The effect of photodynamic treatment of yeast with the sensitizer chloroaluminum phthalocyanine on various cellular parameters.

Photodynamic treatment of Kluyveromyces marxianus with chloroaluminum-phthalocyanine resulted in loss of clonogenicity. Several parameters were studied to identify targets that could be related to loss of colony-forming capacity. Inhibition of various plasma membrane-bound processes was observed, such as substrate transport and plasma membrane ATPase activity. Moreover, K+ loss from the cells was observed. Photodynamic treatment also reduced the activity of various enzymes involved in energy metabolism, thereby decreasing the cellular ATP level. It will be discussed however that none of these processes is likely to be related directly to loss of clonogenicity. Treatment with phthalocyanine and light resulted in a strong inhibition of the incorporation of 14C-phenylalanine in trichloracetic acid-precipitable material. The induction of the beta-galactoside utilization system was also strongly inhibited. The latter two processes did not recover during incubation, subsequent to photodynamic treatment. It is concluded that photodynamically induced inhibition of protein synthesis is a critical factor contributing to the loss of clonogenicity.

The effect of protoporphyrin on the susceptibility of human erythrocytes to oxidative stress: exposure to hydrogen peroxide.

Binding of protoporphyrin caused a perturbation of the erythrocyte membrane, as reflected by a change in cell shape from discoid to echinocyte, and a concomitant increase in mean cellular volume and K(+)-loss. Protoporphyrin-induced changes could be prevented by the presence of BaCl2, whereas binding of protoporphyrin was not affected. Exposure of erythrocytes to hydrogen peroxide leads to K(+)-leakage and lipid peroxidation. In de presence of protoporphyrin, H2O2-induced K(+)-leakage was enhanced, whereas lipid peroxidation was inhibited. The increase in H2O2-induced K(+)-leakage by protoporphyrin was not affected by diamide or various K+ channel blockers, but could be prevented by the addition of BaCl2. The inhibition of lipid peroxidation, on the other hand, was not affected by BaCl2. These results indicate that the enhancement of H2O2-induced K(+)-leakage was most likely caused by the change in cell shape. Addition of chlorpromazine and promethazine, positively charged molecules that induce stomatocytosis, did not cause an enhancement of H2O2-induced K(+)-leakage.

Intracellular damage in yeast cells caused by photodynamic treatment with toluidine blue.

The positively charged photosensitizer toluidine blue (TB) can induce loss of clonogenicity in Kluyveromyces marxianus. Previous studies have revealed that, as a consequence of the localization of this dye at the cell surface, photodynamic action results in extensive damage at the level of the plasma membrane. In this paper, a study is reported on the effect of photodynamic treatment with TB on intracellular enzymes. It is shown that treatment with TB and light resulted in the inhibition of alcohol dehydrogenase, cytochrome c oxidase, glyceraldehyde-3-phosphate dehydrogenase and hexokinase. Photodynamic treatment also lowered the ATP levels. The ATP levels could be partially restored in the presence of glucose but not with ethanol. Toluidine blue binding experiments revealed that photodynamic treatment caused a rapid increase in the amount of cell-associated dye. Moreover, it also appeared that this treatment decreased the binding of TB to the cell surface. It is concluded that TB enters the cell during the first minutes of illumination, whereafter intracellular enzymes are inactivated. The data indicate that photodynamic damage of intracellular sites contributes to the loss of viability.

Relationship between photodynamically induced damage to various cellular parameters and loss of clonogenicity in different cell types with hematoporphyrin derivative as sensitizer.

The possible causal relationship between various forms of photodynamically inflicted damage and reproductive cell death of cultivated cells was evaluated according to three criteria. The probability for the existence of such a relationship is high, when the particular form of cellular damage (i) exhibits a dose-effect curve, comparable to the dose-effect curve of loss of clonogenicity, (ii) is not readily repairable during further incubation of the treated cells and (iii) varies in a way comparable to the loss of clonogenicity under varying experimental conditions. According to these criteria it could be shown that many forms of photodynamically inflicted cellular damage are presumably not directly involved in loss of clonogenicity. Only for a few kinds of cellular damage studied in the present investigations was the probability for a causal relationship with reproductive cell death much higher. For L929 fibroblasts this is either an inhibition of the Na+/K(+)-ATPase activity, or a relatively slight DNA damage combined with a strong inhibition of DNA excision repair. For T24 human bladder carcinoma cells the kinds of cellular damage that may be causally related to reproductive cell death are again inhibition of Na+/K(+)-ATPase activity, inhibition of amino-acid (AIB and glycine) transport activity or impairment of mitochondrial function. Finally, for CHO cells, inhibition of leucine and phenylalanine transport and impairment of mitochondrial function may be crucial for loss of clonogenicity. These results indicate that the pathways leading to photodynamically induced reproductive cell death may be quite different for different cell types.

The pro- and antioxidant properties of protoporphyrin IX.

The effect of protoporphyrin on lipid peroxidation in rat liver microsomes was investigated in the presence and absence of light. Protoporphyrin and light stimulated Fe(3+)-ADP/ascorbate-induced lipid peroxidation, whereas Fe(3+)-ADP/NADPH-mediated lipid peroxidation was inhibited. In the dark, on the other hand, protoporphyrin inhibited lipid peroxidation in both systems in a concentration-dependent manner. Protoporphyrin did not affect the reduction of the Fe(3+)-ADP complex by ascorbate, nor did it affect the activity of the NADPH-cytochrome P-450 reductase under these conditions. Uroporphyrin, a hydrophilic porphyrin and not localized in the membrane, did not inhibit lipid peroxidation in the dark in these systems, whereas bilirubin, a well-known radical scavenger and degradation product of protoporphyrin, inhibited lipid peroxidation in both systems. When peroxyl radicals were generated in solution by the azocompound 2,2'-azobis (2-amidino-propane)dihydrochloride lipid peroxidation was inhibited by both porphyrins and bilirubin. Reaction of bilirubin or protoporphyrin, in the presence of microsomes or human serum albumin, with these peroxyl radicals resulted in degradation of both compounds, which could be determined by a decrease in their absorbance at 460 or 407 nm, respectively. These results indicate that the inhibition of lipid peroxidation is most likely caused by scavenging of peroxyl radicals by protoporphyrin. Massive accumulation of protoporphyrin occurs in livers of erythropoietic protoporphyria patients. The fact that protoporphyrin was able to inhibit lipid peroxidation completely at micromolar concentrations also indicates that the deleterious effects of protoporphyrin, observed in these patients, are most likely not mediated by oxidation of lipids.

Inhibition of transport systems in yeast by photodynamic treatment with toluidine blue.

Photodynamic treatment of yeast with the sensitizer Toluidine blue results in loss of cell viability. In previous investigations it was suggested that plasma membrane damage might be responsible for the loss of colony forming capacity. In this context the influence of photodynamic treatment on transmembrane transport systems was studied. It appeared that the uptake of the sugars glucose, lactose and galactose, the amino acids arginine, phenylalanine, glycine and aspartic acid and of the inorganic compound phosphate was inhibited by photodynamic treatment. The different elements of the energy providing system necessary for active transport, viz. the plasma membrane ATPase and the protonmotive force, were not significantly affected by Toluidine blue and light, indicating that inhibition of transport is not caused by a reduction of the membrane potential or the transmembrane pH gradient. These observations suggest that the transport carriers themselves were damaged by treatment with Toluidine blue and light. This could be confirmed in experiments, in which the lactose and galactose transport proteins of treated and untreated cells were reconstituted in plasma membrane vesicles. It appeared that the carriers, obtained from photodynamically treated Kluyveromyces marxianus cell, had lost their transport capacity.

Hydroxyl radical generation by a light-dependent Fenton reaction.

Illumination of Fe3+, with light of a wavelength varying from 250 to 450 nm, in the presence of the iron chelators ethylenediamine N,N,N',N'-tetraacetic acid (EDTA), ethyleneglycol-bis-(beta-aminoethylether)N,N,N',N'-tetraacet ic acid (EGTA), diethylenetriamine-N,N,N',N',N'-pentaacetic acid (DTPA), or citrate resulted in the reduction of Fe3+ to Fe2+. Fe2+ formation was measured by the formation of its complex with bathophenanthroline disulfonic acid. In all cases Fe2+ formation was completely dependent on the presence of the iron chelator and on the wavelength used for illumination. A correlation was found between the absorption spectrum of the iron-chelator complex and the amount of Fe3+ reduced, suggesting that the absorption of light induced an electron transfer from the chelator to the iron ion. Exposure to oxygen, either during or after illumination, resulted in degradation of the chelator molecule. Illumination of the Fe(3+)-chelator complexes in the presence of H2O2 resulted in the formation of hydroxyl radicals, which could be determined by the formation of the 5,5-dimethyl-1-pyrroline N-oxide (DMPO)-hydroxyl radical adduct, using electron spin resonance spectroscopy. Formation of the spin adduct was inhibited by addition of catalase, mannitol, ethanol, or formate, whereas superoxide dismutase had no effect.

Exocytosis in electropermeabilized neutrophils. Responsiveness to calcium and guanosine 5'-[gamma-thio]triphosphate.

Electropermeabilized neutrophils were used to study the exocytotic response in rabbit neutrophils. Enzyme release from electropermeabilized neutrophils could be induced by elevating the Ca2+ concentration. Ca(2+)-induced secretion was significantly enhanced by guanosine 5'-[gamma-thio]triphosphate (GTP[S]) in a concentration-dependent manner. The effect of GTP[S] could be blocked by guanosine 5'-[beta-thio]diphosphate (GDP[S]) and was not affected by pertussis toxin. GTP[S] did not induce enzyme release in the absence of Ca2+. Induction of an exocytotic response did not require addition of ATP. However, neutrophils permeabilized in the absence of ATP became refractory to stimulation due to a reduction in their affinity for Ca2+. Responsiveness to the effectors Ca2+ or Ca2+ + GTP[S] could be prolonged or restored by ATP. ATP was not the only agent that prolonged responsiveness; other nucleotides and inorganic phosphates were also effective. The protein kinase C inhibitors staurosporine and 1-O-hexadecyl-2-methyl-sn-glycerol did not inhibit exocytosis and had only a small effect on the prolongation and restoration of responsiveness by ATP. A hypothesis is presented suggesting that the loss of responsiveness is caused by dephosphorylation and that the restoration or prolongation of responsiveness is not mediated by protein kinase C. It is possible that an as yet unidentified Ca(2+)-binding protein is dephosphorylated, resulting in a decrease in Ca2+ affinity.

Toxic dark effects of protoporphyrin on the cytochrome P-450 system in rat liver microsomes.

In erythropoietic protoporphyria, accumulation of protoporphyrin has been found in various tissues and liver cirrhosis occurs frequently in this disease, probably due to toxic dark effects of protoporphyrin. We have studied the effect of porphyrins on various enzymic functions in rat liver microsomes. Incubation of microsomes with protoporphyrin resulted in a concentration-dependent inhibition of the oxidation of 7-ethoxycoumarin and aminopyrine by the cytochrome P-450 system. Kinetic analysis showed a decrease in Vmax., whereas the Km was not affected (non-competitive inhibition). Furthermore, reduction of cytochrome c by the NADPH-cytochrome P-450 reductase and by the NADH-cytochrome b5 reductase was inhibited. However, the activity of the reductases was only affected when the microsomes were pre-incubated with protoporphyrin, and it was found that the inhibition was dependent on the duration of the pre-incubation. Kinetic analysis again revealed non-competitive inhibition. When these experiments were repeated with uroporphyrin, no inhibition could be observed. With Stern-Volmer plots it was demonstrated that this was most likely caused by the localization of the porphyrins: protoporphyrin is localized in the membrane, whereas uroporphyrin remains in solution. From these results it is concluded that accumulation of protoporphyrin in the liver may markedly affect the cytochrome P-450 system and thus its detoxification function.

Photodynamic treatment of yeast cells with the dye toluidine blue: all-or-none loss of plasma membrane barrier properties.

Photodynamic treatment of Kluyveromyces marxianus with the sensitizer Toluidine blue leads to the loss of colony forming capacity. In this paper, the influence of this treatment on the barrier properties of the plasma membrane has been studied. Photodynamic treatment with the dye Toluidine blue resulted in efflux of potassium ions and E260-absorbing material. Moreover, cells became stainable with erythrosine. It is concluded that the permeability change induced by photodynamic treatment proceeds in an all-or-none fashion. Treatment of this yeast strain, with the dye and light, also induced a diminution of the cell volume. This process is most likely not coupled to the cellular potassium content, but rather to the integrity of the vacuole. These data suggest that the vacuole has an important function in the maintenance of cell volume. Finally, it was observed that the loss of cell viability was not induced by the all-or-none loss of barrier properties.

Effect of fluoride on inhibition of plasma membrane functions in Chinese hamster ovary cells photosensitized by aluminum phthalocyanine.

Fluoride inhibits photohemolysis induced by chloroaluminum phthalocyanine tetrasulfonate (AlPcS4) when it is added to dye-loaded human erythrocytes prior to light exposure (E. Ben-Hur, A. Freud, A. Canfi, and A. Livne, Int. J. Radiat. Biol. 59, 797-806, 1991). This is due to formation of a complex of F- with Al3+, leading to selective release and/or modified dye binding with some proteins so that the effective photochemical reaction is prevented. In this work we used F- as a probe to evaluate the involvement of the plasma membrane functions of Chinese hamster ovary cells in photocytotoxicity induced by chloroaluminum phthalocyanine (AlPc). Fluoride was found to protect against killing of cells photosensitized by AlPc but not AlPcS4. Plasma membrane damage induced by AlPc photosensitization was manifested by K+ leakage, membrane depolarization, inhibition of glucose and amino acid uptake, and Na+/K(+)-ATPase inactivation. The latter enzyme system was found to be the one most sensitive to inhibition by the combination of AlPc and PDT among the membrane functions studied, and was completely protected by F- in the dose range at which up to 95% of the cells are killed. Of the other membrane functions only glucose transport was slightly protected by F-. It is concluded that damage to the plasma membrane is involved in cell killing induced by AlPc photosensitization and that the plasma membrane enzyme Na+/K(+)-ATPase is a probable candidate as a critical target.

A role for the transient increase of cytoplasmic free calcium in cell rescue after photodynamic treatment.

Chinese hamster ovary (CHO) cells and T24 human bladder transitional carcinoma cells were treated with the photosensitizers aluminum phthalocyanine (AlPc) and hematoporphyrin derivative (HPD), respectively. Exposure of both sensitized cell lines to red light caused an immediate increase of cytoplasmic free calcium, [Ca2+]i, reaching a peak within 5-15 min after exposure and then returning to basal level (approximately 200 nM). The level of the peak [Ca2+]i depended on the light fluence, reaching a maximum of 800-1000 nM at light doses that kill about 90% of the cells. Loading the cells with the intracellular calcium chelators quin2 or BAPTA prior to light exposure enhanced cell killing. This indicates that increased [Ca2+]i after photodynamic therapy (PDT) contributed to survivability of the treated cells by triggering a cellular rescue response. The results of experiments with calcium-free buffer and calcium chelators indicate that both in CHO cells treated with AlPc and with HPD-PDT of T24 cells extracellular Ca2+ influx is mainly responsible for elevated [Ca2+]i. PDT is unique in triggering a cell rescue process via elevated [Ca2+]i. Other cytotoxic agents, e.g., H2O2, produce sustained increase of [Ca2+]i that is involved in the pathological processes leading to cell death.

The effect of fluoride on photodynamic-induced fluorescence changes of aluminium phthalocyanine in Chinese hamster cells.

Fluence-dependent changes in the fluorescence of aluminium phthalocyanine (AlPc) were measured in Chinese hamster ovary (CHO) cells using digital fluorescence microscopy of single cells and spectrofluorimetry of cell suspensions. During illumination the fluorescence initially increased and later progressively decreased. In the presence of fluoride, which protects against phototoxicity of AlPc by forming a fluoroaluminium complex, there was no initial increase in fluorescence: it decreased about 10 times faster than in the absence of fluoride. Qualitatively similar results were observed using single-cell fluorescence microscopy, which also showed the dye to be mostly localized in cytoplasmic organelles and membranes. The pattern of localization did not change during illumination. Concomitant assays of dye extracted from cells revealed little photodegradation that could not account for the fluorescence changes. The absorption spectra of AlPc-loaded cells showed some aggregation of the dye prior to light exposure. During illumination the dye was initially monomerized and subsequently progressively reaggregated. In the presence of fluoride no monomerization was seen, and the aggregation proceeded at a much faster rate. It is concluded that the fluorescence changes are not due to major relocalization of AlPc in the cells, but to light-induced monomerization followed by reaggregation. The protective effect of fluoride may be due to the enhanced aggregation rate, because aggregated dye molecules are photochemically inactive. Because D2(0) affects neither the initial enhanced fluorescence in the absence of fluoride nor the rapid decrease in its presence it appears that 1O2 is not involved in the photodynamic reactions leading to these changes.

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.

Anomalous properties of water in macromolecular gels.

Low molecular weight solutes often exhibit elution characteristics on gel filtration columns which deviate from ideal behaviour. In many previous studies this anomalous behaviour was attributed to the existence of extremely narrow pores in the gel, inaccessible even to very small solute molecules, to explain Kd values lower than unity. Kd values of small solutes higher than unity were usually ascribed to adsorption of the solute to the gel matrix. In the present paper several observations are presented that contradict these suggestions. Experimental evidence indicates that with small solute molecules Kd values differing from unity can be fully explained by the anomalous properties of vicinal water layers at the gel matrix-water interface.

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-.

Phthalocyanine-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.

Potentiation of hyperthermia-induced haemolysis of human erythrocytes by photodynamic treatment. Evidence for the involvement of the anion transporter in this synergistic interaction.

Heat treatment of human erythrocytes led to increased passive cation permeability, followed by haemolysis. K+ leakage was linear up to a loss of about 80% in the temperature range 46-54 degrees C. Kinetic analysis of the results revealed an activation energy of 246 kJ/mol, implicating a transition in the membrane as critical step. Pretreatment of erythrocytes with 4,4'-di-isothiocyano-2,2'-stilbenedisulphonate, chymotrypsin or chlorpromazine caused a potentiation of subsequent heat-induced K+ leakage. Photodynamic treatment of erythrocytes with Photofrin II, eosin isothiocyanate or a porphyrin-Cu2+ complex as sensitizer also induced an increase in passive cation permeability, ultimately resulting in colloid osmotic haemolysis. The combination of photodynamic treatment immediately followed by hyperthermia had a synergistic effect on K+ leakage. Analysis of the results by the Arrhenius equation revealed that both the activation energy and the frequency factor of heat-induced K+ leakage were decreased significantly by preceding photodynamic treatment, suggesting that hyperthermia and photodynamic treatment have a common target for the induction of K+ leakage. Several lines of reasoning indicate that this common target is band 3. A model is thus proposed for the observed potentiation of hyperthermically induced K+ leakage by photodynamic treatment, in which photo-oxidation of band 3 results in increased sensitivity to subsequent thermal denaturation. These phenomena may be of more general significance, as photodynamic treatment and hyperthermia interacted synergistically with respect to K+ leakage with L929 fibroblasts also.

Proton-motive force-driven D-galactose transport in plasma membrane vesicles from the yeast Kluyveromyces marxianus.

Galactose transport was studied in membrane vesicles, prepared by fusion of plasma membranes from the yeast Kluyveromyces marxianus with proteoliposomes containing beef heart cytochrome c oxidase as a proton-motive force-generating system. Sugar transport studies performed under nonenergized conditions revealed that, even at high protein to phospholipid ratios, not all vesicles contained a D-galactose-specific transporter. The amount of vesicles containing an active carrier proved to be proportional to the amount of plasma membrane protein present in the fusion mixture. By addition of a suitable electron donor system a proton-motive force of -160 mV could be generated, inside alkaline and negative. Moreover, D-galactose accumulation was observed. It was found that D-galactose accumulation was highly dependent on the phospholipid composition of the vesicles, whereas generation of a proton-motive force was not. Best results were obtained with vesicles prepared with Escherichia coli phospholipid, giving a galactose accumulation of 14 times. Uphill transport could be established under conditions where only the pH gradient or the electrical gradient was present. Moreover, kinetic analysis of the galactose transport activity in energized vesicles revealed influx with a Km value of 540 microM, which is in good agreement with the apparent affinity constant obtained with whole cells. These results establish that galactose transport of K. marxianus is a proton-motive force-driven process. Moreover it demonstrates that plasma membrane vesicles co-reconstituted with cytochrome c oxidase are a valuable resource for the analysis of proton-motive force-driven sugar transport systems of yeast.