshold, increasing rapidly and then tapering in intensity with the appearance of typical backbone fragment ions. The data suggest the presence of two distinct environments for ion formation. One is associated with molecular desorption at low values of laser fluence that leads to extensive immonium ion formation. The second becomes dominant at higher fluences, is associated initially with backbone type fragments, but, at the highest values of fluence, progresses to immonium fragments. This second environment is suggestive of ion desorption from large pieces of material ablated from the surface. Arrhenius rate law considerations were used to estimate temperatures associated with the onset of these two processes.     

Photohemolysis sensitized by the furocoumarin imperatorin and its oxyfunctionalized derivatives

The dark and photosensitized (366 nm) hemolytic effects of imperatorin and its photooxidation products, the hydroperoxides I and II as well as the corresponding alcohol of the hydroperoxide I (imperatorin alcohol), were studied on human erythrocytes. Imperatorin was shown to photosensitize hemolysis, its fluence (D) dependence of the rate of photohemolysis (V) followed the equation V = V0 + aD2 + bD1/2, in which V0 is the dark hemolysis rate and a and b are constants. At fluences below 200 kJ/m2, the main hemolytic contribution derives from the bD1/2 component, which is due to the in situ formation of the imperatorin hydroperoxides, while at fluences higher than 200 kJ/m2, the main contribution corresponds to the aD2 component due to the two-photon damage of cell membranes. Hydroperoxides I and II induce oxyhemoglobin cross-linking, as well as its conversion to methemoglobin and hemichrome. These reactions involve hydroxyl and alkoxy radicals, as the hemolysis and oxyhemoglobin conversion could be inhibited by t-butanol and butylated hydrotoluene. For comparison, the dark hemolytic effect of the imperatorin alcohol was approximately 10-fold less than of the hydroperoxides.     

AUGMENTATION OF ULTRAVIOLET ERYTHEMA BY INDOMETHACIN IN ACTINIC PRURIGO: EVIDENCE OF MECHANISM OF PHOTOSENSITIVITY

Abstract— The effect of topical indomethacin on the intensity of erythema induced by ultraviolet radiation was measured by reflectance spectrophotometry in six patients with actinic prurigo. The intensity of UV-C erythema was decreased by indomethacin in five patients. The intensity of UV-B erythema was increased by indomethacin in five patients, and UV-A erythema was increased by indomethacin in all patients. The increased inflammatory response induced by UV-B and UV-A with indomethacin application was related to erythemal sensitivity at these wavelengths. Topical indomethacin caused no change in the intensity of UV-A erythema in a group of non-photosensitive subjects.
That inhibition of cyclo-oxygenase augments the inflammatory response to ultraviolet radiation suggests that lipoxygenase metabolites of arachidonic acid may be involved in the mechanism of photosensitivity in actinic prurigo.     

Multiplex polymerase chain reaction analysis of UV-A- and UV-B-induced delayed and early mutations in V79 Chinese hamster cells

We previously reported that approximately 10% of V79 Chinese hamster fibroblast populations clonally derived from single cells immediately after irradiation with either ultraviolet B (UV-B, 290-320 nm, mainly 311 nm) or ultraviolet A (UV-A, 320-400 nm, mainly 350-390 nm) radiation exhibit genomic instability. The instability is revealed by relatively high mutation frequencies in the hypoxanthine phosphoribosyl transferase (hprt) gene up to 23 cell generations after irradiation. These delayed mutant clones exhibited higher levels of oxidative stress than normal cells. Therefore, persistently increased oxidative stress has been proposed as a mechanism for UV-induced genomic instability. This study investigates whether this mechanism is reflected in the deletion spectrum of delayed mutant clones. Eighty-eight percent of the delayed mutant clones derived from UV-A-irradiated populations were found to have total deletion of the hprt gene. Correspondingly, 81% of UV-A-induced early mutations (i.e. detected shortly after irradiation) also had total deletions. Among delayed UV-B-induced mutant clones, 23% had total deletions and 8% had deletion of one exon, whereas all early UV-B events were either point mutations or small deletions or insertions. In conclusion, the multiplex polymerase chain reaction deletion screen showed that there were explicit differences in the occurrence of large gene alterations between early and delayed mutations induced by UV-B radiation. For UV-A radiation the deletion spectra were similar for delayed and early mutations. UV-A radiation is, in contrast to UV-B radiation, only weakly absorbed by DNA and probably induces mutation almost solely via production of reactive oxygen species. Therefore, the present results support the hypothesis that persistent increase in oxidative stress is involved in the mechanism of UV-induced genomic instability.     

THE EFFECT OF PSORALENS AND ANGELICINS ON PROTEINS IN THE PRESENCE OF UV-A IRRADIATION

Abstract— The photobinding to proteins of furocoumarins with linear and angular structure (psoralens and angelicins) has been found to occur at relatively high fluences of UV-A irradiation (66.5 kJm²). The extent of photobinding between serum albumin and the investigated furocoumarins (psoralen, 8-methylpsoralen, 8-methoxypsoralen, angelicin and 4,5'-dimethylangelicin) varies largely with the furocoumarin structure and is correlated with the extent of photodegradation of the same furocoumarins when irradiated alone in aqueous solution. On the other hand, for each furocoumarin, the extent of photobinding varies considerably with different proteins.     

AN EVALUATION OF NALIDIXIC ACID FILM AS A UV-A RADIATION DOSIMETER

Nalidixic Acid (NA)* incorporated into a polyvinyl chloride film has recently been proposed as a UV-A radiation dosimeter. A trial, in which two different batches of NA film were exposed to both solar simulating and monochromatic 330 nm radiation is described. With both batches of film a linear relationship was observed between the UV-A fluence derived from the NA film and the fiuence determined with conventional electronic radiometric equipment. There was, however, considerable batch variation in response to UV-A radiation. Corrections may be made for batch differences. If the batch response can be standardized, our results would support the use of NA film as a simple routine method of measuring UV-A radiation.