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E Ben-Hur

Publications and source records attributed to E Ben-Hur.

At least 55 records · Page 3Linked to original sources
PubMed1993

Validity of chromium-51 labelling of human erythrocytes as an assay for their survival after photodynamic therapy in vitro for blood sterilization.

Labelling of human erythrocytes with 51CrO42- is used as an assay for survival of red blood cells (RBC) in the circulation. To ascertain whether the assay is valid for RBC treated with sulfonated aluminium phthalocyanine and red light, a procedure designed for viral deactivation in RBC concentrates, we followed release of 51Cr and haemolysis after photosensitization. The results shows that 51Cr release does not occur until about 3 h prior to haemolysis. The absence of 51Cr loss from cells not committed to lysis indicates that the label technique is valid for determination of survival photosensitized RBC in the circulation.

Blood↗
PubMed1993

Inhibition of phthalocyanine-sensitized photohemolysis of human erythrocytes by quercetin.

Photohemolysis of erythrocytes in the presence of aluminum phthalocyanine tetrasulfonate as a sensitizer is inhibited by quercetin. D2O (98.5%) stimulated photohemolysis regardless of quercetin presence, suggesting the participation of singlet oxygen in the process. Since it has been shown that this flavonoid reacts with singlet oxygen, the protective effect might be attributed, at least partially, to its competitive reaction with singlet oxygen. At the molecular level, the alterations of membrane proteins that escort the process of photohemolysis, such as cross-linking of spectrin monomers and of other membrane proteins, were selectively inhibited by quercetin. This effect was qualitatively similar to that induced by NaF, suggesting that quercetin may, like NaF, also inhibit type I photooxidations, which contribute to hemolysis. The lipophilicity of quercetin seems to be an essential factor in the inhibition process; rutin, a water-soluble 3-rutinoside of quercetin, had only a negligible protective effect on photohemolysis.

Erythrocytes↗
PubMed1993

Phthalocyanine-induced photohemolysis: structure-activity relationship and the effect of fluoride.

Phthalocyanine (Pc) containing A1, Ga or Zn as central metal ligand and substituted with a varying number of sulfonic acid residues as well as additional benzene rings were synthesized and their photodynamic activity was assayed using photohemolysis of human erythrocytes as an endpoint. The Pc derivatives varied > 300-fold in their photodynamic activity. Activity correlated with binding of the dye to the cell, with the exception of some of the amphiphilic dyes where cell uptake was an order of magnitude higher than expected from the observed activity. Fluoride was shown to inhibit A1PcSn-induced photohemolysis. This effect occurred also with other A1Pc and GaPc derivatives, but the concentration of F- required to slow photohemolysis by a factor of two (Ki) varied between 4 microM and 10 mM. Fluorescence spectral studies indicated complex formation between F- and the dye, which was stronger for A1Pc than GaPc derivatives. Ultrastructural studies using scanning electron microscopy showed that the photosensitized cells were converted to spherocytes and that F- prevented this to a large extent.

Erythrocytes↗
PubMed1992

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.

Aluminum↗
PubMed1992

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.

Aluminum↗
PubMed1992

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.

Animals↗
PubMed1992

Ascorbate-assisted, phthalocyanine-sensitized photohaemolysis of human erythrocytes.

The rate of photohaemolysis of human red blood cells sensitized by chloroaluminium phthalocyanine sulphonate is increased by ascorbate, with or without added FeCl3. Stimulation of haemolysis by ascorbate without addition of metal salt, and in the presence of a strong chelator such as desferrioxamine, is an unexpected phenomenon. Lysis rate and ascorbate concentration were directly related, suggesting that ascorbate acts as a reactant and not as a catalyst. The process also requires oxygen; azide and D2O tests indicate some participation of singlet oxygen, although to a lesser extent than in the photosensitized haemolysis in the absence of ascorbate. Kinetic considerations suggest a reaction path initiated by excited sensitizer and ascorbate, parallel to the singlet oxygen-mediated process. Because of the ubiquitous presence of ascorbate in human tissues in concentrations comparable to those of dissolved oxygen, it is quite possible that in photodynamic therapy a fraction of the photodynamic damage proceeds via a Type I, ascorbate-assisted, mechanism.

Ascorbic Acid↗
PubMed1992

Protection by the fluoride ion against phthalocyanine-induced photodynamic killing of Chinese hamster cells.

When a dilute F- solution was added to a culture of Chinese hamster cells that had been preincubated with an aluminium phthalocyanine sensitizer derived from AlPcCl, the photosensitivity of the cells was markedly reduced compared to control cells not treated with F-. Under the same treatment conditions, the reduction in [3H]thymidine incorporation into cellular DNA caused by light and this sensitizer and the production of DNA-protein crosslinks caused by light and this sensitizer were also inhibited by F-. In contrast, the killing of Chinese hamster cells, the reduction of thymidine incorporation by the cells, and the production of DNA-protein crosslinks in the cells caused by the combination of light and either Photofrin II or the silicon phthalocyanine HOSiPcOSi(CH3)2(CH2)3-N(CH3)2 were not inhibited by F-. We conclude that the aluminium phthalocyanine sensitizer used is largely or completely AlPc(OH)(H2O), that it is converted to a fluoro complex by F-, and that this compound probably is a less efficient generator of photochemical damage at a critical cellular target(s) than is AlPc(OH)(H2O). The inhibition of thymidine incorporation and DNA-protein crosslink formation indicates that the effects of F- can be expressed at intracellular sites. It is further concluded that the silicon phthalocyanine sensitizer and Photofrin II do not interact significantly with F-.

Animals↗
PubMed1992

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.

Animals↗
PubMed1991

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.

Chromatography, Gel↗
PubMed1991

Phthalocyanine-sensitized lipid peroxidation in cell membranes: use of cholesterol and azide as probes of primary photochemistry.

Various phthalacyanine (Pc) derivatives of phototherapeutic interest have been shown to be efficient type II (singlet oxygen, 1O2) sensitizers in aqueous and non-aqueous solutions. However, primary Pc photochemistry in biological environments, e.g. cell membranes, has not been studied in a definitive manner. To address this question, we used endogenous cholesterol in the erythrocyte ghost as a mechanistic reporter lipid Membranes sensitized with chloroaluminum Pc tetrasulfonate (AlPcS) and exposed to white light at 10 degrees C underwent lipid peroxidation, as indicated by the accumulation of hydroperoxides and thiobarbituric acid reactivity. Specific analysis of cholesterol photo-products by thin layer chromatography and high performance liquid chromatography revealed the presence of 3 beta-hydroxy-5 alpha-cholest-6-ene-5-hydroperoxide (5 alpha-OOH), with much smaller amounts of 3 beta-hydroxycholest-5-ene-7 alpha-hydroperoxide (7 alpha-OOH) and 5 alpha-cholest-6-en-3 beta, 5-diol and cholest-5-en-3 beta, 7 alpha-diol (5 alpha-OH and 7 alpha-OH). Identification of 5 alpha-OOH as a major photoproduct provides unambiguous evidence for large scale 1O2 intermediacy. Azide inhibited lipid peroxidation in a dose-dependent fashion, providing additional support for a type II mechanism. However, the 1O2 quenching constant from Stern-Volmer analysis was approximately 50 times lower than that determined for a non-membrane probe, lactate dehydrogenase. The latter value agreed with literature values. A probable explanation is that membrane-bound dye generates most of the 1O2 involved in lipid peroxidation. Although azide can intercept any 1O2 escaping into (or formed in) the medium, it has limited access to 1O2 generated on the membrane and reacting (or being quenched) near its site of origin.

Azides↗
PubMed1991

The role of glycolysis and univalent ions in phthalocyanine-sensitized photohaemolysis of human erythrocytes.

Phthalocyanines sensitize human erythrocytes to red light in vitro and in vivo. The mechanism of photosensitization of haemolysis of red blood cells was studied using haemoglobin release in vitro as an endpoint. We have previously shown that the cation present in the incubation medium determines the rate of photohaemolysis, with the rate increasing in the order Li+, Na+, K+, Cs+, Rb+. With halogen anions, the rate increases in the order F-, Cl-, Br-. With F- in isotonic solution the rate was about two orders of magnitude slower than with Cl-, so that haemolysis was practically prevented. Fluoride slowed haemolysis at very low concentrations (less than 1 mM) with an apparent K1 of 0.1 mM in isotonic NaCl. Most of the effect disappeared when F- was added after light exposure, with a half-time of 1 min. The effect of F- was specific to phthalocyanine. Neither Photofrin-induced photohaemolysis nor gramicidin-induced haemolysis were inhibited by fluoride. Addition of 10 mM deoxyglucose prior to photosensitization enhanced haemolysis and reduced cellular ATP levels by about 50% compared to controls containing glucose. Haemolysis was preceded by a reduction in ATP levels in the presence of both glucose and deoxyglucose. No significant decrease in ATP levels was found following light exposure in the presence of 0.75 mM F-. It is concluded that glycolysis and ATP are important in preventing photohaemolysis. The protective effect of F- may be related to its inhibition of a fast early reaction which triggers the events leading to photohaemolysis induced by phthalcyanine.

Anions↗
PubMed1991

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.

Aluminum↗
PubMed1991

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-.

Aluminum↗
PubMed1990

Cellular NAD+ and ATP levels in alkylation-induced cytotoxicity enhanced by an inhibitor of poly(ADP-ribose) synthesis.

Alkylating agents cause a marked depletion of cellular NAD+ levels by activating nuclear ADP-ribosyl transferase (ADPRT), which utilizes NAD+ as a substrate in the synthesis of poly(ADP-ribose). As a consequence of NAD+ depletion, it is possible that cellular ATP pools could be depleted. Because of this, exogenously supplied NAD+ had been proposed as a way to counteract some of the effects of an alkylator. We found that exogenously supplied NAD+ significantly increased intracellular levels of NAD+ in MMS- and MNNG-treated V79 Chinese hamster cells. Cytotoxicity was not changed by the exogenously supplied NAD+, however. 3-Aminobenzamide (3-ABA), an ADPRT inhibitor, prevented the depletion of intracellular NAD+ by MMS or MNNG treatment and potentiated cytotoxicity. As was the case without 3-ABA, exogenously supplied NAD+ plus 3-ABA did not change the cytotoxicity, even though NAD+ levels were increased. Intracellular ATP levels were also measured and were found to be unaffected following MMS treatment, and only slightly depleted following MNNG treatment. Exogenously supplied NAD+ raised these levels above those for their respective controls. Because survival was unaffected by elevated levels of NAD+ and ATP, our results suggest that depletion of cellular NAD+ pools following MMS and MNNG treatment is not a critical factor in determining cytotoxicity for these V79 cells. The energy reserves of V79 cells, at doses of MMS or MNNG which kill 99% of the cells, are apparently adequate to maintain normal levels of ATP.

Adenosine Triphosphate↗
PubMed1989

Inhibition of human lymphocyte stimulation by visible light and phthalocyanine photosensitization: mitogen and wavelength dependency.

Mitogenic stimulation of human peripheral blood lymphocytes is inhibited by phthalocyanine photosensitization using visible light. The mechanism of inhibition was studied using stimulating agents differing in their mode of action. Stimulation by the plant lectin phytohemagglutinin (PHA) was the least sensitive to inhibition by photosensitization, followed by the tumor promoter phorbol myristate acetate (PMA) and the calcium ionophore A23187. Mitogenic stimulation could also be inhibited by light only, but higher fluences were needed. The order of efficacy was blue greater than red greater than green light, and the response to A23187 was least affected, followed by an increased inhibition of the response to PHA and PMA stimulation. Possible targets and mechanisms for these effects are discussed.

Calcimycin↗
PubMed1989

Synthesis and photocytotoxicity of some new substituted phthalocyanines.

Some new, ring-substituted phthalocyanines have been synthesized and underwent preliminary testing for photodynamic activity using the colony forming ability of Chinese hamster cells in culture as an endpoint. Using 4-(3-N,N-diethylaminopropyl)phthalonitrile as a precursor, the previously unknown metal-free 2,9,16,23-tetra-(3-N,N-diethylaminopropyl)phthalocyanine was prepared and converted to its zinc (II) and its cationic water-soluble 2,9,16,23-tetramethylammonium zinc (II) iodide derivatives. Other new phthalocyanine derivatives tested, include a 2,9,16,23-tetra(2-hydroxymethyl-2-methylbutoxy)phthalocyaninato zinc (II) and 2,3,9,10,16,17,23,24-octahydroxyphthalocyaninato zinc (II) derivatives. Boron tribromide cleavage of the newly prepared and fully characterized 2,3,9,10,16,17,23,24-octamethoxyphthalocyanine gave the unstable metal-free octahydroxyphthalocyanine, which could only be characterized by ultraviolet-visible, NMR and IR spectroscopy.

Animals↗