EFFECTS OF UV IRRADIATION ON A LIVING SKIN EQUIVALENT

Abstract— The Living Skin Equivalent (LSE™) is an organotypic coculture composed of human dermal fibroblasts interspersed in a collagen-containing matrix and overlaid with human keratinocytes forming a stratified epidermis. The LSE has a dry, air-exposed epidermal surface suitable for the application of oils, creams and emulsions. These features suggested its feasibility as an in vitro skin model for studying the protective effects of sunscreens. Using the thiazolyl blue (MTT) conversion assay as a measure of mitochondrial function, the extent of cytotoxicity induced by various doses of UV-R (280–400 nm) or UV-A (320–400 nm) was evaluated in the LSE. The doses of UV radiation that caused 50% reductions in MTT conversion (UV-R₅₀ or UV-A₅₀) in different lots of LSE were 0.053 ± 0.021 J/cm² (n = 29) and 11.6 ± 4.9 J/cm² (n = 17) for UV-R and UV-A, respectively. The protective effects of an 8% homosylate standard and of five UV-A sunscreens, topically applied to the LSE, were determined and compared with their reported protection factors in human skin. Morphological changes and the release of proinflammatory mediators (interleukin-1-α, tumor necrosis factor-α and prostaglandin E2) implicated in UV-induced erythema were also demonstrated in the LSE exposed to UV-A or UV-B. The data suggest that the LSE can be used for studying the effects of U V radiation on skin and may have utility for assessing the efficacy of certain sunscreens against UV-B and UV-A.     

Photomodulation of Oxidative Metabolism and Electron Chain Enzymes in Rat Liver Mitochondria

Abstract— Low-level laser irradiation has been applied in a variety of laboratory studies and clinical trials for photobiostimulation over the last three decades. Considerable skepticism exists regarding the concept of photostimulation within the medical community. One of the major difficulties with photoirradiation research is that it lacks experimentally supportable mechanisms for the alleged photobiostimulatory effects. This study was undertaken to determine whether oxidative metabolism and electron chain enzymes in rat liver mitochondria can be modulated by photoirradiation. Oxygen consumption, phosphate potential, and energy charge of rat liver mitochondria were determined following photoirradiation. Activities of mitochondrial enzymes were analyzed to assess the specific enzymes that are directly involved with the photostimulatory process. An argon-dye laser at a wavelength of 660 nm and at a power density of 10 mW/cm² was used as a photon source. Photoirradiation significantly increased oxygen consumption (0.6 J/cm² and 1.2 J/cm², P < 0.05), phosphate potential, and the energy charge (1.8 J/cm² and 2.4 J/cm², P < 0.05) of rat liver mitochondria and enhanced the activities of NADH: ubiquinone oxidoreductase, ubiquinol: ferricytochrome C oxidoreductase and ferrocytochrome C: oxygen oxidoreductase (0.6 J/cm², 1.2 J/cm², 2.4 J/cm² and 4.8 J/cm², P < 0.05). The activities of succinate ubiquinone oxidoreductase, ATPase, and lactate dehydrogenase were not affected by photoirradiation.     

Lutetium Texaphyrin (PCI-0123): A Near-Infrared, Water-Soluble Photosensitizer

Lutetium texaphyrin, PCI-0123, is a pure, water-soluble photosensitizer with a large broad absorption band centered at 732 nm. The compound was tested for photodynamic therapy (PDT) effectiveness in a murine mammary cancer model. The texaphyrin macrocycle as illustrated by magnetic resonance imaging and ¹⁴C-radiolabeled texaphyrin studies was shown to be tumor selective; a tumor-to-muscle ratio of 10.55 was seen after 5 h. Lutetium texaphyrin, at a drug dose of 20 μmol/kg with irradiation 5 h postinjection at 150 J/cm² and 150 mW/cm², had significant efficacy (P < 0.0001) in treating neoplasms of moderate size (40 ± 14 mm³) and also had significant efficacy ( P < 0.0001) in treating larger neoplasms (147 ± 65 mm³). The PDT efficacy was correlated with the time interval between PCI-0123 administration and light exposure. A 100% cure rate was achieved when photoirradiation took place 3 h postinjection compared to 50% for 5 h using 10 μmol/kg and 150 J/cm² at 150 mW/cm². The PDT efficacy was attributable to the selective uptakehetention of the texaphyrin photosensitizer in addition to the depth of light penetration achievable at the 732 nm laser irradiation.     

COMPARATIVE EFFECT OF UVA AND UVB ON CULTURED RABBIT LENS

Abstract Effects on lens physiology of UVB and UVA used separately and sequentially were investigated using 4 week old rabbit lenses in organ culture. Narrowband UVB at 0.3 J/cm²= joules/lens (1 h exposure) has little effect on sodium and calcium concentrations in the lens interior or transparency of lenses subsequently cultured for 20 h after a 1 h exposure. With an incident energy of 3 J/cm² of broadband UVB (295–330 nm), lenses become opaque and slightly swollen with significant ion imbalances during culture over a 1 day period. In contrast, lenses exposed to approximately 6–24 J/cm² of UVA (330–400 nm) remain transparent after 1 day of culture. Extended culture up to 4 days reveals no signs of opacification. Ion homeostasis and normal lens hydration are also maintained in UVA-irradiated lenses. The presence of 95% oxygen during UVA irradiation is also without effect. Broadband UVA irradiation is damaging, however, if lenses are first exposed to subthreshold doses of narrowband UVB (307 ± 5 nm) irradiation, viz . 0.3 J/cm². Thus, sequential UVB/UVA irradiation at subthreshold doses causes impaired active cation transport and accumulation of sodium and calcium accompanying lens opacification.     

THE EFFECT OF LASER IRRADIATION ON THE RELEASE OF bFGF FROM 3T3 FIBROBLASTS

Studies have shown that low-level laser irradiation increases the proliferation of fibroblasts in cell culture. The mechanism of action is unknown. Basic fibroblast growth factor (bFGF) is a multifunctional polypeptide that has been detected in most tissues and which supports cell proliferation and differentiation. The purpose of this study was to determine whether laser irradiation (660 nm) can stimulate production of bFGF from fibroblast cells in cell culture. Our study showed that fibroblasts irradiated with laser energy at 2.16 J/cm² demonstrated increased cell proliferation and enhanced production of bFGF, whereas fibroblasts irradiated with laser energy at 3.24 J/cm² neither demonstrated increased cell proliferation or an enhanced release of bFGF as compared to the control group. These results provide direct evidence that the proliferation of fibroblasts as a result of stimulation by low level laser irradiation may be associated with the autocrine production of bFGF from fibroblasts.     

DETECTION OF DNA-PSORALEN PHOTOADDUCTS in situ

Abstract— An immunological method, with the use of specific immune serum, has been developed for detection of 8-methoxypsoralen (8-MOP) photoadducts to DNA, formed in situ in cell nuclei, after combined treatment with 8MOP and UV-A irradiation (Zarçbska et al. , 1978). Lymphocytes fixed on slides or in suspension, and cryostat sections of different mammalian tissues, served as antigenic substrate, after treatment with 8-MOP and UV-A in vitro. Specific fluorescence in these substrates was detected in the nuclei after treatment with 30 ˜ 140 kJ/m² UV-A in the presence of 0.1-0.3 μg/cm² 8-MOP. PHA-stimulated-lymphocytes appeared to be the most sensitive substrate.
However, hairless mice treated with high doses of UV-A in vivo , 70 ˜ 360 kJ/m² did not reveal a specific fluorescence of epidermal nuclei, unless a high local concentration of 8-MOP was attained.
The apparent discrepancy in the level of photoadduct detection between the in vitro and in vivo treated specimens was explained by the low number of DNA-8-MOP-photoadducts formed in vivo under these experimental conditions. The relevance of these findings to the role of DNA-8-MOP-photoadducts formed during PUVA photochemotherapy is discussed.     

UNDERSTANDING PHOTOSYNTHESIS, PIGMENT AND GROWTH RESPONSES INDUCED BY UV-B AND UV-A IRRADIANCES

Abstract—Plant response to UV-B (0.290–0.320 μm) irradiation in controlled environments has been difficult to assess, possibly because plants also respond to UV-A (0.320–0.400 μm) and visible radiation. Photosynthetic dysfunction is often reported, but effects on photosynthetic pigments have been equivocal. Because UV-A/blue radiation is involved in pigment synthesis, the experimental UV-A irradiation was controlled and this study was conducted under high ambient photosynthetic photon flux (mid-day PPF > 1400 pmol m ^–2 s^–1). Two biologically effective UV-B irradiances (10.7 and 14.1 kJ m^-2 day^-I) were utilized and the UV-A irradiances were matched in controls (˜5 and 9 kJ m^-2 day^-1). Normal and two mutant pigment isolines (chlorophyll-deficient, flavonoid-deficient) of soybean cultivar Clark were utilized for comparisons. Many pigmedgrowth variables exhibited a statistical interaction between spectral quality and quantity. UV-A/blue photoregulation was demonstrated in the UV-A controls. The pigmentlgrowth pattern observed at the lower UV-B irradiance was interpreted as a photosystem II response similar to shade adaptation, suggesting phytochrome involvement in UV-B irradiation responses. On the other hand, two variables most commonly observed to manifest UV-B-induced effects—decreased photosynthesis and increased leaf flavonoid content—exhibited no interactions due to UV exposure or spectral quality. In general, the observed response patterns indicated either moderation of UV-B-induced responses by UV-A/blue radiation, or coaction between them, and provides an explanation for the common failure to demonstrate fluence-related responses in UV-B experiments.     

Effect of Photosensitizer Delivery System and Irradiation Parameters on the Efficiency of Photodynamic Therapy of B16 Pigmented Melanoma in Mice

Abstract— Previous studies (Biolo et al., Photochem. Photobiol. 59, 362-365, 1994) showed that liposome-delivered Si(IV)-na-phthalocyanine (SiNc) photosensitizes B16 pigmented melanoma subcutaneously transplanted in C57 mice to the action of 776 nm light. However, the efficacy of the phototreatment was limited by a lack of selectivity of tumor targeting by SiNc as well as by incomplete necrosis of the neoplastic mass. The present investigations show that the use of a different delivery system (Cremophor emulsion vs liposomes of dipalmitoylphosphatidylcholine) causes no significant increase in the selectivity of tumor targeting for three injected doses of SiNc (0.5, 1, 2 mg/kg). However, upon 776 nm light irradiation (300 mW/cm²; 520 J/cm²), the delay in the rate of tumor growth was maximal (7-8 days) for the highest naphthalocyanine dose. On the other hand, a remarkable improvement in the tumor response was obtained by inducing an intratumoral temperature increase to 44°C immediately after PDT. The thermal effect appeared to be due to photoexcitation of melanin by 776 nm light (550 mW/cm²; 520 J/cm²) and subsequent partial conversion of absorbed energy into heat.     

Comparison of changes in metal toxicity following exposure of water with high dissolved organic carbon content to solar, UV-B and UV-A radiation

This study examines the effects of natural solar radiation on the metal-binding capacity of dissolved organic matter (DOM). Newington Bog water (35.5 mg L⁻¹ dissolved organic carbon [DOC]) was irradiated for 20 days under UV-B lamps in the laboratory and under natural solar radiation. In the presence of irradiated DOM, IC₅₀ (contaminant concentration required to reduce algal growth by 50%) was significantly decreased with UV-B treatment for four metals: Pb, 64%; Cu, 63%; Ni, 35% and Cd, 40%. Solar radiation also significantly decreased IC₅₀ of Pb (58%) and Cu (49%), DOC concentration (11%), DOM fluorescence (DOMFL, 33%) and DOC-specific UV absorbance. Further experiments on Raisin River water (20.7 mg DOC L⁻¹) exposed to 20 days of artificial UVA and UV-B radiation produced significant decreases in IC₅₀ for Cu (48%) with UV-A and for Pb (43%) with UV-B. DOC concentration was decreased 20% by UV-B and 24% by UV-A. DOMFL decreased 51.5% in the first 5 days of UV-A exposure, an effect that was not observed with the UV-B treatment. The UV-A treatment decreased UV absorbance more at longer wavelengths and over a broader wavelength band than did the UV-B treatment. Change in toxicity with UV irradiation was inconsistent among the metals tested in this study, indicating that some organic metal-binding ligands were more quickly removed or altered than others. The DOM remaining after irradiation appears to be qualitatively different from the unirradiated DOM. The much greater irradiance of UV-A makes its contribution to the removal and/or alteration of DOM at least as important as the influence of higher energy UV-B.     

EXCISION REPAIR OF PYRIMIDINE DIMERS IN MARSUPIAL CELLS

Monodelphis domestica was further characterized as a model for photobiological studies by measuring the excision repair capabilities of this mammal's cells both in vivo and in vitro. Excision repair capability of the established marsupial cell line, Pt K2 ( Potorous tridactylus ), was also determined. In animals held in the dark, we observed that ˜50% of the dimers were removed by 12 and 15 h after irradiation with 400 J m⁻² and 600 J m⁻², respectively, from an FS-40 sunlamp (280–400 nm). Cells from primary cultures of M. domestica excised ˜50% of the dimers by 24 h after irradiating with 50 J m⁻² and 36 h after exposure to 100 J m⁻² with no loss of dimers observed 24 h following a fluence of 300 J m⁻². Pt K2 cells were observed to have removed -50% of the dimers at -12 h after 50 J m⁻² with only -10% of the dimers removed at 24 h following 300 J m⁻². The observed loss of pyrimidine dimers from epidermal DNA of UV-irradiated animals and from fibroblasts in culture, held in the dark, suggests that these marsupial cells are capable of DNA excision repair.     

THYMIDINE UPTAKE, INCORPORATION AND DNA POLYMERASE ACTIVITY IN MURINE BLADDER TUMOR CELL,MBT–2, EXPOSED TO UV ACTIVATED DIHEMATOPORPHYRIN ETHER

Abstract— Photodynamic therapy (PDT) is a new modality for treatment of malignancy. In this paper, we reported the effect of UV activated dihematoporphyrin ether (DHE) on [³H] thymidine uptake and DNA synthesis in murine bladder tumor cells,MBT–2. Exponentially growing cells were pretreated with 0.05–5 μg/ml of DHE for 30 min in complete darkness prior to irradiation with 0.15-0.90 J/cm² of UV light (265 nm). The rates of thymidine uptake and DNA synthesis were suppressed in a DHE concentration and photic energy dependent manner. Double reciprocal analysis on the kinetics of the thymidine uptake and DNA synthesis indicated that the inhibition was non-competitive, i.e. decrease in both the apparent K^m value and maximum velocity in DHE plus UV light treated cells. The activities of DNA polymerase a and (3 were determined by [*H] dATP incorporation into DNA of permeabilizedMBT–2 cells. DNA polymerase a activity was approximately 60% of the control after 0.45 J/cm² of UV light exposure; a further inhibition of DNA polymerase a was observed when 0.5–5ng/W of DHE and UV photoradiation were combined. In contrast, a slight stimulation of DNA polymerase fJ was noted after a similar treatment. This study demonstrates that photodynamic therapy-induced suppression of DNA synthesis inMBT–2 cells is a complex process involving in reduction of thymidine transport as well as the perturbation of the enzymes involved in DNA synthesis.     

NORMAL BRAIN TISSUE RESPONSE TO PHOTODYNAMIC THERAPY USING ALUMINUM PHTHALOCYANINE TETRASULFONATE IN THE RAT

Abstract— The effects of photodynamic therapy (PDT) on normal brain tissue and depth of brain necrosis were evaluated in rats receiving 2.5 mg/kg aluminum phthalocyanine tetrasulfonate. Twenty-four hours later brains were irradiated with 675 nm light at a power density of 50 mW/cm² and energy doses ranging from 1.6 to 121.5 J/cm². Brains were removed 24 h after PDT and evaluated microscopically. When present, brain lesions consisted of well-demarcated areas of coagulation necrosis. When plotting the depth of necrosis against the natural log of energy dose, the data fit a piecewise linear model, with a changepoint at 54.6 J/cm² and an x intercept of 7.85 J/cm². The slopes before and after the changepoint were 2.04 and 0.21 mm/In J cm^-2, respectively. The x intercept suggests a minimum light dose below which necrosis of normal brain will not occur, whereas the changepoint indicates the energy density corresponding to an approximate maximum depth of necrosis.     

Photodynamic Therapy of Human Glioma (U87) in the Nude Rat

Abstract— We measured the response of normal brain and the human U87 glioma implanted in the brain of rats (n = 65) to photodynamic therapy (PDT) using Photofrin as the sensitizer. Normal brain and U87 tumor implanted within brain of athymic (nude) rats were subjected to PDT (12.5 mg/kg of Photofrin) at increasing optical energy doses (35 J/cm², 140 J/cm², 280 J/cm²) of 632 nm light. Photofrin concentration in tumor, brain adjacent to tumor and normal brain were measured in a separate population of rats. Twenty-four hours after PDT, the brains were removed, sectioned, stained with hematoxylin and eosin (H&E), and the volumes of the PDT-induced lesion measured. Photofrin concentration in tumor greatly exceeded that of normal brain and brain adjacent to tumor (>20×). Both normal brain and U87 tumor exhibited superficial tissue damage with PDT at 35 J/cm². However, both normal and tumor-implanted brain exhibited tissue damage with increasing optical dose. A heterogeneous pattern of pannecrosis along with a uniform volume of pannecrosis was detected in the tumor. In contrast, normal brain exhibited a uniform sharply demarcated volume of necrosis. Our data indicate that the U87 human brain tumor model and the normal brain in the athymic rat are sensitive to PDT and Photofrin with an optical dose-dependent response to treatment.     

Measurements of the Protective Effect of Topically Applied Sunscreens using in vitro Three-dimensional Dermal and Skin Equivalents

Abstract— For preventing or minimizing acute and chronic skin damage caused by UV radiation, the use of sunscreens is probably the most important measure. To screen the protective efficacy of new sunscreen molecules or formulations against UV rays, we evaluated as in vitro testing methods the use of two three-dimensional models, a dermal equivalent (DE) and a skin equivalent (SE). The DE is composed of a porous collagen-glycosaminoglycans-chitosan matrix populated by normal human fibroblasts. The SE is comprised of a fully differentiated epidermis realized by seeding keratinocytes onto the DE. In this study, we demonstrated that the DE and SE models react to the deleterious effects of UVA and UVB. Then, we extended our research to the evaluation of their usefulness for photoprotection trials. Sunscreen agents (Euso-lex 8020 and 6300) and commercially available sunscreens (chemical and physical filter formulations) that protect the skin against either UVA or UVB were evaluated. The tested products were applied (n = 6) topically (10 μL) and incubated for 30 min prior to irradiation over a range of UVA (0-50 J/cm²) or UVB (0-5 J/cm²). The photoprotection provided by the tested sunscreen molecules and formulations was evaluated by measurement of residual cellular viability 24 h postirradiation using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT) test and assessment of the inflammation response by interleukin-la release assay. When sunscreens were applied prior to UV exposure, a higher residual cellular viability versus control was obtained, demonstrating the photoprotective effects of the tested products. These in vitro models could be used for screening tests to evaluate the protective effects of sunscreen molecules and formulations, especially for UVA trials because there is a lack of consensus for an in vivo method.     

In vitro GRANULOMA FORMATION BY SPLEEN CELLS TREATED WITH PSORALEN PLUS LONG-WAVE ULTRAVIOLET RADIATION

Abstract— In vitro gransulomas ar induced by culturing murine spleen cells with artificial microparticles, dextran beads. In the presence of 0.5 μg/mL 8-methoxypsorlen, UVA radiation (0.2-2.0 J/cm² suppressed granuloma formatin in a UVA dose- dependent manner. The dose of PUVA did not affect the cell viability as assessed by trypan blue exclusion. The time course of granuloma formation in 0.5 J/cm² PUVA-treated cells was similar to that of normal spleen cells, with a miximum granuloma index at day 3 of culture, althought a49–63% suppression of granuloma formation was observed in PUVA-treated, adherent cells even when they were cultured with normal nonadherent cells. These data suggests that PUVA alters macrophages, resulting in the suppression of granuloma formation in vitro     

LONG-WAVE ULTRAVIOLET RADIATION INDUCES PROTEIN KINASE C IN NORMAL HUMAN KERATINOCYTES

Abstract Skin tumor promotion by phorbol ester is believed to be mediated by the phospholipid-dependent ser/ thr kinase, protein kinase C (PKC). Long-wave ultraviolet radiation (320-400 nm, UVA), which has also been shown to promote skin tumors, induces elevated levels of PKC in murine fibroblasts, suggesting that UVA may promote the development of basal and squamous cell skin cancers by a mechanism involving PKC. To examine UVA effects on PKC in a model relevant to skin, we maintained normal human epidermal keratinocytes (NHEK) in serum-free medium and exposed the cultured cells to various doses of UVA or to the phorbol ester, 12- O -tetradecanoylphorbol-13-acetate (TPA). Fifty minutes after exposure to UVA (5-20 J/cm²), PKC activity was elevated up to three-fold in NHEK cytosolic fractions, and membrane-associated PKC activity was elevated up to two-fold by UVA. The TPA treatment induced a 10-fold increase in membrane-associate PKC activity only. Immunoblot analysis suggested that a UVA-induced increase in PKC protein occurred. Both UVA and TPA reduced the cell number by 50-75% in the first 24-48 h; however, irradiated cultures began to recover at 72 h post-UVA due to an increased proliferative rate beginning after 48 h. Treatment with TPA induced a high level of differentiation as measured by cornified envelope formation. Ultraviolet A irradiation exposure was not followed by increased differentiation. These findings suggest that acute UVA exposure elevates PKC activity in human keratinocytes and may act through PKC to promote actinic skin cancer. The molecular mechanism is like to differ from that of the phorbol esters, however.     

INVESTIGATIONS ON BIOLOGICAL EFFECT OF POLARIZED LIGHT

Abstract— The biological effects of single and 4-time irradiation of primary human embryo fibroblasts with 4 J/cm² polarized light emitted by a halogen light source were investigated. The functional state of the plasma membrane was examined by means of lectin-binding and polycationized ferritin-binding techniques. It was established that the Con A binding of the cells did not change, whereas the number of negatively charged binding sites increased to a significant degree in relation to the untreated (control) samples and cell cultures exposed to diffuse (non-polarized) light. The micromorphological examinations showed no ultrastructural deviations. The quantitative increase of negative surface charges may be regarded as an indication of the biological effect of polarized light exerted on the cell membrane. The modifying effect of polarized light on the survival of E. coli exposed to the ionizing radiation was manifested in decreased anoxic radiation response.     

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.     

PHOTOCHEMICAL ALTERATIONS IN DNA REVEALED BY DNA-BASED LIQUID CRYSTALS

Abstract The preparations of chicken erythrocyte linear double-stranded DNA and superhelical plasmid pBR322 DNA were irradiated by continuous low-intensity UV radiation (I = 25-50 W/m², λ= 254 nm) as well as by highintensity picosecond laser UV radiation (I = 10¹¹-10¹³ W/m², λ= 266 nm). The effect of DNA secondary structure alterations on the formation of liquid-crystalline dispersions from UV-irradiated DNA preparations was studied. It was shown that in the case of linear DNA, watching the disappearance of abnormal optical activity characteristic for cholesteric liquid crystal we managed to detect the presence of photochemical alterations in DNA irradiated by low-intensity UV radiation at an absorbed energy of more than 20 quanta per nucleotide. In the case of superhelical DNA using enzyme treatment of liquid-crystalline dispersions and monitoring the appearance of abnormal optical activity, we detected the presence of photochemical alterations in DNA molecules after low-intensity UV irradiation at an absorbed energy of less than 4 quanta per nucleotide. Under the latter approach using picosecond UV laser irradiation at three different light intensities we were able to distinguish the different mechanisms of fine alterations in DNA secondary structure at an absorbed energy value of about 3 quanta per nucleotide.     

INACTIVATION OF Trypanosoma cruzi TRYPOMASTIGOTE FORMS IN BLOOD COMPONENTS BY PHOTODYNAMIC TREATMENT WITH PHTHALOCYANINES

Abstract— -Three phthalocyanine dyes HOSiPcOSi(CH₃)₂(CH₂)₃N(CH₃)₂ (Pc 4), HOSiPc-OSi(CH₃)₂(CH₂)₃N+(CH₃)3I- (Pc 5) and aluminum tetrasulfophthalocyanine hydroxide (AlOHPcS₄) were evaluated for their ability to inactivate the trypomastigote form of Trypanosoma cruzi in fresh frozen plasma (FFP) and red blood cell concentrates (RBCC). The compound Pc 4 was found to be highly effective in killing T. cruzi, Pc 5 less effective and AlOHPcS₄ ineffective. With FFP as the medium, a complete loss of parasite infectivity in vitro (≥5 log₁₀) was found to occur with 2 μ M Pc 4 after irradiation with red light (>600 nm) at a fiuence of 7.5 J/cm², while with RBCC as the medium, a complete loss was found to occur at a fiuence of 15 J/cm². Even without illumination, Pc 4 at 2 μ M also killed about 3.7-4.1 log₁₀ of T. cruzi in FFP during 30 min. Observed differences in T. cruzi killing by the various phthalocyanines may relate to differences in binding; Pc 4 binds to the parasites about twice as much as Pc 5. Ultrastructural analysis of treated parasites suggests that mitochondria are a primary target of this photodynamic treatment. The data indicate that Pc 4 combined with exposure to red light could be used to eliminate bloodborne T. cruzi parasites from blood components intended for transfusion. The inactivation of T. cruzi by Pc 4 in the dark suggests a possible therapeutic application.