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.     

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.     

MEASUREMENT OF THE RATE OF UPTAKE AND SUBCELLULAR LOCALIZATION OF PORPHYRINS IN CELLS USING FLUORESCENCE DIGITAL IMAGING MICROSCOPY

Abstract A fluorescence imaging system incorporating a cooled slow-scan charge-coupled device camera was used to study the rate of uptake and subcellular localization of porphyrins in living cells. Measurements were carried out on human dermal fibroblasts (D532) using two different porphyrins meso -tetra(4- N -methylpyridyl)porphine (TMPP) and meso -tetra(4- N -hexylpyridyl)porphine (THPP). It was observed that TMPP was rapidly taken up by cells and principally located in the nucleus. The THPP, on the other hand, internalized more slowly and exhibited a particulate distribution in the cytoplasm.     

A TEST OF DIFFERENT PHOTOSENSITIZERS FOR PHOTODYNAMIC TREATMENT OF CANCER IN A MURINE TUMOR MODEL

Abstract The efficiency of different sensitizers for photodynamic therapy (PDT) was tested using a model system with a C3H mammary carcinoma growing subcutaneously on the dorsal side of mouse feet. Growth curves were constructed from which growth delay and doubling time in the regrowth phase were calculated. As PDT induced oedema in the mouse foot, this model system also allowed assessment of normal tissue response.
The following sensitizers were tested: hematoporphyrin derivative (HpD), Photofrin II (PII), tetraphenylporphinetetrasulfonate (TPPS₄), acridine orange (AO), phthalocyanine tetrasulfonate (PCTS), Al- and Zn-phthalocyanine tetrasulfonate (A1PCTS and ZnPCTS). For tumor control, the following sensitizer efficiencies were found: PII > HpD > AIPCTS > TPPS₄ >>> ZnPCTS, PCTS, AO. With regard to sensitizing normal-tissue damage: PII > AIPCTS, TPPS₄ > HpD, ZnPCTS, PCTS. The results suggest that AIPCTS should be further evaluated for use in PDT.     

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.     

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.     

IMPAIRED REPAIR OF UVC-INDUCED DNA DAMAGE IN L5178Y-R CELLS: DNA UNWINDING STUDIES WITH THE USE OF 1-β-D-ARABINOFURANOSYL CYTOSINE

Abstract— L5178Y-R (LY-R) and L5178Y-S (LY-S) cells differ in sensitivity to UVC radiation (D₀: 2.8 and 9.0 J/m²respectively, for cells exposed in Fischer's medium). We used these cells and a DNA unwinding technique in conjunction with 1-β-D-arabinosyl cytosine to determine DNA strand breaks accumulating as a result of enzymatic incision during DNA repair. Following UVC exposure DNA strand break accumulation was observed in LY-S cells but not in LY-R cells. The repair defect in LY-R cells is accompanied by a delayed recovery of [³H]thymidine incorporation.     

Irradiation of DNA with 193 nm Light Yields Formamidopyrimidine-DNA Glycosylase (Fpg) Protein-Sensitive Lesions

Abstract— Irradiation of aqueous solutions of plasmid DNA (pUC18) at pH 7.6 with 193 nm laser light results in low yields of prompt single strand breakage (air-saturated sample φ_ssb= [1.5 ± 0.1] ± 10⁻⁴, argon-saturated sample φ_ssb= [0.9 ± 0.1] ± 10⁻⁴). Treatment of the irradiated DNA samples with Escherichia coli formamidopyrimi-dine-DNA glycosylase (Fpg) protein results in an approximate 20-fold increase in the yield of single strand breakage (air-saturated sample φ_fpg= [33.1 ± 3.1] ± 10⁻⁴, argon-saturated sample φ_fpg= [23.8 ± 2.6] × 10 ⁴). This result indicates that 193 nm light induces other modification) (most likely of the purine moieties) that are 20 times more abundant than prompt strand breakage within the DNA matrix.     

A PHOTOSYSTEM I-PHENOSAFRANINE SOLAR CELL

Abstract Bacteriophage λ_vir was inactivated when it was irradiated with near-UV light in the presence of chlorpromazine. DNA strand breakage in the treated phage was indicated by alkaline sucrose gradient centrifugation. The number of the breaks was increased with increasing fluence. Although the inactivation rate was enhanced with a decreasing salt concentration in the reaction mixture and under a nitrogen atmosphere, the number of the strand breaks was not altered in either case. Therefore, the DNA strand breakage is not a sole lethal damage in the treated phage. The addition of NaN₃ repressed the inactivation and the reaction in a D₂O medium enhanced the inactivation even if the reaction mixture was irradiated under anaerobic conditions. Under anaerobic conditions, the inactivation occurs presumably via a radical mechanism.     

REPAIR OF ULTRAVIOLET-DAMAGED TRANSFORMING DNA IN A MISMATCH REPAIR-DEFICIENT STRAIN OF HAEMOPHILUS INFLUENZAE

Abstract— Ultraviolet inactivation of Haemophilus influenzae transforming DNA followed inverse square root kinetics in both mismatch repair-proficient(hex₊) and deficient (hex-1) recipients. No DNA concentration effect was seen with UV-excision repair-deficient(uvr_-) strains. Low-efficiency genetic markers remained more sensitive than high-efficiency ones when they were assayed on excision repair-deficienthex₊ uvr_- strains. They were equally resistant whenhex₊ uvr_- recipients were used. We explain this by assuming that recombinational repair of UV lesions in the donor strand and mismatch repair of the recipient strand may overlap and cause double strand interruptions. This will eliminate low-efficiency transformants.     

ACTION OF HYDROGEN PEROXIDE ON HUMAN FIBROBLAST IN CULTURE

Abstract— Human fibroblasts in culture lose the capacity of proliferating when exposed to hydrogen peroxide in the concentration range of 1 to 10 μ M . The toxicity of H₂O₂ to xeroderma pigmentosum cells (XP12RO). defective in excision repair of lesions produced by UV-irradiation, was about twice as high as to cells proficient in excision repair (VA13). This compound produces single-strand breaks in intracellular DNA but not in purified DNA. These breaks are in situ physical discontinuities rather than alkali-labile bonds, and their generation occurs at the same extent at 4°C and 37° indicating that they are not produced by an endonuclease. The results favor the hypothesis that H₂O₂ reacts in the cell producing a radical species which brings about the formation of DNA single-strand breaks. These breaks are effectively repaired by both XP12RO and VA13 fibroblasts. The possible reason for the lethality of H₂O₂ is discussed.     

THE PHOTODEGRADATION OF PORPHYRINS IN CELLS CAN BE USED TO ESTIMATE THE LIFETIME OF SINGLET OXYGEN

NHIK 3025 cells were incubated with Photofrin II (PII) and/or tetra (3-hydroxyphenyl)porphyrin (3THPP) and exposed to light at either 400 or 420 nm, i.e. at the wavelengths of the maxima of the fluorescence excitation spectra of the two dyes. The kinetics of the photodegradation of the dyes were studied. When present separately in the cells the two dyes are photodegraded with a similar quantum yield. 3THPP is degraded 3-6 times more efficiently by light quanta absorbed by the fluorescent fraction of 3THPP than by light quanta absorbed by the fluorescent fraction of PII present in the same cells. The distance diffused by the reactive intermediate, supposedly mainly 1O2, causing the photodegradation was estimated to be on the order of 0.01-0.02 micron, which corresponds to a lifetime of 0.01-0.04 microsecond of the intermediate in the cells. PII has binding sites at proteins in the cells as shown by an energy transfer band in the fluorescence excitation spectrum at 290 nm. During light exposure this band decays faster than the Soret band of PII under the present conditions. Photoproducts (1O2 etc.) generated at one binding site contribute significantly in the destruction of remote binding sites.     

MECHANISMS FOR DYE-MEDIATED PHOTODYNAMIC ACTION: SINGLET OXYGEN PRODUCTION, DEOXYGUANOSINE OXIDATION AND PHAGE INACTIVATING EFFICIENCIES

Abstract The production of singlet oxygen (¹O₂) upon irradiation of several dyes in aqueous solution at pH 9.0, was quantitatively analyzed on the basis of the appearance of stable nitroxide radicals using the amine 2,2,6,6-tetramethyl-4-piperidone as ¹O₂ acceptor. The dyes were checked for purity, their concentrations uniformized in terms of absorbance values and a correction factor was introduced which took into account the amount of photons absorbed. The rates of ¹O₂ production (in arbitrary units per min) were: 71 with rose Bengal, 70 with methylene blue, 61 with eosin Y, 18 with thiopyronine, 10 with proflavine and 9 with acridine yellow. Production of ¹O₂ was not observed with 9-aminoacridine, acridine orange, quinacrine and ethidium bromide. Irradiated lumichrome initiated, with the same amine, another type of reaction. The rates of two other photoreactions were also determined under similar experimental conditions by following (i) the deoxyguanosine decomposition in which case the reaction was found to be less sensitive but largely parallel to the ¹O₂ production and (ii) the bacteriophage ØX174 inactivation in which case the dyes showed differences in their relative efficiencies. The proteinic capsid of the phage appeared as an effective impermeability barrier towards externally generated ¹O₂. Moreover, some of the dyes studied intercalated into the phage DNA, a process known to favor radicalar reactions.     

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.     

INDUCTION OF SISTER CHROMATID EXCHANGES IN Allium cepa MERISTEMATIC CELLS EXPOSED TO meso- TETRA(4-PYRIDYL) PORPHINE and HEMATOPORPHYRIN PHOTORADIATION

The photodynamic effect of two porphyrin derivatives, meso-tetra (4-pyridyl)porphine (T₄PyP) and hematoporphyrin (HP), and activating red light (647.1 nm) was tested, measuring the number of sister chromatid exchanges induced by this treatment in Allium cepa chromosomes. A significant increase in the frequencies of sister chromatid exchanges was observed when chromosomes substituted with 5-bromo-2'-deoxyuridine were treated for 4 h (G₁ of the second cycle of division) with T₄PyP or HP followed by an irradiation with red light for 5 min.     

OBSERVATIONS ON THE PHOTOSENSITIZED BREAKAGE OF DNA BY 2-THIOURACIL AND 334-nm ULTRAVIOLET RADIATION

Abstract— Breaks induced in purified DNA by 334-nm ultraviolet (UV) radiation are enhanced 30 times when 2-thiouracil (s²Ura) is present during aerobic irradiation. This enhancement by s²Ura is maximally effective at a concentration of about 1 m M. Anoxic irradiation reduces the s²Ura-enhanced breakage by 90%, indicating a Type II photosensitization. Benzoate, glycerol, diazabicyclo[2.2.2.]octane (DABCO) and histidine all inhibit formation of s²Ura photosensitized breaks, unlike diethylenetriaminepenta-acetic acid (DETAPAC) and catalase, which do not. The relationships between the concentration of DABCO. benzoate and histidine and their protection against induction of single strand breaks (SSBs) were similar, with little inhibition below 10 m M and maximal inhibition near 0.1 M for all compounds. Irradiation of the DNA-s²Ura mixture dissolved in D₂O instead of H₂O enhanced the rate of induction of SSBs in DNA by 334-nm light almost five times. Addition of superoxide dismutase (40, 80 and 200 μg/ml) decreased the rate of induction of breaks in DNA by 334-nm radiation plus s²Ura (in H₂O) by about 40%. Boiled superoxide dismutase had no effect.     

MECHANISM OF FORMATION OF FORMALDEHYDE IN THE FLASH PHOTOLYSIS OF METHYL FORMATE

Abstract —From flash photolyses of methyl formate (HCOOCH₃), d -methyl formate (DCOOCH₃), methyl formate- d ₃ (HCOOCD₃) and fully ***deuterated methyl formate (DCOOCD₃) in the vapour phase at room temperature, the relative efficiencies of the formyl and methoxyl radicals in producing formaldehyde have been determined.     

PHOTOTOXIC POTENTIALITIES OF TARTRAZINE: SCREENING TESTS

Abstract Tartrazine (TRT) a cosmetics, food and drug additive, was tested with respect to phototoxic potentialities. Although, using the cholesterol technique, TRT did not appear to produce any ¹O₂ upon visible light irradiation, the EPR spin trapping experiments with 5,5-dimethyl-1-pyrroline-N-oxide, as spin trap, were in favour of activated oxygen species (O₂^˙, OH^˙) and hydrated electron production by photoexcited TRT.
Irradiation of TRT with complementary biological systems (nucleic acids, bacteria, biological membranes) showed that few of them can be damaged by this photosensitizer. Tartrazine could induce a weak deoxyribose degradation but did not produce any DNA strand breaks on isolated φXRFI DNA. Tartrazine was detected as mutagen in the Salmonella microsome (Ames) assay, only in conjunction with visible light. In the presence of photoexcited TRT, erythrocyte membranes were damaged by covalently cross-linking proteins.
The tests developed here seem thus to be suitable for detecting any unwanted phototoxic activity associated with potential photosensitizers.     

NECROSIS OF MURINE TAIL SKIN FOLLOWING PHOTODYNAMIC TREATMENT WITH meso-tetra-(p-SULPHOPHENYL) PORPHINE (TPPS)

Abstract. Albino mice were injected intravenously with 2 mg of meso-tefra-(p -sulphophenyl) porphine (TPPS) and subsequently their tails were exposed to a range of doses of full-spectrum visible light. At a power density of 75 mW cm^-2, a 50% incidence of gross necrosis of the tail skin (ED₅₀) occurred at a light dose of 130 J cm^-2. For haematoporphyrin derivative at the same administered dose, the ED₅₀ was 33 J cm^-2. Increasing the interval between TPPS and light from 1 to 34 days did not spare the tail skin, in terms of an increase in ED₅₀. The slopes of curves of incidence of tail necrosis vs light dose were steep: 1/slope ranged between 20 and 50 J cm^-2.     

PHOTOBINDING OF PSORALENS TO BACTERIAL MACROMOLECULES IN SITU AND INDUCTION OF GENETIC EFFECTS IN A BACTERIAL TEST SYSTEM. EFFECTS OF SINGLET OXYGEN DIAGNOSTIC AIDS D2O AND DABCO

The photobinding of radiolabeled psoralen and 8-methoxypsoralen (8-MOP) to biological macromolecules under conditions that affect the lifetime of singlet oxygen (¹O₂) is reported. These conditions are: increase of ¹O₂ lifetime in D₂O and ¹O₂ quenching with DABCO. The photobinding to calf thymus DNA was studied in vitro and the covalent photobinding to DNA and other biological macromolecules (RNA, proteins) was also studied in intact bacteria. The results of the DNA photobinding experiments have been related to the induction of genetic damage in a bacterial test system. In addition, laser flash photolysis has been used to measure the effect of D₂O and DABCO on the psoralen and 8-MOP triplet lifetimes. In general D₂O increases the triplet lifetimes and DABCO quenches the triplet states with the probable formation of radicals. The results suggest that the covalent photobinding of 8-MOP to various biological macromolecules in situ is a basis for cell damage occurring at various cellular targets. Analysis of the results of the mutagenicity test suggests that in the presence of D₂O the mechanism of induction of genetic lesions is not changed and therefore largely seems to be independent of singlet oxygen.