THE EFFECT OF FLUORIDE ON BINDING and PHOTODYNAMIC ACTION OF PHTHALOCYANINES WITH PROTEINS

Fluoride inhibits chloroaluminum phthalocyanine tetrasulfonate (AlPcS)-induced photohemolysis when added to dye loaded cells prior to light exposure. The mechanism by which F- exerts this effect was studied by measuring the binding of phthalocyanine (Pc) to various proteins in the absence and presence of F-. Parallel measurements were made of the photodynamic action under these conditions. Fluoride reduced the binding to proteins of AlPcS and CoPcS. The binding of CuPcS, ZnPcS and H2PcS was not affected. When bound to bovine serum albumin and exposed to light, H2Pc, ZnPc and AlPcCl were bleached at a biphasic rate. Only the photobleaching of AlPcCl was affected by F-. The effect of F- was to inhibit the initial rapid phase without affecting the slower phase. In the presence of D2O only the second phase of photobleaching was enhanced, in the absence or presence of F-. No effect of F- was observed on tryptophan photooxidation or glyceraldehyde-3-phosphate dehydrogenase photoinactivation by AlPcS. Crosslinking of spectrin monomers photosensitized by AlPcS was inhibited by F- in parallel with the reduced binding of dye to the protein. It is concluded that F- exerts its effect by complexing with metal ligands of Pc. As a result, the dye may be released from the protein or the binding mode may be changed in such a way that effective photochemistry is prevented. Primary photophysical processes of Pc most probably are not affected by F-.     

MODEL STUDIES ON PHOTODYNAMIC CROSS-LINKING

Abstract— A model system is described for studying photodynamic protein cross-linking. It appeared that several different types of cross-links are possible between photooxidized amino acid residues—especially histidine—and functional groups in proteins.     

PHOTODYNAMIC EFFECTS INDUCED BY THE LUCIFERIN/LUCIFERASE SYSTEM

Abstract— Luciferin from Photinur pyralis is an effective sensitizer, when excited with UV light in the range of 310–390 nm. With histidine or dithiothreitol as substrate, a type II photooxidation occurs, as judged from the inhibitory effect of sodium azide. During the ATP-driven luciferin-luciferase reaction, the resulting bioluminescence does not induce photodynamic reactions, as there is no overlap between the bioluminescence spectrum and the excitation spectrum of luciferin. However, in the presence of a second sensitizer, excitable by the bioluminescent light, photodynamic reactions can take place in the absence of exogenous light. As a consequence several photosensitizers can thus provoke photodynamic inactivation of luciferase.     

PHOTODYNAMIC EFFECTS OF HEMATOPORPHYRIN DERIVATIVE ON THE UPTAKE OF RHODAMINE 123 BY MITOCHONDRIA OF INTACT MURINE L929 FIBROBLASTS AND CHINESE HAMSTER OVARY Kl CELLS

Abstract— It was shown that the cationic fluorescence probe rhodamine 123 accumulates in mitochondria of murine L929 fibroblasts and Chinese hamster ovary Kl epithelial cells due to the driving force of both plasma membrane and mitochondrial membrane potentials. Photodynamic treatment of L929 cells with hematoporphyrin derivative resulted in an increased uptake of rhodamine 123 and a diminished uptake of 1,1,3,3,3',3'-hexamethylindocarbocyanine iodide. This indicates a considerably increased mitochondrial membrane potential, which most likely is the result of a direct or secondary inhibition of the ATP-synthetase, and a decreased plasma membrane potential. The oxygen consumption rate and the ATP level decreased due to photodynamic treatment. Post-incubation of L929 cells subsequent to photodynamic treatment revealed that the uptake of rhodamine 123. the ATP content and the oxygen consumption rate were restored. For all parameters similar results were obtained with CHO-K1 cells, with the exception that during post-incubation the intracellular ATP content remained at the level reached after illumination. These results indicate that photodynamically induced disturbance of mitochondrial functions and the ATP level are not crucial for the loss of clonogenicity of L929 cells. In CHO-K1 cells however, the continuously lowered ATP level may have detrimental consequences for cell survival. The photodynamic stimulation of the rhodamine 123 uptake may be a rather general phenomenon. Because rhodamine 123 exhibits a much higher toxicity towards carcinoma cells than towards other cells, a synergistic interaction between this drug and photodynamic therapy (PDT) may be anticipated, if PDT also stimulates mitochondrial rhodamine 123 accumulation in carcinoma in vivo.     

PHOTODYNAMIC GENERATION OF HYDROXYL RADICALS BY HEMATOPORPHYRIN DERIVATIVE AND LIGHT

In a reaction mixture containing hematoporphyrin derivative, deoxyribose, Fe³⁺-EDTA and either methionine or tryptophan, hydroxyl radicals were formed during illumination with visible light. When either hematoporphyrin derivative, Fe³⁺-EDTA or the amino acid was omitted from the reaction mixture, the generation of hydroxyl radicals ceased. These observations suggest an iron-catalyzed Haber-Weiss reaction, involving superoxide and hydrogen peroxide in the generation of hydroxyl radicals. It could be shown that with methionine H₂O₂ was indeed an essential intermediate in the reaction sequence. With tryptophan, however, H₂O₂, was not generated. Apparently a photooxidation product of tryptophan could replace H₂O₂ in the OH-generating reaction with Fe²⁺-EDTA. Although superoxide was generated in the reaction mixture, it was not an indispensable intermediate. Apparently a porphyrin radical, formed via photoexcitation of hematoporphyrin derivative, could replace superoxide in the Haber-Weiss reaction.     

Photodynamic effects of hematoporphyrin-derivative on enzyme activities of murine L929 fibroblasts

Photodynamic treatment of murine L929 fibroblasts with hematoporphyrin-derivative resulted in the inactivation of cytosolic, mitochondrial and lysosomal enzymes and in a decrease in cellular adenosine triphosphate and reduced glutathione concentrations. Comparison of these results with those of previous studies revealed that transmembrane transport systems and DNA repair enzymes are inactivated after much shorter illumination periods than are intracellular enzymes. Although the pattern of photodynamic damage altered by varying the protocol of preincubation with hematoporphyrin-derivative and washing, it appeared that under all experimental conditions the plasma membrane was much more sensitive to photodynamic damage than were the intracellular enzymes. Lysosomal membrane disruption with subsequent detrimental release of lysosomal enzymes has been implicated previously in certain forms of porphyrin-induced photodynamic cell destruction. Cytochemical studies on enzyme localization virtually exclude such a mechanism in hematoporphyrin-derivative-induced cell inactivation in L929 fibroblasts.     

The Role of Protein Kinase C Activity in the Killing of Chinese Hamster Ovary Cells by Ionizing Radiation and Photodynamic Treatment

In several recent studies it has been shown that protein kinase C (PKC) activity may either potentiate or antagonize cell killing by different cytotoxic agents. These apparently conflicting observations suggest that the effects of PKC activity on cell survival may depend on the different properties of different cell types but do not exclude the possibility that the effects may also depend on the nature of the cytotoxic agent. In this context the effects of PKC activation and PKC inhibition or down-regulation on Chinese hamster ovary (CHO) cell survival after photodynamic treatment and ionizing radiation were studied. It appeared that PKC activation by short-term incubation with 12-0-tetradecanoyl-phorbol-13-acetate (TPA) protected CHO cells against ionizing radiation but, in contrast, sensitized the cells to photodynamic treatment. Conversely, inhibition of PKC by H7 and down-regulation of PKC activity by prolonged incubation with TPA sensitized CHO cells to ionizing radiation but protected the cells against photodynamic treatment. These results demonstrate that in one particular cell type PKC activity may have opposite effects on cell survival following cellular damage, depending on the nature of the cytotoxic agent .     

Photodynamic inactivation of retroviruses by phthalocyanines: the effects of sulphonation, metal ligand and fluoride.

The photodynamic inactivation of retroviruses was investigated using aluminium and zinc phthalocyanine (Pc) derivatives. The N2 retrovirus packaged in either of the two murine cell lines, Psi2 and PA317, was used as a model for enveloped viruses. AlPc derivatives were found to be more effective photodynamically for inactivation of the viruses than the corresponding ZnPc derivatives. Sulphonation of the Pc macrocycle reduced its photodynamic activity progressively for both AlPc and ZnPc. Fluoride at 5 mM during light exposure completely protected viruses against inactivation by AlPc. In the presence of F-, inactivation by the sulphonated derivatives AlPcS1 and AlPcS4 was reduced 2.5- and twofold respectively. In a biological membrane (erythrocyte ghosts), F- had no significant effect on AlPcS4-sensitized lipid peroxidation. Under similar conditions, cross-linking of spectrin monomers in ghosts is drastically inhibited (E. Ben-Hur and A. Orenstein, Int. J. Radiat. Biol., 60 (1991) 293-301). Since Pc derivatives do not inactivate non-enveloped viruses, it is hypothesized that inactivation occurs by photodynamic damage to envelope protein(s). Substitution of sulphonic acid residues reduces the binding of Pc derivatives to the envelope protein(s), thereby diminishing their photodynamic efficacy and the ability of F- to modify it.     

PTHALOCYANINE-INDUCED PHOTODYNAMIC CHANGES OF CYTOPLASMIC FREE CALCIUM IN CHINESE HAMSTER CELLS

Exposure to light of Chinese hamster cells preloaded with chloroaluminum phthalocyanine causes an immediate increase of cytoplasmic free calcium, [Ca2+], from about 0.2 microM to 1 microM within 5 min after illumination. This increase was dose-dependent within the biological dose range, reaching a plateau at a dose that kills 99.5% of the cells. Fluoride addition prior to light exposure protected against cell killing and reduced the increase of [Ca2+]i. These findings raise the possibility that changes in [Ca2+]i after photodynamic treatment may be relevant to cell killing and/or other biological responses of the cells, e.g. release of eicosanoids.