PHOTOSENSITIZING EFFECTS OF HEMATOPORPHYRIN DERIVATIVE IMMOBILIZED ON SEPHAROSE

Abstract— The cytotoxicity that ensues following photosensitization by hematoporphyrin derivative (Hpd) is attributed to production of singlet oxygen. Many of the cellular end points reported to be affected are localized to membranes, hydrophobic environments conducive to partitioning of hydrophobic porphyrins in Hpd. In order to test the hypothesis that efficacy of Hpd-induced photosensitization is enhanced by its ability to freely enter cells or subcellular organelles, we immobilized Hpd on a sepharose support. This immobilized reagent was found to produce ¹O₂ when photoradiated, in yields similar to those observed for Hpd in solution, as evidenced by the bleaching of p -nitrosodimethylaniline in the presence of imidazole. The immobilized Hpd was capable of photosensitizing, i.e. inhibit, cytochrome c oxidase activity in intact mitochondrial membranes and in aqueous solution. However, enzymes located on the interior of mitochondrial membranes (F₀F₁ ATP synthase and succinate dehydrogenase), in the mitochondrial matrix (malate dehydrogenase), or on the inside of the plasma membrane, (Na++ K+)- ATPase, were unaffected by immobilized Hpd plus photoradiation compared to free Hpd. The results suggest that photosensitization by Hpd most likely arises from entry of the photosensitizer into the biological membrane, although proteins on the exterior membrane surface may be susceptible to damage by ¹O₂ produced in proximity to their location.     

PHOTOSENSITIZED LYSIS OF LIPOSOMES BY HEMATOPORPHYRIN DERIVATIVE

The spectral properties and efficiency for photosensitizing the lysis of phosphatidylcholine liposomes have been measured for the components of hematoporphyrin derivative (Hpd) after alkaline hydrolysis and fractionation by polyacrylamidc gel chromatography. Two major and two minor Hpd fractions have been identified whose spectral properties correlate with the anoxic sensitizing efficiency and the oxygen enhancement ratio (OER). The fastest moving fraction, which is the putative biologically active component, comprised one-third of the starting material and had OER = 2.7. Liposome lysis by this fraction was inhibited in the presence of human serum albumin at concentration ratios comparable to those employed for photoradiation therapy. The present results show that Hpd can act as an oxic and anoxic photosensitizer of a model biomembrane and suggest that separation from serum proteins is required for in vivo photosensitization.     

The effects of 1,2,3,4,6-penta-O-galloyl-beta-D-glucose on rat liver mitochondrial respiration

The inhibitory effects of pure galloylglucose (1,2,3,4,6-penta-O-galloyl-beta-D-glucose) on the respiratory chain of rat liver mitochondria were investigated. The respiratory control ratio (RCR) decreased by 50% on addition of 20 microM pentagalloylglucose to highly coupled mitochondria, but the adenosine-5'-diphosphate/oxygen (ADP/O) ratio decreased only slightly. The RCR disappeared and the ADP/O ratio could not be measured at concentrations of pentagalloylglucose above 30 microM. On the other hand, the uncoupler-induced oxygen consumption was also inhibited. These findings suggest that pentagalloylglucose at low concentrations inhibits the electron transport system to decrease the RCR, but scarcely impairs the membrane, practically retaining the coupled reaction, while at high concentrations it impairs the structural integrity of the mitochondrial membrane. Pentagalloylglucose competitively inhibited succinate dehydrogenase activity, and noncompetitively inhibited reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase and ubiquinol-1 oxidase activities of submitochondrial particles (SMP). However, it did not show significant inhibition of the cytochrome c oxidase activity of SMP. It is thus concluded that pentagalloylglucose, which is the lowest-molecular-weight component of tannic acid, exerts its effect on mitochondrial respiration and oxidative phosphorylation through action on the membrane and on succinate dehydrogenase, NADH dehydrogenase and cytochrome bc1 complex of mitochondria.     

IS RHODAMINE 123 A PHOTOSENSITIZER?

Because of conflicting reports on the photoxicity of rhodamine 123 (Rh 123), we have undertaken a study of Rh 123 photosensitization in several in vitro systems. First, Rh 123 is not a photodynamic agent and does not react with singlet oxygen. Second, when bound to cytochrome c (Cyt c), Rh 123 photosensitizes ferro Cyt c but not ferri Cyt c degradation by an oxygen-independent process. When delivered to skin fibroblasts where it specifically stains mitochondria, Rh 123 photosensitizes membrane damage. These results are consistent with the hypothesis that Rh 123 is a phototoxic stain. The lack of photosensitivity of Rh 123-stained mitochondria in some cell lines might therefore be due to specific structural features.     

EVIDENCE AGAINST THE PRODUCTION OF SUPEROXIDE BY PHOTOIRRADIATION OF HEMATOPORPHYRIN DERIVATIVE

Abstract Experiments were performed to ascertain whether superoxide anion (O₂⁻) was produced by the photodynamic activation of hematoporphyrin derivative (HPD). Three different systems were utilized to detect formation of O₂⁻, oxidation of epinephrine to adrenochrome, reduction of cytochrome c and reduction of nitro blue tetrazolium (NBT). The effects on these detectors under identical conditions for HPD + h ν were compared to those obtained with two O₂⁻ generating systems, riboflavin + by and xanthine-xanthine oxidase, and to a singlet oxygen generating system, photoradiation of methylene blue. The results indicated that HPD + hv differed from the two O₂⁻ generating systems in failing to reduce cytochrome c or NET, and that HPD + h ν was similar to the behavior of methylene blue + h ν . In addition, HPD + h ν but not the O₂⁻ generating systems could inhibit mitochondrial cytochrome c oxidase activity. We conclude that the photodynamic activation of HPD does not produce O₂⁻ as a major oxygen radical and that the effects of HPD + h ν on mitochondrial cytochrome c oxidase are not caused by O₂⁻.     

EFFECTS OF HEMATOPORPHYRIN DERIVATIVE ON THE CELLULAR ENERGY METABOLISM IN THE ABSENCE AND PRESENCE OF LIGHT

Effects of Photofrin II on energy metabolism and metabolic viability were studied in a mammalian transformed cell line (BHK-21) in dark and after photo-irradiation with visible light. Cells were allowed to accumulate Photofrin by incubating for 4 h in buffer containing Photofrin (5-60 micrograms/ml). The results show that Photofrin significantly affects the cellular energy metabolism even in the absence of light; activity of cytochrome c oxidase is decreased and glucose utilization and lactate production (glycolysis) are increased. Irradiation with light resulted in a significant decrease in the activity of cytochrome c oxidase, glycolysis, ATP content, energy charge, ratios of adenine nucleotides like ATP/ADP, ATP/AMP and cell viability (dye exclusion test). Presence of inhibitors of energy metabolism, potassium cyanide (respiration) and 2-deoxyglucose (glycolysis), further enhanced the cytotoxic effects induced by hematoporphyrin derivative and light.     

FLUORESCENCE SPECTRAL CHANGES OF HEMATOPORPHYRIN DERIVATIVE UPON BINDING TO LIPID VESICLES, Staphylococcus aureus AND Escherichia coli CELLS

Abstract— The binding of hematoporphyrin derivated (Hpd) to lipid vesicles and bacterial membranes was determined by fluorescence spectroscopy. The fluorescence measurements of Hpd in aqueous solutions showed two bands at 613 and 677 nm. In lipid environments of lecithin vesicles the fluorescence spectrum was shifted to 631 and 692 nm, respectively. Hpd was rapidly bound to the cell membrane of Staphylococcus aureus while much less binding occurred in the presence of Escherichia coli. At the same time, spheroplasts of both bacteria were shown to bind Hpd to a similar extent. These results are well correlated with the photoinactivation of the gram positive bacteria with Hpd while the gram negative cells were shown to be resistant. The pH dependence of both Hpd binding to S. aureus as well as the photodynamic inhibitory effect of the same bacteria are similar. It is concluded that the segregation of Hpd to the cell membrane is a prerequisite for its photodynamic effect.     

THE MECHANISM OF PHOTOSENSITIZATION IN PHOTODYNAMIC THERAPY: CHEMILUMINESCENCE CAUSED BY PHOTOSENSITIZATION OF PORPHYRINS IN SALINE CONTAINING HUMAN SERUM ALBUMIN

Chemiluminescence (CL) caused by photosensitization of porphyrins in phosphate buffered saline (PBS) solution containing 3% human serum albumin (HSA) was observed for the first time. Irrespective of porphyrins concerned, CL shows a spectrum ranging from 380 to 520 nm with a peak near 450 nm and decays almost single-exponentially with a lifetime of about 15 s. The intensity of CL depends on concentrations of porphyrins and HSA in PBS solution. We have examined a number of porphyrins and observed CL for the compounds with triplet lifetimes longer than 0.1 ms. The appearance and quenching of CL by photosensitization of porphyrin-HSA systems indicate that type II reaction by singlet oxygen occurs significantly in photodynamic therapy resulting in hypoxic regions in environments surrounding the sensitizer.     

Mitochondrial signaling in mammalian cells activated by red and near-IR radiation

Abstract Mitochondrial signaling is an information channel between the mitochondrial respiratory chain and the nucleus for the transduction signals regarding the functional state of the mitochondria. The present review examines the question whether radiation of visible and near-IR (IR-A) radiation can activate this retrograde-type cellular signaling pathway. Experimental data about modulation of elements of mitochondrial retrograde signaling by the irradiation (mitochondrial membrane potential DeltaPsi(m), reactive oxygen species ROS, Ca(2+), NO(*), pH(i), fission-fusion homeostasis of mitochondria) are reviewed. The terminal enzyme of the mitochondrial respiratory chain cytochrome c oxidase is considered as the photoacceptor. Functions of cytochrome c oxidase as a signal generator as well as a signal transducer in irradiated cells are outlined.     

LIGHT-DEPENDENT GENERATION OF SUPEROXIDE FROM HUMAN ERYTHROCYTES

Using the cytochrome c reduction method, we investigated light-dependent erythrocytic superoxide production. After 4 h light and dark exposure of erythrocytes from eight healthy human subjects, an average of 18.6% more superoxide was generated by erythrocytes exposed to light. Pretreatment of erythrocytes with the superoxide dismutase inhibitor N,N-diethyldithiocarbamate increased detection of superoxide while pretreatment with the anion channel blocker 4-acetamido-4'-isothiocyano-2,2'-disulfonic acid stilbene decreased detection. These findings indicate that substantially more spontaneously generated superoxide is produced and escapes from normal erythrocytes at ambient oxygen tensions on exposure to light. This excess generation and escape of superoxide from erythrocytes exposed to light may result in tissue photosensitization, especially in the retina of the eye, where high oxygen tension, blood and chronic light exposure occur simultaneously.     

EFFECTIVE PHOTODYNAMIC ACTION BY RHODAMINE 123 LEADING TO PHOTOSENSITIZED KILLING OF CHINESE HAMSTER OVARY CELLS IN TISSUE CULTURE AND A PROPOSED MECHANISM

The effectiveness of rhodamine 123 (R123) as a photosensitizer of cell killing is relatively low and correlates with its inefficient production of singlet oxygen. The known selective retention of R123 in the mitochondria of epithelially derived carcinoma cells, however, is a selective feature that could lead to a more useful therapeutic ratio if photosensitizing effectiveness could be increased. Chinese hamster ovary (CHO) cells in tissue culture were therefore exposed to R123 shortly before and during illumination under conditions controlled for oxygen concentration and temperature. Effective photosensitization of cell killing, as judged by colony formation, was produced by 95% but not by 19% O₂ during illumination of cells at 5d?C or 37d?C, and this was additionally enhanced at the sublethal temperature of 42d?C. Two CHO cell lines were examined; one line, CHO-AA8, was proficient in the repair of DNA damage and the parent to the second line, CHO-EM9, that was deficient in the repair of DNA strand breaks. Cells of both lines incorporated R123 to a similar degree and were similarly photosensitized by the presence of igh oxygen concentration. Furthermore, plasma membrane damage as judged by teh exclusion of trypan blue was not observed immediately after illumination in the presence of R123, but was seen in the presence of meso-tetra-(4-sulfonatophenyl)-porphine (TPPS₄). The extent of damage to the plasma membrane by TPPS₄ was greater in the presence of 95% compared to 19% O₂ during illumination. Photodynamic action at the level of teh plasma membrane appears to contribute to photosensitization by TPPS₄ but not by R123 soon after exposure of cells to these sensitizers. It is hypothesized that photodynamic action by R123 is the primary mechanism causing the observed photosensitization of cell killing, and that mitochondria are teh site of photosensitized damage responsible for this killing.     

CHEMILUMINESCENT DIPHENYLACETALDEHYDE OXIDATION BY MITOCHONDRIA IS PROMOTED BY CYTOCHROMES and LEADS TO OXIDATIVE INJURY OF THE ORGANELLE

Plant and animal mitochondria promote the aerobic oxidation of diphenylacetaldehyde (DPAA). This process is accompanied by chemiluminescence and rotenone-insensitive oxygen uptake. Tn rat liver and potato tubers, mitochondrial swelling is concurrently detected. Light emission and oxygen consumption decreased (about 50%) in cytochrome c-depleted mitochondria. A model system–cytochrome c or b5/dihexadecylphosphate liposomes–was also able to oxidize DPAA with parallel reduction of the cytochrome. Reduction of respiratory complex I or I plus II by addition of rotenone or antimycin A, respectively, did not prevent DPAA oxidation. However, when all cytochrome was reduced by addition of cyanide, aldehyde oxidation was completely suppressed. Altogether these data indicate that respiratory cytochromes are responsible for DPAA oxidation with production of excited species and consequent mitochondrial permeabilization.     

TYPE I AND TYPE II MECHANISMS IN THE PHOTOSENSITIZED LYSIS OF PHOSPHATIDYLCHOLINE LIPOSOMES BY HEMATOPORPHYRIN

Abstract The lysis of phosphatidylcholine (PC) liposomes was sensitized to visible light (>500nm) by hematoporphyrin (HP) incorporated in the liposomes (0.09-1.5%, wt/wt) or in the external buffer (1-15 μM). The lytic mechanism changed from the Type II pathway mediated by singlet oxygen (¹O₂) at low HP concentrations to the anoxic, Type I pathway at high HP concentrations. Spectral measurements of HP in aqueous and organic solvents indicate that the HP was not aggregated (monomers and/or dimers) for Type II sensitization and aggregated for Type I conditions. High concentrations of azide (>0.1 M) or DABCO (>0.5 M) were protective with high HP concentration under oxic and anoxic conditions, which cannot involve the scavenging of ¹O₂. Feasible protective mechanisms are quenching of the HP triplet state by high azide and repair of the damaged membrane by DABCO via an electron transfer process. There was significant protection against lysis under Type I conditions by low concentrations of ferricyanide (>1 mM), indicative of an electron transfer mechanism. The incorporation of 22 mol % cholesterol in PC liposomes with 1% HP had no effect on the lytic efficiency for oxic and anoxic conditions. Dipalmitoylphosphatidylcholine liposomes incorporating 1% HP showed negligible photosensitized lysis at 50°C compared with PC liposomes with 1% HP at 25°C. The promotion of photosensitized lysis by hydrodynamic agitation observed in prior work with methylene blue (Grossweiner and Grossweiner, 1982) was significant with HP sensitization for both Type I and Type II conditions. Actinometry with PC liposomes incorporating 1% HP indicated that photosensitized lysis was very inefficient, requiring many absorbed quanta per lysed liposome. Preliminary experiments with crude hematoporphyrin derivative (Hpd) showed similar concentration effects on lytic efficiency, where PC liposomes incorporating 0.1% (wt/wt) Hpd were strongly sensitized by oxygen, whereas sensitization by oxygen was insignificant with 3.1% Hpd. The results with HP and crude Hpd indicate that lytic damage in a biomembrane does not necessarily require oxygenation.     

Helium-neon laser irradiation of rat liver mitochondria gives rise to a new subpopulation of mitochondria: isolation and first biochemical characterization.

An experiment was performed to isolate the small atypical mitochondria produced during the irradiation of normal mitochondria with an He-Ne laser. Rat liver mitochondria were irradiated with a low-power continuous-wave He-Ne laser (energy dose, 5 J cm-2), followed by isolation using a sucrose gradient. In the irradiated sample, two bands were observed, one corresponding to normal mitochondria and the other to atypical mitochondria. Certain biochemical features of the mitochondria were investigated: mitochondrial enzyme activity and the presence of DNA and RNA were demonstrated. Hybridization experiments carried out with labelled mitochondrial probes, containing the genes for cytochrome oxidase subunit I and 12S rRNA, confirmed the mitochondrial nature of the isolated RNA.     

Determination of cyanide using a microbial sensor

A microbial cyanide sensor was prepared, consisting of immobilizedSaccharomyces cerevisiae and an oxygen electrode. When the electrode was inserted into a solution containing glucose, the respiration activity of the microorganisms increased. The change in the respiration activity is monitored with the oxygen electrode. When cyanide is added to the sample solution, the electron transport chain reaction of the respiration system in the mitochondria is inhibited, resulting in a decrease in respiration. The inhibition is caused by cyanide binding with respiration enzymes such as the cytochrome oxidase complex in the mitochondrial inner membrane. Therefore, the cyanide concentration can be measured from the change in the respiration rate. When the sensor was applied to a batch system at pH 8.0 and 30°C, the cyanide calibration curve showed linearity in the concentration range between 0.3 μM and 150 μM CN-.     

LIGHT-INDUCED LEAKAGE OF SPIN LABEL MARKER FROM LIPOSOMES IN THE PRESENCE OF PHOTOTOXIC PHENOTHIAZINES

Abstract— Liposomes prepared from dipalmitoyl lecithin, cholesterol and dicetyl phosphate and containing a trapped spin label marker were exposed to long wavelength UV light in the presence of a series of phenothiazine tranquilizers. EPR spectroscopy was used to detect spin label marker released from liposomes, taking advantage of the disappearance of line broadening from electron spin exchange which occurred on spin label release. The minimum effective phototoxic dose in mice of these phenothiazines was also determined. Kinetic studies of light-induced spin label release from phenothiazine-sensitized liposomes showed that membrane damage was rapidly induced and that the damaging species were short-lived. The damage process was oxygen dependent and could be temporarily prevented by cysteamine or α-tocopherol added immediately before irradiation. Only those phenothiazines which mediated light-dependent liposomal membrane damage had phototoxic activity in mice and the degree of photosensitization was parallel in the two systems. In both photosensitization phenomena, the nature of the substituent at the phenothiazine 2-position was more important than the phenothiazine side chain.     

Der Mensch,ein elektrisches Wesen

The cells of our body, and the organelles within the cells, are surrounded by a biological membrane. The membranes are composed of a lipid bilayer which has no conductivity for the electric current. Across most biological membranes of living cells exists an electric tension or an electrochemical potential. All expressions of human life require energy which is supplied by the molecule ATP. The chemical energy of ATP originates from “cold combustion" of nutrients in mitochondria, accompanied by the intermediate formation of an electrochemical potential. The ATP‐synthase uses the energy of the potential to form ATP. If the tension across the membrane exceeds a certain value, deleterious oxygen radicals are formed. High ATP‐values prevent the formation of oxygen radicals by inhibition of cytochrome c oxidase, the last step of cell respiration. During stress situations of the organism this ATP‐inhibition is switched off so that the electric tension increases and oxygen radicals are produced. These have been shown to cause multiple degenerative diseases.     

PHEOPHORBIDE a-INDUCED PHOTO-OXIDATION OF CYTOCHROME c: IMPLICATION FOR PHOTODYNAMIC THERAPY

Pheophorbide a-induced photo-oxidation, in vitro, of cytochrome c oxidase and cytochrome c results in irreversible modifications to both protein components. Photo-oxidation of cytochrome c, as exhibited by change in its heme oxidation state, displays exponential kinetics and is detected with a lag period. Both the photo-induced inactivation of the enzyme, and destruction of the substrate ability of cytochrome c occur as complex multi-process events. Under similar experimental conditions, the loss of the substrate capability of cytochrome c develops approximately three times faster than inactivation of the enzyme. The slight lag in the photo-oxidation of cytochrome c is due to pheophorbide a-induced superoxide production. However, the relative amount of photo-oxidant produced is considerably more effective than the cytochrome c reducing capacity of the superoxide. Neither hydroxyl radical nor hydrogen peroxide are involved in the photo-oxidation of the heme function. The possibilities of heme oxidation by a singlet oxygen mediated pathway or direct electron abstraction involving the heme or apoprotein are not excluded. It is proposed that a multi-site oxidation of numerous reduced energy cofactors within cells may augment collateral enzyme inactivation in maximizing photosensitizer-induced cytotoxicity. Accordingly, amphipathic photosensitizers, capable of accessing both lipid and aqueous compartments containing reduced cofactors, may be more effective agents for photodynamic therapy than those which exhibit a high specificity of subcellular localization.     

THE ROLE OF OXYGEN IN PHOTOSENSITIZATIONS WITH POLYACETYLENES AND THIOPHENE DERIVATIVES

Abstract— The mechanism of photosensitization by polyacetylenes and biosynthetically derived thiophenes from species of the plant family Asteraceae was examined. With thiophenes photosensitization of yeast and E. coli occurred under aerobic but not anaerobic conditions. For similar experiments with polyacetylenes, both photodynamic and non-photodynamic mechanisms were observed. While the relative toxicities of thiophenes and polyacetylenes under near UV radiation was in general similar, the in vitro generation of singlet oxygen was considerably less for polyacetylenes than thiophenes, which is additional evidence for the existence of an alternate mechanism of action in polyacetylene photosensitization. Rates of photodegradation of polyacetylenes are higher than for thiophenes suggesting that bond breaking/formation processes are more favored relative to energy transfer to oxygen for polyacetylenes than thiophenes.