PRIMARY DNA DAMAGE, HPRT MUTATION AND CELL INACTIVATION PHOTOINDUCED WITH VARIOUS SENSITIZERS IN V79 CELLS

DNA strand breaks and hypoxanthine guanine phosphoribosyl transferase (HPRT) mutants were measured in parallel in photochemically treated (PCT) cells and compared at the same level of cell survival. Chinese hamster fibroblasts (V79 cells) were either incubated with the lipophilic dyes tetra(3—hydroxyphenyl)porphyrin (3THPP) and Photofrin II (PII), the anionic dye meso -tetra(4—sulfonatophenyl)porphine (TPPS₄) or the cationic dye meso -tetra( N -methyl-4-pyridyl)porphine ( p -TMPyPH₂ before light exposure. In the cells, the lipophilic dyes were localized in membranes, including the nuclear membrane, while the hydrophilic dyes were taken up primarily into spots in the cytoplasm. In addition, the hydrophilic TPPS₄ was distributed homogeneously throughout the whole cytoplasm and nucleoplasm. According to the HPRT mutation test, the mutagenicity of light doses survived by 10% of the cells was a factor of six higher in the presence of 3THPP than of PII, whereas for X-rays it was a factor of three higher than for PCT with 3THPP. Light exposure in the presence of the hydrophilic dyes TPPS₄ and p -TMPyPH₂ was not significantly mutagenic. There was no correlation between the induced rates of HPRT mutants and of DNA strand breaks. Thus, TPPS₄ was the most efficient sensitizer with regard to DNA strand breaks when compared at the same level of cell survival, followed by 3THPP, PII and p -TMPyPH₂. Hence, the rate of DNA strand breaks cannot be used to predict the mutagenicity of PCT.     

PHOTOSENSITIZING EFFICIENCIES, TUMOR- and CELLULAR UPTAKE OF DIFFERENT PHOTOSENSITIZING DRUGS RELEVANT FOR PHOTODYNAMIC THERAPY OF CANCER

Abstract Several parameters of the following dyes, all relevant as sensitizers for photochemotherapy of cancer, have been studied: Photofrin II (PII), hematoporphyrin (HP)-di-hexyl-ether, HP-di-ethyl-ether, tetra (3-hydroxyphenyl) porphyrin, (3THPP), tetraphenyl porphine tetrasulphonate (TPPS₄) aluminium phthalocyanine tetrasulfonate (A1PCTS), aluminium phthalocyanine (A1PC), chlorin e, (Chi e₆) and merocyanine 540 (MC 540). The following parameters and features of these dyes were studied: (1) Tumor uptake in C3H mouse mammary carcinomas. (2) Skin/tumor concentration ratio in the same animal system. (3) Triton X-114/H₂0 partition coefficients at different pH-values. (4) Uptake of the dyes by human cells of the line NHIK 3025. (5) Relative fluorescence quantum yields of the dyes bound to cells. (6) Absorption-, fluorescence-excitation- and fluorescence-emission spectra of the cell-bound dyes. (7) Relative quantum yields for photoinactivation of cells after 18 h incubation with the dyes. (8) Relative quantum yields of photodegradation of the singlet oxygen trap 1,3-diphenylisobenzofuran (DPBF) in cells after 18 h incubation with the dyes. The following main conclusions were drawn: (1) 3THPP was the best and most selective tumor localizer of the dyes tested, followed by AIPCTS, TPPS₄, PII and Chi e.,. (2) The Triton X-114/H₂0 partition coefficient of most of the dyes decreased with increasing pH. (3) The cellular uptake of the dyes (18 h incubation in medium with 3% serum) increased with increasing Triton X-114/H₂0 partition coefficient. (4) HP-di-hexyl-ether had the highest quantum yields both for photoinactivation of cells and degradation of cell-bound DPBF, followed by the other lipophilic porphyrins and Chi e₆. The water-soluble dyes TPPS₄ and AIPCTS had quantum yields of the order of ten times lower than those of the lipophilic porphyrins. (5) There was a clear correlation between the quantum yields for cell-inactivation and those for photodegradation of DPBF, suggesting that the same reactive photoinduced species is involved in both processes. This suggestion was strengthened by the observation that DPBF reduced the quantum yield of cell inactivation. Thus, all the tested dyes seem to act via a type II process. (6) All of the dyes, even the water-soluble TPPS₄ and AIPCTS, are aggregated in aqueous solutions, and the cells bind both monomers and aggregates. (7) A significant fraction of the cell-bound dyes was located close to tryptophan-containing proteins. (8) Cell-bound Chi e,₆ had the highest fluorescence quantum yield of the dyes.     

CELLULAR DELIVERY AND RETENTION OF PHOTOFRIN: III. ROLE OF PLASMA PROTEINS IN PHOTOSENSITIZER CLEARANCE FROM CELLS

Abstract— Human plasma proteins, albumin, globulins and low density (LDL), high density (HDL) and very low density (VLDL) lipoproteins were tested for their effects on retention of Photofrin and three other photosensitizers in cultured cells. This was assessed by incubating the cells, subsequent to the exposure to Photofrin, in the photo-sensitizer-free medium containing various concentrations of different plasma proteins. Photofrin clearance levels differed with individual plasma proteins and also were dependent on concentration of these proteins in the incubation medium. All of the proteins except VLDL promoted clearance of Photofrin taken up by the cells in the presence of 5% human serum. Subsequent to some Photofrin exposure conditions (in the presence of 5% fetal bovine serum, or in protein-free medium), albumin, in contrast to LDL, HDL and globulins, exhibited decreased capacity for promoting the photosensitizer clearance from the cells. The VLDL showed very little or no effect in promoting cellular clearance of Photofrin, tetraphenyl porphine tetrasulfonate (TPPS₄), and di- and tetrasulfonated chloroaluminum phthalocy-anine (AlPcS₂ and AlPcS₄, respectively). The LDL seem to be particularly effective in promoting clearance of Photofrin and AlPcS₂ from the cells, whereas albumin and globulins were shown to be more effective than LDL and HDL in promoting the cellular clearance of TPPS₄.     

SINGLET OXYGEN GENERATION BY PORPHYRINS AND THE KINETICS OF 9,10-DIMETHYLANTHRACENE PHOTOSENSITIZATION IN LIPOSOMES

Abstract— Two new sensitizers are introduced for a potential use in photodynamic therapy: Zn²⁺- and MG²⁺-tetrabenzoporphyrin (ZnTBP and MgTBP). A comparative study of the quantum yields of singlet oxygen generation (Φ_Δ) of hematoporphyrin derivative (HpD), Photofrin II (PF-II), Zn²⁺-phthalocyanine tetrahydroxyl [ZnPC(OH)₄] and the newly introduced sensitizers ZnTBP and MgTBP in liposomes, as well as the kinetics of a photochemical reaction sensitized by them, was made by employing the fluorescent membrane probe 9,10-dimethylanthracene (DMA). We followed the photosensitization of DMA in real time by monitoring its fluorescence decrease at 457 nm and found that DMA's photosensitization is oxygen mediated. The kinetic traces of the photosensitization reactions were fitted to an analytical function, and the Φ_Δ values were evaluated. At 10 μ M sensitizer in an aqueous suspension of 2 mg/mL egg phosphatidylcholine (EPC), HpD was found to have the largest value of Φ_Δ (0.215), followed by PF-II (0.191), ZnTBP (0.023), MgTBP (0.019) and ZnPC(OH)₄ (0.005). As a test of the method, Φ_Δ for methylene blue in ethanol was measured and found to be 0.45 as compared to 0.52 reported in the literature. Due to difference in the sensitizers' absorbances at the laser's wavelength, the reaction photosensitized by ZnTBP was the fastest with a time constant of 6.7 min, followed by MgTBP (8.7), PF-II (11.9), HpD (17.1) and ZnPC(OH)₄ (31.2), all at equal sensitizers' concentrations and laser intensities. The binding constants of the sensitizers to EPC liposomes are also reported.     

THE EFFECT OF DIFFERENTIATION ON PHOTOSENSITIZER UPTAKE BY HL60 CELLS

The capability of human promyelocytic leukemia cells HL60 to be induced to differentiate to various stages along the monocytic or myelocytic pathway was exploited for investigation of the uptake of selected photo-sensitizers by diverse types of cells of the same origin. The results showed that there was no substantial difference in photofrin uptake between noninduced HL60 cells, immature monocytes, immature neutrophils and cells differentiated along the eosinophilic pathway. In contrast, HL60 cells differentiated into macrophages (HL609) exhibited markedly increased photofrin uptake, which was further enhanced by their pretreatment with bacterial lipopolysaccharide. Similar results were obtained with other photosensitizers tested: di-and tetrasulfonated aluminum phthalocyanines (AIPcS₂ and AIPcS₄), tetrasulfonated zinc phthalocyanine (ZnPcS₄), tetraphenylporphine tetrasulfonate (TPPS₄) and benzoporphyrin derivative monoacid (BPD). Despite marked differences in the state of self-aggregation and other chemical properties of these compounds, the degree of their preferential uptake by HL60 PH cells showed very little variation. In a typical experiment, the uptake of these photosensitizers by HL60 PH cells was four to five times higher than the uptake by noninduced HL60 cells. In addition to the fluorometric assay employed in most of the experiments, cellular concentration of AlPcS₄ was determined by measurement of elementary aluminum using atomic absorption spectroscopy.     

STRUCTURE-ACTIVITY RELATIONSHIP OF PORPHINES FOR PHOTOINACTIVATION OF BACTERIA

Abstract— The antibacterial photodynamic effects of uncharged ( o -tetrahydroxyphenyl porphine [THPP], m -THPP and p -THPP), cationic (5,10,15,20-tetra[4- N -methylpyridyllporphine [TMPyP]) and anionic (5,10,15,20-tetra[4-sulfonatophenyl porphine] [TPPS₄]) porphines on Staphylococcus aureus and Escherichia coli bacteria inactivation were examined. The results show that uncharged porphines provoked antibacterial photodynamic activity on S. aureus, and also on E. coli in the presence of the membrane-disorganizing peptide polymixin B nonapeptide (PMNP). The TMPyP compound was highly photoactive toward gram-positive bacteria but only marginally effective on gram-negative cells, whereas TPPS₄ showed no activity on either gram-positive or gram-negative bacteria. The photoactivity of TMPyP is due to the electrostatic attraction between the positively charged sensitizer molecule and the negatively charged membrane of the gram-positive target cells. For TPPS₄, the inactivity toward gram-positive bacteria is due to electrostatic repulsion between the charged sensitizer molecule and the cell membrane. For gram-negative bacteria, the inactivity is conceivably due to preferential (electrostatic) binding to the positively charged PMNP, which is an adjuvant for membrane disorganization, but has no effect on cell viability. For hydrophobic sensitizers, the photoactivity depends on the state of aggregation. The extent of deaggregation of the different THPP isomers was determined by fluorescence measurements of bound sensitizers and could be positively correlated with their photoinactivation capacity. We conclude that the structure-activity relationships of these porphines are affected by their net charge and by aggregation.     

PHOTOOXYGENATION OF METHYL LINOLEATE SENSITIZED BY PORPHYRINS AND DYES IN ACETONITRILE SOLUTION AND AQUEOUS EMULSION SYSTEMS

Photooxygenation reaction of an unsaturated fatty acid ester, methyl linoleate (methyl 9- cis. 12- cis -octadecadienoate, ML-H), sensitized by porphyrins and several types of dyes has been studied in aqueous emulsion and acetonitrile solution under air at 40°C. The oxygen (O₂) uptake proceeded slowly in the absence of sensitizers upon irradiation of an aqueous emulsion and an acetonitrile solution of ML-H (20 m M ) at ℷ_ex > 290 nm (11.4 and 6.1 μmol h^-1, respectively). The rate of O₂ uptake was enhanced by a catalytic amount (0.1 m M ) of porphyrins and dyes; hematoporphyrin (HP), zinc tetrakis(N-methyl-4-pyridiniumyi)porphyrin (ZnTMPyP), methylene blue (MB), rose bengal (RB), acridine orange (AO), and acriflavine (AF). In both systems, the sensitized photooxidation of ML-H by O₂ proceeded equimolarly to produce isomeric mixture of C9 and C13 hydroperoxides having the trans,cis and trans,trans conjugated diene configurations, independent of the types of the sensitizers used. The yield ratio of trans,trans/ trans,cis products in the MB-sensitized photooxygenation in acetonitrile and aqueous emulsion were almost equal (0.32 and 0.35. respectively). The sensitizing activity of the sensitizers, as measured by the quantum yield of O₂ uptake, increased in the order: MB (≃ 0) < ZnTMPyP < RB < HP < AF < AO in the aqueous emulsion and AO < AF < HP < RB = MB in the acetonitrile solution. The order in homogeneous acetonitrile solution was interpreted by the sensitizing ability of the dyes to produce singlet oxygen, while that in heterogeneous aqueous emulsion was correlated to the lipophilicity of dyes as well as the singlet-oxygen-producing ability.     

The Effect of Biological Substrates on the Ultrafast Excited-state Dynamics of Zinc Phthalocyanine Tetrasulfonate in Solution

Zinc phthalocyanine tetrasulfonate (ZnPcS₄), a potential photosensitizer for photodynamic therapy (PDT), has been studied using femtosecond laser spectroscopy. The excited-state dynamics in water have been found to be fast (<80 ps) and dominated by intermolecular aggregation. Since the proposed mechanism for PDT is energy transfer from the triplet excited state of the photosensitizer to triplet O₂ creating singlet O₂, the short lifetime is expected to be unfavorable for producing singlet O₂. This leads to the suggestion that the presence of biological substrates may have an effect on the excited-state dynamics. To test this hypothesis, the lifetimes of the ex-cited states of ZnPcS₄ have been directly measured in the presence of a model membrane, n-hexadecyltrimethylammonium bromide (CTAB). The excited-state dynamics of ZnPcS₄ in buffer solutions and with human serum albumin (HSA) have also been measured. The presence of HSA and CTAB increases the excited-state lifetime significantly relative to that observed in water. The longer lifetime of ZnPcS4 in CTAB (>1 ns) indicates that the micellar surface favors monomer formation. By increasing the excited-state lifetime, the surface substantially in-creases the photosensitizing potential of ZnPcS₄.     

8-Oxodeoxyguanosine Formation in the DNA of Cultured Cells After Exposure to H2O2 Alone or with UVB or UVA Irradiation

Abstract— The purpose of the study was to determine if aluminum phthalocyanine tetrasulfonate (A1PcS₄) photodynamic therapy (PDT) induced the formation of micronuclei in vitro and whether micronuclei formation was dependent on fiuence or cell type. NIH-3T3 and EMT-6 monolayer cultures were incubated in AlPcS₄ (0 or 1 μg/mL) for 24 h, received 0.0, 0.5,1.0 or 1.5 J/cm ² of 675 nm light, then reincubated and harvested at either 24, 48 or 72 h. The micronucleus frequency was determined in binucleated cells using the cytochalasin-block method. Cytotoxicity was assessed by using the 3(4,5-dimenthylthiazoyl-2-yl)2,5(diphenyl-tetrazolium bromide) (MTT) method. The effect of treatment on cell cycle progression was determined by calculating a proliferative index.
Aluminum phthalocyanine tetrasulfonate PDT induced a fluence-dependent increase in the frequency of micro-nuclei in NIH-3T3 and EMT-6 cells. The maximal effect of PDT was obtained in both cell lines 24 h after treatment. NIH-3T3 and EMT-6 cells exposed to a low fiuence of 0.5 J/cm² had a significantly lower number of micro-nuclei per cell 48 h following PDT treatment compared to the number of micronuclei per cell observed 24 h following treatment; however, when cells were exposed to a fluence (1.0 or 1.5 J/cm²) the number of micronuclei per cell did not diminish until 72 h after PDT treatment. The results obtained from the micronucleus assay paralleled those results obtained from the MTT assay.     

PHOTOBLEACHING OF MONO-l-ASPARTYL CHLORIN e6 (NPe6): A CANDIDATE SENSITIZER FOR THE PHOTODYNAMIC THERAPY OF TUMORS

Abstract— Most sensitizers used for the photodynamic therapy (PDT) of tumors photobleach on illumination. Thus, it is of interest to examine the photobleaching behavior of new sensitizers proposed for use in PDT. This report surveys the quantum yields and kinetics of the photobleaching of mono- l -aspartyl chlorin e₆ (NPe6), a hydrophilic chlorin that has many of the photoproperties desirable in a sensitizer for clinical PDT. It is a very effective sensitizer for the PDT of several types of model tumors in animals and is now in Phase I clinical trials. The quantum yield of NPe6 photobleaching in pH 7.4 phosphate buffer in air was 8.2 × 10⁻⁴; this is greater than the yields for typical porphyrin photosensitizers. For example, the yields for hematoporphyrin and uroporphyrin are 4.7 × 10 ⁵ and 2.8 × 10⁻⁵, respectively. The yield decreased significantly in organic solvents of low dielectric constant. The Sn derivative of NPe6 was more light stable than NPe6 (yield = 5.7 × 10 ⁻⁶), while the Zn derivative was more sensitive (yield = 1.9 × 10⁻²). Oxygen appeared to be necessary for the photobleaching of NPe6; however, bleaching was not inhibited by 100 mM azide, an efficient quencher of singlet oxygen. The photooxidizable substrates cysteine, dithiothreitol and furfuryl alcohol increased the quantum yield of photoblcaching two- to four-fold, while the electron acceptor, met-ronidazole, increased it almost six-fold. Photobleaching yields for several other chlorins were also measured.     

EFFICIENT PHOTOREDUCTION OF METHYLVIOLOGEN BY METALLOPHTHALOCYANINE SENSITIZERS

Abstract— Metallophthalocyanines (MPc) and tetrasubstituted magnesium phthalocyanines (MgPcR₄) were investigated as sensitizers for the photoreduction of methylviologen (MV²⁺) under visible light(420–800 nm) irradiation using disodium ethylenediaminetetraacetic acid (Na₂EDTA) as an electron donor in dimethyl formamide (DMF)-water mixture. Magnesium phthalocyanine and aluminium phthalocyanine were found to be the most efficient sensitizers among nine MPc's examined (M = 2Li, 2Na, Mg, Ca, Al(Cl), Fe, Ni, Co, and Zn). The following sequence of the efficiency among MgPc and MgPcR₄'s was obtained.     

PHOTODYNAMIC EFFECTS OF NEW SILICON PHTHALOCYANINES: In vitro STUDIES UTILIZING RAT HEPATIC MICROSOMES AND HUMAN ERYTHROCYTE GHOSTS AS MODEL MEMBRANE SOURCES

Abstract— Photodynamic therapy (PDT) of cancer is a modality that relies upon the irradiation of tumors with visible light following selective uptake of a photosensitizer by the tumor tissue. There is considerable emphasis to define new photosensitizers suitable for PDT of cancer. In this study we evaluated six phthalocyanines (Pc) for their photodynamic effects utilizing rat hepatic microsomes and human erythrocyte ghosts as model membrane sources. Of the newly synthesized Pc, two showed significant destruction of cytochrome P-450 and monooxygenase activities, and enhancement of lipid peroxidation, when added to microsomal suspension followed by irradiation with ∼ 675 nm light. These two Pc named SiPc IV (HOSiPcOSi[CH₃]₂[CH₂]₃N[CH₃]₂) and SiPc V (HOSiPcOSi[CH₃]₂[CH₂]₃N[CH₃]₃¹ I) showed dose-dependent photodestruction of cytochrome P-450 and monooxygenase activities in liver microsomes, and photoenhancement of lipid peroxidation, lipid hydroperoxide formation and lipid fluorescence in rnicrosomes and erythrocyte ghosts. Compared to chloroaluminum phthalocyanine tetrasulfonate, SiPc IV and SiPc V produced far more pronounced photodynamic effects. Sodium azide, histidine, and 2,5-dimethylfuran, the quenchers of singlet oxygen, afforded highly significant protection against SiPc IV- and SiPc V-mediated photodynamic effects. However, to a lesser extent, the quenchers of superoxide anion, hydrogen peroxide and hydroxyl radical also showed some protective effects. These results suggest that SiPc IV and SiPc V may be promising photosensitizers for the PDT of cancer.     

Rapid Initiation of Apoptosis by Photodynamic Therapy

Abstract— Photodynamic therapy (PDT) of neoplastic cell lines is sometimes associated with the rapid initiation of apoptosis, a mode of cell death that results in a distinct pattern of cellular and DNA fragmentation. The apoptotic response appears to be a function of both the sensitizer and the cell line. In this study, we examined photodynamic effects of several photosensitizers on murine leukemia P388 cells. Two drugs, a porphycene dimer (PcD) and tin etiopurpurin (SnET2), which localized at lysosomal sites, were tested at PDT doses that resulted in 50% loss of viability (LD₅₀), measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. An oligonucleosomal pattern of DNA degradation was observed within 1 h after irradiation. Neither sensitizer antagonized PDT-mediated internucleosomal DNA cleavage by the other. Very high PDT doses with either agent abolished this rapid internucleosomal cleavage. Exposure of cells to high concentrations of either sensitizer in the dark also resulted in rapid DNA fragmentation to nucle-osomes and nucleosome multimers; this effect was not altered by the antioxidant 6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid (trolox), although the latter could protect cells from cytotoxicity and apoptotic effects caused by LD₅₀ PDT doses. Photodamage from two cat-ionic sensitizers, which localized at membrane sites, caused rapid DNA cleavage to 50 kb particles; however, no further fragmentation was detected after 1 h under LD₁₀, LD₅₀ or LD₉₅ PDT conditions. Moreover, the presence of either cationic sensitizer inhibited the rapid internucleosomal cleavage induced by SnET2 or PcD photodamage. The site of photodynamic action may therefore be a major determinant of the initiation and rate of progression of apoptosis.     

SITES OF PHOTODYNAMICALLY INDUCED DNA REPAIR IN HUMAN CELLS

Abstract Human REH cells were incubated with the photosensitizers meso -tetra(4-sulfonatophenyl)porphyrin (TSPP=TPPS₄) or meso -tetra(3-hydroxyphenyl)porphyrin (3-THPP). The relatively hydrophilic TSPP was partly found in the cytoplasm and partly in the nuclei, whereas the lipophilic 3-THPP was found apparently in membranes and not inside the nuclei. After illumination, sites of DNA repair were labeled by means of a monoclonal antibody against proliferating cell nuclear antigen (PCNA) bound in the nuclei. The amount of bound PCNA in non-S-phase cells was proportional to the light dose. The bound PCNA was homogeneously distributed in the nuclei 0.5 h after photodynamic treatment (PDT) with TSPP. In contrast, for cells given PDT with 3-THPP, the periphery of the nuclei was selectively labeled, indicating that the initial DNA damage was localized close to the sensitizer at the nuclear membrane.     

Association of Ceramide Accumulation with Photodynamic Treatment-Induced Cell Death

Abstract— Ceramide, a stress-induced second messenger, has been associated with apoptosis in several malignant and non-malignant cell lines. We have shown that photodynamic treatment (PDT), using the phthalocyanine photosensitiz-er Pc 4 (HOSiPcOSi[CH₃]₂[CH₂]₃N[CH₃]₂), causes increased ceramide generation and subsequent induction of apoptosis in L5178Y-R (LY-R) mouse lymphoma cells. To test further if ceramide generation accompanies photocytotoxicity, we treated various cell lines with a PDT dose producing a 99-99.9% loss of clonogenicity. Like LY-R cells, human leukemia (U937) cells underwent rapid DNA fragmentation initiating within 1 h after PDT. Similarly, Chinese hamster ovary (CHO) cells showed rapid DNA laddering, beginning 1 h following the treatment. In contrast, mouse radiation-induced fibrosarcoma (RIF-1) cells showed no apoptosis within 24 h post-PDT, as judged by the absence of 50 kbp and oligonucleosome-size DNA fragments, as well as no annexin V binding to cells with preserved membrane integrity. Using the same doses of PDT, we observed a time-dependent ceramide accumulation in all three cell lines. While a significant increase in ceramide levels was reached within 1 and 10 min in U937 and CHO cells, respectively, elevated ceramide production was measured only after 30 min in RIF-1 cells. In addition, exogenous N-acetyl-sphingosine was able to mimic PDT-induced apoptosis in U937 and CHO cells. We suggest that ceramide accumulation is associated with PDT-induced apoptosis and photocytotox-icity.     

PHOTOSENSITIZERS IN ORGANIZED MEDIA: SINGLET OXYGEN PRODUCTION AND SPECTRAL PROPERTIES

Abstract— Measurement of singlet oxygen production by porphyrin-type photosensitizers in simple buffer solutions show that TPPS is more efficient than PII (Photofrin II) or than hematoporphyrin. This behavior was observed using three independent analytical detection methods for ¹O₂: RNO bleaching, tryptophan degradation, and oxygen consumption. Similar results were obtained when irradiating with a classical light source or with a pulsed dye laser. Addition of EPC liposomes to the buffer solution caused a decrease of efficiency for TPPS and an increase for PII. Addition of BSA shows the same relative pattern: a small increase of efficiency for TPPS and a tenfold increase for PII. These changes can be ascribed to specific interactions between the sensitizer molecules and the organized medium. Since changes in the fluorescence properties are also due to interactions with the medium, we monitored the emission of the sensitizers as a function of the environment. The fluorescence peak positions (at 648, 705 nm for TPPS and at 615, 677 nm for PII) were all red shifted. The intensities show an increase, particularly for PII in liposomes, due to a marked change in the microviscosity.     

ACID-BASE EQUILIBRIA OF THE EXCITED SINGLET AND TRIPLET STATES AND THE SEMI-REDUCED FORM OF ACRIDINE ORANGE

Abstract— The acid-base equilibria of the excited singlet and triplet states of acridine orange (AO) were studied by flash-photolysis and fluorometric methods. The dye is a stronger base in the first excited singlet state (pK_s= 13.3) than in the triplet and ground states (pK_r= 10.3: pK_c, = 10.2); acridine orange follows the trend observed with some other heterocyclic compounds, viz. pK_s > pK_r= pK,_c. At room temperature, an anomalous fluorescence occurs from the dye in basic media: the assignment of this emission is discussed.
The semi-reduced dye was studied as a function of pH. In a large pH range (3–14), only the protolytic equilibrium between the cationic (AOH₂⁺) and the neutral (AOH) radicals was observed; the pK value corresponding to this equilibrium was found to be in the range of pH 5–6.     

A Role for Hydrogen Peroxide in the Pro-apoptotic Effects of Photodynamic Therapy

Although the first reactive oxygen species (ROS) formed during irradiation of photosensitized cells is almost invariably singlet molecular oxygen (¹O₂), other ROS have been implicated in the phototoxic effects of photodynamic therapy (PDT). Among these are superoxide anion radical (^•O₂⁻), hydrogen peroxide (H₂O₂) and hydroxyl radical (^•OH). In this study, we investigated the role of H₂O₂ in the pro-apoptotic response to PDT in murine leukemia P388 cells. A primary route for detoxification of cellular H₂O₂ involves the peroxisomal enzyme catalase. Inhibition of catalase activity by 3-amino-1,2,4-triazole led to an increased apoptotic response. PDT-induced apoptosis was impaired by addition of an exogenous recombinant catalase analog (CAT- skl) that was specifically designed to enter cells and more efficiently localize in peroxisomes. A similar effect was observed upon addition of 2,2'-bipyridine, a reagent that can chelate Fe⁺², a co-factor in the Fenton reaction that results in the conversion of H₂O₂ to ^•OH. These results provide evidence that formation of H₂O₂ during irradiation of photosensitized cells contributes to PDT efficacy.     

MECHANISM OF PHYSICAL QUENCHING OF SINGLET MOLECULAR OXYGEN BY CHLOROPHYLLS and RELATED COMPOUNDS OF BIOLOGICAL INTEREST

Abstract— The rate constant k₅/ > for physical quenching of singlet oxygen O₂(δ₁;) by the sensitizer in dye-sensitized photooxygenations is determined in the case of chlorophylls a and b (7.3 times 10⁸, 4.2 times 10⁸ M^-1 s^-1 respectively), pheophytins a and b (7.4 times 10⁷, 3.0 times 10⁷ M^-1 s^_1 respectively), tetraphenylporphyrin (4.4 times 10⁷ M^-1 s^_1), magnesium tetraphenylporphyrin (5.0 times 10⁸ M^-1 s^_1), zinc tetraphenylporphyrin (1.5 times 10⁸ M^-1 s^_l) and protoporphyrin IX-dimethylester (9.1 times 10⁷ M ^-1 s^_1) in benzene. These sensitizers show a linear correlation between log k_s^O , and their half-wave oxidation potentials and the value of the slope is similar to that observed for various compounds such as phenols. It is concluded that (i) the interaction between chlorophylls and related compounds with singlet oxygen may involve an exciplex as for phenols, and (ii) physical quenching may be envisaged as a spin-orbit-induced intersystem crossing within the exciplex.     

THE EFFECT OF PORPHYRIN PHOTOSENSITIZERS ON THE TOXICITY OF l,3-BIS(2-CHLOROETHYL)-1- NITROSOUREA IN C57BL/6J MICE

Abstract The most widely used agents for photodynamic therapy are the porphyrin photosensitizers. It has been shown that hematoporphyrin derivative (HpD) can cause murine marrow hypercellularity and splenic hypertrophy. We have examined the effect on survival and marrow cellularity of high dose l,3-bis(2-chloroethyl)-l-nitrosourea (BCNU) after HpD or dihematoporphyrin ether (DHE) pretreatment in C57BL/6J mice.
The lethal toxicity of the LD_S0+ 10% dose of BCNU (60 mg kg⁻¹) was significantly reduced by pretreatment with HpD when the HpD was administered at least 3 days prior to the BCNU. HpD administered 1 or 2 days prior to BCNU or after BCNU had no effect. The percent death rate was reduced from 80 to 0% when HpD was administered 7 and 5 days prior to BCNU.
No alteration of the lethal toxicity rate of BCNU at doses of 80 mg kg⁻¹ were identified with DHE pretreatment although some increase in median survival was noted in two groups. Some reduction in lethal toxicity was noted when 60 mg kg⁻¹ BCNU was used and the pretreatment dose of DHE was 10 or 25 mg kg⁻¹ given twice 3 days apart. Furthermore, a significant reduction of BCNU induced marrow cell depletion was found when low doses of DHE were used as pretreatment. High doses of DHE resulted in marrow depletion. Both HpD and DHE altered the toxicity of BCNU.
Should porphyrin photosensitizers, which alone have little toxicity, prove to protect against nitrosourea toxicity then an important dose limiting factor (myelotoxicity) could be altered if not reduction in the tumouricidal activity occurs.