EFFECTS OF UV-B IRRADIATION ON PHOTOMOVEMENT IN THE DESMID, Cosmarium cucumis

Abstract Monochromatic UV-B irradiation affects neither the absorption nor the fluorescence of the bulk pigments in the desmid Cosmarium cucumis but it impairs photomovement of these organisms at fluence rates which are not higher than the ambient level of solar UV-B irradiation. Photoaccumula-tions and phototaxis are strongly inhibited especially at wavelengths ≤ 300 nm while photodispersal at higher white light fluence rates is hardly affected by supplementary UV-B. This effect has important consequences for the growth and survival of populations in their natural environment: these photosyn-thetic organisms utilize photomovement to find and stay in areas of suitable visible light fluence rates. The UV-B component of solar irradiation both impairs the strategy of the organisms to find a suitable position and the escape mechanism by which the cells move out of areas with too strong white illuminances which photooxidize the bulk pigments and bleach the population within a few days.     

Biphasic fluence-response curves and derived action spectra for light-induced absorbance changes in Phycomyces mycelium

The photoresponses of Phycomyces, including phototropism and photocontrol of sporangiophore development, are mediated primarily by blue and UV light. Recent results on these two responses indicated a subsidiary role for green light. We have measured in vivo light-induced absorbance changes (LIAC) in mycelial samples of a caroteneless (carB) strain to compare the effectiveness of UV, blue, and green light. In the dual-wavelength kinetic mode of the spectrophotometer, measuring wavelengths of 445 and 470 nm were chosen, because green light produces substantial absorbance changes between these two wavelengths. Fluence-response curves were measured for 13 wavelengths between 365 and 530 nm, and for variable exposure times between 0.5 and 320 s. With one exception (365 nm), the curves were biphasic. The low fluence component was generally sigmoidal with an abrupt rise. The high fluence component failed to reach saturation for the fluences tested (less than 70 μmol m⁻² s⁻¹). Using the inferred threshold fluences of these two components as criterion effects, we obtained two action spectra. For the low fluence component, the action spectrum showed major peaks at 394, 450, and 530 nm and a minor peak at 416 nm. The high fluence component action spectrum showed very little sensitivity in the blue region. The major sensitivity was in the near UV, and a relatively small peak appeared in the green part of the spectrum at 507 nm. The biphasic character of the fluence-response curves suggests that two photosystems are responsible for the absorbance changes. The low fluence photosystem is sensitive mainly to blue and UV light and may thus represent a physiological blue-light photoreceptor. The high fluence photosystem is clearly not of this type. It (and perhaps the low fluence system as well) may mediate some of the subsidiary physiological effects of green light.     

Equal-quantum action spectra indicate fluence-rate-selective action of multiple photoreceptors for photomovement of the thermophilic cyanobacterium Synechococcus elongatus

Unicellular thermophilic cyanobacterium Synechococcus elongatus displayed phototaxis on agar plate at 55 degrees C. Equal-quantum action spectra for phototactic migration were determined at various fluence rates using the Okazaki Large Spectrograph as the light source. The shapes of the action spectra drastically changed depending on the fluence rate of the unilateral monochromatic irradiation: at a low fluence rate (3 mumol/m2/s), only lights in the red region had significant effect; at a medium fluence rate (10 mumol/m2/s), four major action peaks were observed at 530 nm (green), 570 nm (yellow), 640 nm (red) and 680 nm (red). At high fluence rates (30-90 mumol/m2/s), the former two peaks remained, while red peaks at 640 nm and 680 nm disappeared and, interestingly, an action peak around 700-740 nm (far-red) newly appeared. These results indicate that two or more distinct photoreceptors are involved in the phototaxis and that suitable photoreceptors are selectively active in response to the stimulus of light fluence rates. Far-red or red background lights irradiated vertically from above drastically inhibited phototaxis toward red light or far-red light, respectively. These results indicate involvement of some phytochrome(s).     

LIGHT-INDUCED CHEMOTACTIC REACTIONS IN THE DINOFLAGELLATE, Peridiniopsis berolinensis

Abstract The photosensitizer riboflavin induces accumulation of the colorless flagellate Peridiniopsis berolinensis in a light field at low photon fluence rates but dispersal from a light field at high photon fluence rates. The wavelength dependence of the accumulation resembles the absorption spectrum of riboflavin in the visible range. The photodynamic effects are caused by a positive or negative chemotactic response to photoproducts in the medium which are produced by photochemical reactions of the dye.     

FLUENCE-RATE DEPENDENCE OF MONOPHOTONIC REACTIONS OF NUCLEIC ACIDS IN VITRO AND IN VIVO

The Bunsen-Roscoe law, also known as the reciprocity law ( E = f(F) with F = I t ) has only limited validity for monophotonic reactions of nucleic acids. Especially at low fluence rates, the extent of in vitro and in vivo photoreactions of nucleic acids in the far-UV and near-UV range is a function of the fluence and of the fluence rate ( E = f (F;I)). In vitro experiments with poly(dA)poly(dT) clearly show that the far-UV (254 nm) response, indicated by the changes of the ellipticity at 315 nm, does not obey the Bunsen-Roscoe law at low fluence rates in the range between 1 W m^-2 and 20 W m^-2. In vivo experiments with Escherichia coli revealed very similar anomalies. Studying the growth delay after irradiation with far-UV light at 280 nm or near-UV light at 334 nm, we have confirmed the lack of reciprocity in both spectral ranges. The failure of the Bunsen-Roscoe law for the 280 nm and 334 nm UV irradiation effect at low fluence rates was in the range O < I < 40 W m^-2. In both cases reciprocity occurred at higher fluence rates (40 < I < 100 W m^-2).     

In Vivo Fluence Rate Effect in Photodynamic Therapy of Early Cancers with Tetra(m-hydroxyphenyl)chlorin

Abstract— Several parameters affect clinical trials in photodynamic therapy and influence the therapeutic outcome. Beside drug dose, light dose, drug-light interval and other variables, the fluence rate is a parameter that can influence the therapeutic results. In this study we have evaluated the fluence rate effect with a second-generation photosensitizer, tetra( m -hydroxyphenyl)chlorin (mTHPC) using a 7,12-dimethylbenz(a)anthracene induced early squamous cell carcinoma of the Syrian hamster cheek pouch as a tumor model. Following injection of 0.5 mg/kg of mTHPC, irradiation tests were performed at two drug-light intervals, 4 and 8 days. Wavelength and light dose were adapted from those applied routinely in clinical trials. Irradiations at 652 nm were carried out with fluences ranging from 8 to 20 J/cm² delivered at fluence rates of 15 and 150 mW/cm². Similar tests were also performed at 514 nm with a fluence of 80 J/cm² delivered at fluence rates ranging from 25 to 125 mW/cm². At both wavelengths and drug-light intervals for a given fluence, the higher fluence rates resulted in less tissue damage in tumor and healthy mucosae. However, the lower fluence rates yielded slightly less therapeutic selectivity. This study confirms that the fluence rate is of major importance in clinical PDT.     

MULTIPLICATIVE ACTION OF A UV-B PHOTORECEPTOR and PHYTOCHROME IN ANTHOCYANIN SYNTHESIS

Using 290-nm light, which excites only a UV-B photoreceptor, and 385- and 660-nm light, which activate only phytochrome, the fluence rate-response curves of monochromatic irradiations for anthocyanin synthesis in the first internodes of broom sorghum (Sorghum bicolor Moench, cv. Acme Broomcorn) were analyzed. Although the two photoreceptors absorbed light independently, they multiplicatively increased the action of each other. Accordingly, when the fluence rates of both wavelengths were changed together, the resulting slopes of the fluence rate-response curves of double-log plots were steep compared with the slopes obtained with the respective monochromatic irradiations. The slopes of fluence rate-response curves for monochromatic irradiations at 325 to 345 nm were steeper than those at other wavelengths. This difference was shown to be due to the multiplicative actions of both photoreceptors.     

LIGHT DOSIMETRY FOR PHOTODYNAMIC THERAPY BY WHOLE BLADDER WALL IRRADIATION

Abstract In Photodynamic Therapy (PDT) there is a need for accurate quantitative light dosimetry. This has become particularly apparent in the treatment of superficial bladder cancer, either by focal or by whole bladder wall irradiation. We have studied this problem using a spherical model of the bladder, consisting of two concentric thin-walled plastic spheres, 8 and 10 cm in diameter. The inner sphere was filled with water or with a light-scattering medium. The space between the spheres was filled with an optically tissue equivalent liquid. An isotropic light source was placed at the center of the spheres. Light energy fluence rates (light "dose rates") during PDT of the bladder simulated in this manner, were measured using a specially developed miniature light detector and were also calculated using a mathematical model. These data were confirmed by measurements in vivo (dog bladder). In the case of focal irradiation at a wavelength of 630 nm, the ratio (R) between the true light fluence rate at the bladder surface and the fluence rate due to the primary light beam is somewhat larger than 1, depending on the diameter of the primary beam. The maximum ratio is 2, for a beam diameter of several centimeters. In the case of whole bladder wall PDT, R is larger than 5. This is due to the strong scattering of (red) light by tissue and indicates that the intensity of the primary beam, which is usually reported, is not a good measure of the true fluence rate for whole bladder wall PDT. When the light source is moved away from the center of the spheres, the rate of change of the fluence rate at the bladder wall is more than a factor of 2 larger when the bladder cavity is filled with a light-scattering suspension, as compared with water. The use of a light-scattering medium may therefore not be advantageous to achieve a uniform light distribution across the bladder wall.     

LETHALITY IN REPAIR-PROFICIENT ESCHERICHIA COLI AFTER 365 nm ULTRAVIOLET LIGHT IRRADIATION IS DEPENDENT ON FLUENCE RATE

Abstract Reciprocity (total applied fluence produces the same response, regardless of the fiuence rate) for the lethal effects caused by 365 and 254 nm ultraviolet light (UV) was studied for repair-proficient and -deficient Escherichia coli strains. In the repair-proficient strain, E. coli WP2 uvrA * recA *, reciprocity after 365 nm UV was only observed at fluence rates of about 750 Wm^-2 and above. Below this rate, the cells became increasingly sensitive as the fluence rate was decreased. Similar lack of reciprocity was obtained whether the cells were exposed at 0 or 25°C. The double repair-defective mutant, E. coli WP100 uvrA recA , showed complete reciprocity after 365 nm UV over the same range of fluence rates measured for the repair-proficient strain. For 254 nm UV, complete reciprocity occurred in both strains over a range of fluence rates differing by an order of magnitude.     

Intraperitoneal Photodynamic Therapy: Comparison of Red and Green Light Distribution and Toxicity

Abstract— The aim of this study was to compare red (652 nm) and green (514 nm) light for photodynamic therapy (PDT) of the peritoneal cavity with emphasis on light distribution and toxicity. Red-light PDT was limited by intestinal toxicity and it was hypothesized that less penetrating green light would allow higher light doses to be used in the peritoneal cavity. Female non-tumor-bearing rats were photosensitized with mTHPC (meta-tetrahydroxyphenylchlorin, Foscan®) intravenously or intraperitoneally and the peritoneum was illuminated using a minimally invasive technique. For both red and green light, the time of illumination was varied to give the required dose. Light fluence rate was measured in situ at multiple sites within the abdominal cavity. The toxicity experiments were carried out with a total of 160 J incident red or 640 J incident green light and a drug dose of 0.15 mg/kg Foscan® For red light a mean fluence rate of 55.2 38.5 mW cm ₂ was measured, with a peak fluence rate of 128 mW cm ₂ on the intestines. For green light the mean and peak fluence rates were 8.2 9.0 (i.e. including zero fluence rate measurements) and 28 mW cm ₂, respectively. Intestines were most vulnerable to red light illumination. The intravenous injection route resulted in increased toxicity for red light, but for green light there were no major differences between intravenous and intraperitoneal routes. The 4 h interval between drug and illumination resulted in very little toxicity for both wavelengths. We conclude that for intraperitoneal PDT green light allows higher light doses than red light, but the light distribution over the peritoneum is much less favorable and may not be suitable for whole peritoneal illumination using a minimal-access technique.     

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.     

Topical photodynamic therapy at low fluence rates--theory and practice

Photodynamic Therapy (PDT), with topically applied 5-aminolaevulinic acid as the photosensitiser, is an effective treatment for various malignant and pre-malignant skin conditions. Several studies have shown the importance of fluence rate as well as fluence in the efficacy of PDT. We propose a measure of PDT efficacy, Photodynamic Damage Dose (PDD), which uses the product of instantaneous fluence rates, photosensitiser concentrations and oxygen concentrations in its calculation. We derive a qualitative numerical model of PDT and verify it by demonstrating an inverse fluence rate effect, increased efficacy of fractionated PDT, PDT induced hypoxia, and the dependence of photobleaching on fluence rate under certain circumstances. We recommend that fluence, fluence rate and any fractionation regime used should be detailed when reporting a trial as altering any of these has significant effects on PDT efficacy. The model predicts that low fluence rate irradiations should be as effective as high fluence rate irradiations if carried out over the same length of time. To test this we build a light emitting diode-based lamp (fluence rate of 7 mW cm(-2) at 635 nm) and used it to treat 32 superficial basal cell carcinomas on 22 patients (30 min treatment time, fluence 12.6 J cm(-2)). The complete response rate at one year was 84%, which is comparable to that achieved using higher fluence rate sources for similar treatment times. We conclude that this robust, inexpensive light source is effective for topical PDT.     

PHYTOCHROME MACRO-DISTRIBUTION, LOCAL PHOTOCONVERSION AND INTERNAL PHOTON FLUENCE RATE FOR Cucurbita pepo L. COTYLEDONS

Abstract— Using 7-day-oId cotyledons of Cucurbita pepo L., local phytochrome photoconversions could be measured for blue, red and far-red light. For this purpose, after nonsaturating irradiation, cotyledons were sliced into discs 0.3 to 0.5 mm thick and signals measured. This method also yielded the internal phytochrome distribution of the cotyledons with maximal concentration near the adaxial surface, dropping to about 50% in the center and reaching again about 90% at the abaxial surface. Local phytochrome conversion rates were used to calculate internal fluence rates across the cotyledons. Relative internal fluence rates were also derived from measured reflectances and transmittance according to the Kubelka-Munk theory. The general shape of the internal fluence distribution calculated on the basis of these two methods coincided well. It was observed that the internal local photoconversion is proportional to the penetration depth over a wide range of incident fluences and for all wavelengths tested, showing in addition that reciprocity holds. A method to calculate internal fluence rates by a simplified procedure assuming either linear or exponential functions is described.     

ARE THERE SEVERAL PHOTORECEPTORS INVOLVED IN PHOTOTROPISM OF Phycomyces blakesleeunus? KINETIC STUDIES OF DICHROMATIC IRRADIATION*

Abstract— Photogeotropic equilibrium angles were measured for Phycomyces blakesleeanus wild type firstly by means of dichromatic fluence rate response curves using simultaneous irradiation with near threshold 450 nm reference light (constant at 1.2 × 10^?8 W m^?2) and variable fluence rates of test light (498–630 nm) from the same side. These curves showed minima for test light fluence rates that were close to the photogeotropic threshold for these wavelengths. Secondly, the time course of this inhibitory effect was studied with both the inductive reference 450 nm light (2 × 10^?-⁷ W m^?2) and the test light (606 or 450 nm) given as light pulses of 2 s duration (2 s light/48 s dark periods for 6 h). The dark period between the onset of the inductive reference light and test light pulses was varied between 0 and 48 s. No inhibitory effects were observed for simultaneous pulses; however, inhibitory effects were demonstrated for delay times of 2 s and 20 s for 606 nm as well as 450 nm test light. If the test light pulses were given immediately before the inductive reference light, only 606 nm test light was effective in producing a significant inhibitory effect. The results are discussed with regard to a multichromophoric photoreceptor system and to the wavelength dependence of the effects observed. The data and conclusions favor a photoreceptor system with at least two separate chromophoric absorptions of the blue light receptor type, one acting positively, the other acting inhibitorily, and at least one other photoreceptor of presumably minor influence.     

Light Dosimetry for Intraperitoneal Photodynamic Therapy in a Murine Xenograft Model of Human Epithelial Ovarian Carcinoma

Few studies have been published to date measuring spatially resolved fluence rates in complex tissue geometries. Here the light distributions of three different intraperitoneal light delivery geometries in a murine ovarian cancer model were investigated to assess their influence on the tumorcidal efficacy of photodynamic therapy (PDT). In vivo fluence rate measurements in the peritoneal cavities of mice, with the light intensity being mapped in three transverse planes, were performed using fiber-optic detectors. Three different source fiber designs and placements were tested for their ability to provide uniform irradiation of the peritoneal cavity. The biological response to a PDT protocol comprising three separate treatments administered at 72 h intervals, each consisting of a 0.25 mg kg intraperitoneal injection of benzopor-phyrin derivative-mono acid ring A followed 90 min later by delivery of 15 J of 690 nm light, was measured. The tissue response was evaluated by measuring the number of remaining visible lesions and the total residual tumor mass. Fluence rate measurements showed large variations in the fluence rate distribution for similar intended treatments. The most uniform and reproducible illumination was achieved using two 18 mm long cylindrical emitting optical fibers. The biological response was comparable to that produced when a flat-cleaved end optical fiber is used to illuminate the four quadrants of the abdomen sequentially. While a good reproducibility in tumor induction in this animal model exists, no correlation was found between the fluence rate distribution measured in one group of animals and the biological response in a separate group of similarly treated animals. Due to the large intra-animal variability in fluence rate distribution, representative fluence rate mapping in complex tissue geometries is of limited value when applied to an individual PDT treatment. Thus, surveillance of the fluence rate during individual treatments will be required for acceptable PDT dosimetry. To improve the versatility of this particular animal model for PDT research, a large number of extended sources are required to increase uniformity of the illumination in order to reduce unwanted cytotoxic side effects resulting from foci of high fluence rates. In this way, subsequent increase of the total energy delivered to the tumor may be possible.     

Fluence Rate Determines PDT Efficiency in Breast Cancer Cells Displaying Different GSH Levels

Photodynamic therapy (PDT) appears as a promising alternative in the treatment of breast cancer since it can be highly effective in curing cancer while preserving normal tissue. However, predicting outcomes in PDT still constitutes a great challenge. One of the parameters that are usually empirically determined is the rate of photon flux delivered to the tissue (light fluence rate). In the present study, we intended to understand why monolayers of human cells derived from mammary adenocarcinomas (MDA-MB-231 and MCF-7) respond quite differently to fluence rates (cells were irradiated either for 6 or for 16 min) at a fixed light dose (4.5 J cm⁻²) delivered with an array of LEDs in a typical methylene blue PDT protocol. While death rates of MDA-MB-231 cells were insensitive to the fluence rate, MCF-7 cells showed a quite impressive (three times) decrease in cell death levels in the shorter irradiation protocol. Independent on cell type cell death was invariably correlated with the depletion of reduced glutathione intracellular levels and consequently with widespread redox misbalance. Our data show the potential to optimize fluence rates to provide exhaustion of the cell antioxidant responses in order to circumvent therapy resistance of breast tumors.     

Fractionated Illumination at Low Fluence Rate Photodynamic Therapy in Mice

Photodynamic therapy (PDT) for actinic field cancerization is effective but painful. Pain mechanisms remain unclear but fluence rate has been shown to be a critical factor. Lower fluence rates also utilize available oxygen more efficiently. We investigated PDT effect in normal SKH1-HR mice using low and high fluence rate aminolevulinic acid (ALA) PDT and a fractionated illumination scheme. Six groups of six mice with different light treatment parameters were studied. Visual skin damage was assessed up to 7 days post-PDT. Fluorescence and reflectance spectroscopy during illuminations provided us with real-time information about protoporphyrin IX (PpIX) photobleaching. A novel dosing approach was introduced in that we used a photobleaching percentage instead of a preset fluence. Data show similar total and maximum damage scores in high and low fluence rate groups. Photobleaching of PpIX in the low fluence rate groups shows a trend toward more efficient photobleaching. Results indicate that low fluence rate PDT is as effective as and more efficient than high fluence rate PDT in normal mouse skin. Low fluence rate PDT light protocols need to be explored in human studies in search for an effective and well-tolerated treatment for actinic field cancerization.     

Protoporphyrin IX fluorescence kinetics in UV-induced tumours and normal skin of hairless mice after topical application of 5-aminolevulinic acid methyl ester

Accumulation of protoporphyrin IX (PpIX) was investigated in normal skin and UV-induced tumours in hairless mice after topical application of a cream containing 2, 8 or 16% of 5-aminolevulinic acid methyl ester (ALA-Me). Higher levels of PpIX were measured in tumours compared to normal skin. The maximal amount of PpIX was reached at 1.5, 3 and 4 h after 2, 8 and 16% ALA-Me application, respectively. Higher tumour to normal skin PpIX fluorescence ratios were measured after application of 8 and 16% ALA-Me than after application of 2%. After irradiation with a broad spectrum of visible light from a slide projector, more than 90% of PpIX was bleached by fluences of 36 and 48 J/cm2, at fluence rates of 10 and 40 mW/cm2 respectively. At these fluences, the PpIX photobleaching rate was significantly higher (P<0.05) in normal mouse skin than in tumours. In addition, for a given fluence, more PpIX was photobleached at the lower fluence rate (10 mW/cm2) than at the higher fluence rate (40 mW/cm2) in normal skin (P<0.001) as well as in tumours (P<0.05) after exposure to 24 J/cm2 of light. In conclusion, the highest tumour to normal skin PpIX ratio was observed 3 h after application of 8% ALA-Me, suggesting that light exposure should be performed at this time in order to achieve an optimal PDT effect in this tumour model.     

Photodynamic effectiveness and vasoconstriction in hairless mouse skin after topical 5-aminolevulinic acid and single- or two-fold illumination

Several options were investigated to increase the efficacy of photodynamic therapy (PDT) using protoporphyrin IX (PpIX) induced by topically applied 5-aminolevulinic acid (ALA). Hairless mice with normal skin or UVB-light-induced skin changes were used as a model. In the first part of the study animals were illuminated immediately (t = 4) or 6 h (t = 10, PpIX fluorescence maximum) after the end of a 4 h ALA application. A total incident light fluence of 100 J/cm2 (514.5 nm) was delivered at a fluence rate of 100 or 50 mW/cm2. The PDT-induced damage to normal skin was more severe after treatment at t = 10 than at t = 4. Illumination at 50 mW/cm2 caused significantly more visible damage than the same light fluence given at 100 mW/cm2. For UVB-illuminated skin, different intervals or fluence rates made no significant difference in the severity of damage, although some qualitative differences occurred. In situ fluence rate measurements during PDT indicated vasoconstriction almost immediately after the start of the illumination. A fluorescein exclusion assay after PDT demonstrated vasoconstriction that was more pronounced in UVB-treated skin than in normal skin. The second part of the study examined the effect of two illuminations. The first illumination bleaches the PpIX fluorescence. At the start of the second illumination, new PpIX had been formed. Light of 514.5 nm was delivered at 100 mW/cm2 to a total incident light fluence of 200 J/cm2 at t = 4 (single illumination) or 100 J/cm2 at t = 4 plus 100 J/cm2 at t = 10. There was no visual difference in skin damage between 100 and 200 J/cm2 single illumination. Two-fold illumination (100 + 100 J/cm2) caused significantly more skin damage, indicating a potentially successful option for increasing the efficacy of topical ALA-PDT.