The laws of delayed photohaemolysis sensitized by chlorin e6.

The relationships between the rate of post-irradiated photohaemolysis sensitized by chlorin e6 and parameters such as the light fluence (time of irradiation) and sensitizer concentration were studied. On the basis of the single-parametric approach proposed by Valenzeno and Pooler, it was found that the haemolytic rate varies with the square of both the light fluence and the sensitizer concentration. Thus it can be concluded that, in a single erythrocyte lesion, two chlorin e6 molecules participate, each absorbing one photon. The possibility of suppression of post-irradiation haemolysis was also studied using the lipophilic antioxidant, butylated hydroxytoluene (BHT), and scavengers of 1O2, O2.- and HO. radicals. It was found that BHT inhibits, to a considerable extent, the post-irradiation lysis of cells, by about a factor of 2.5 at a BHT concentration of 9 microM. The addition to the medium of NaN3 (a scavenger of 1O2), superoxide dismutase (a scavenger of O2.- radicals), ethanol and D-mannitol (scavengers of HO. radicals), when irradiation was interrupted, did not produce a marked influence on the kinetics of subsequent haemolysis. On the basis of the results obtained, the nature of erythrocyte targets, which are crucial for the photodynamic effect of chlorin e6, is discussed.     

PHOTOSENSITIZATION BY 2-CHLORO-3, 11-TRIDECADIENE-5, 7, 9-TRIYN-1-OL: DAMAGE TO ERYTHROCYTE MEMBRANES, Escherichia coli, AND DNA

The natural product 2-chloro-3,11-tridecadiene-5,7,9-triyn-1-ol (1) photosensitized the inactivation of Escherichia coli in the presence of near-ultraviolet light (320-400 nm; NUV) under both aerobic and anaerobic conditions. A series of E. coli strains differing in DNA repair capabilities and catalase proficiency exhibited indistinguishable inactivation kinetics following treatment with the chemical plus NUV. The presence of carotenoids did afford some protection to E. coli against inactivation under aerobic conditions, consistent with the involvement of singlet oxygen. The photosensitized hemolysis of human erythrocytes occurred more rapidly in the absence than in the presence of oxygen. Aerobically, the onset of hemolysis was partially inhibited by NaN3 and by 2,6-di-t-butyl-4-methylphenol (BHT) but not by superoxide dismutase (SOD). The aerobic lipid peroxidation observed in the membranes of erythrocyte ghosts was completely inhibited by BHT, and partially by NaN3, but not by SOD. These results suggest that either lipid peroxidation of the membrane is not the main cause of photohemolysis or that BHT has insufficient access to intact erythrocyte lipids to protect them. Aerobically, crosslinking of membrane proteins was also observed; it was not affected by SOD, but was partially inhibited by BHT and NaN3. The anaerobic photosensitized hemolysis of erythrocytes was more rapid; a radical mechanism was suggested since BHT inhibited the hemolysis to a greater extent than under aerobic conditions. Neither lipid peroxidation nor protein crosslinking was observed under conditions believed to be anaerobic. A light-dependent electron transfer to cytochrome c was obtained under argon but not under oxygen. Although induced mutations were not observed in the experiments with E. coli, 1 was capable of damaging both supercoiled pBR322 and Haemophilus influenzae transforming DNA in a manner that seemed to be equivalent under aerobic and anaerobic conditions. In conclusion, 1 can behave as typical photodynamic molecule under aerobic conditions but, in contrast to most photodynamic molecules, it is also phototoxic under anaerobic conditions. The extent to which the radical reactions detected under anaerobic reactions compete with the photodynamic processes when oxygen is present is not known.     

Hypericin-mediated photocytotoxic effect on HT-29 adenocarcinoma cells is reduced by light fractionation with longer dark pause between two unequal light doses

The present study demonstrates the in vitro effect of hypericin-mediated PDT with fractionated light delivery. Cells were photosensitized with unequal light fractions separated by dark intervals (1 or 6 h). We compared the changes in viability, cell number, survival, apoptosis and cell cycle on HT-29 cells irradiated with a single light dose (12 J/cm(2)) to the fractionated light delivery (1 + 11 J/cm(2)) 24 and 48 h after photodynamic treatment. We found that a fractionated light regime with a longer dark period resulted in a decrease of hypericin cytotoxicity. Both cell number and survival were higher after light sensitization with a 6-h dark interval. DNA fragmentation occurred after a single light-dose application, but in contrast no apoptotic DNA formation was detected with a 6-h dark pause. After fractionation the percentage of cells in the G1 phase of the cell cycle was increased, while the proportion of cells in the G2 phase decreased as compared to a single light-dose application, i.e. both percentage of cells in the G1 and G2 phase of the cell cycle were near control levels. We presume that the longer dark interval after the irradiation of cells by first light dose makes them resistant to the effect of the second illumination. These findings confirm that the light application scheme together with other photodynamic protocol components is crucial for the photocytotoxicity of hypericin.     

Hypochlorous acid-induced membrane pore formation in red blood cells

The hyperproduction of hypochlorous acid (HOCl), an extremely toxic biological oxidant generated by neutrophils and monocytes, is involved in the pathogenesis of many diseases. In these studies, we attempted to determine the membrane and cellular events associated with HOCl-induced erythrocyte impairment and haemolysis. In vitro human erythrocyte exposure to HOCl (0.1-1.0 mM) resulted in rapid oxidation of reduced glutathione, an increase in cell osmotic fragility and the formation of transient membrane pores. The process of glutathione oxidation depended on the [oxidant]/[cell number] ratio. The HOCl-induced haemolysis observed was apparently mediated by pore formation and altered membrane electrolyte permeability. The estimated pore radius was approximately 0.7 nm and the average number per cell was 0.01. The rate constant of HOCl-produced haemolysis depended on pH. There were significant differences in haemolysis of HOCl-treated erythrocytes which had maximal stability at pH 7.2-7.3.     

Electrical breakdown of human erythrocytes: a technique for the study of electro-haemolysis

This paper describes a technique suitable for investigating the electromechanical breakdown properties of erythrocyte cells. The cells were exposed to square wave electric pulses of precise duration and voltage. The erythrocytes were suspended in normal isotonic saline between two opposing platinum electrodes. A red LED light source and photodiode detector system were positioned orthogonally to the electrodes to record changes in the light transmission that occur immediately after applying an electric pulse. The light transmitted through the electrically treated erythrocyte suspension could be monitored continuously. Experiments were conducted to explore the inter-relationship between the critical voltage and pulse length for haemolysis. Human blood taken from "healthy" donors underwent haemolysis at a critical field strength of 304 kV/m for a 5 micros pulse and 292 kV/m for a 50 micros pulse. The relationship of critical pulse length and critical voltage for the blood samples was found to be inversely linear.     

A Bifunctional Photosensitizer for Enhanced Fractional Photodynamic Therapy: Singlet Oxygen Generation in the Presence and Absence of Light

The photosensitized generation of singlet oxygen within tumor tissues during photodynamic therapy (PDT) is self‐limiting, as the already low oxygen concentrations within tumors is further diminished during the process. In certain applications, to minimize photoinduced hypoxia the light is introduced intermittently (fractional PDT) to allow time for the replenishment of cellular oxygen. This condition extends the time required for effective therapy. Herein, we demonstrated that a photosensitizer with an additional 2‐pyridone module for trapping singlet oxygen would be useful in fractional PDT. Thus, in the light cycle, the endoperoxide of 2‐pyridone is generated along with singlet oxygen. In the dark cycle, the endoperoxide undergoes thermal cycloreversion to produce singlet oxygen, regenerating the 2‐pyridone module. As a result, the photodynamic process can continue in the dark as well as in the light cycles. Cell‐culture studies validated this working principle in vitro.     

Effect of polyethylenimine, a cell permeabilizer, on the photosensitized destruction of algae by methylene blue and nuclear fast red

The present study reports the effect a cell permeabilizer, polyethylenimine (PEI) has on the photodynamic effect of methylene blue (MB) and nuclear fast red (NFR) in the presence of hydrogen peroxide (H2O2). The photosensitized destruction of the algae Chlorella vulgaris under irradiation with visible light is examined. The photodynamic effect was investigated under aerobic and anaerobic conditions. The presence of a permeabilizer during the photosensitized destruction of C. vulgaris does not enhance the activity of the MB, MB/H2O2 system or the NFR, NFR/H2O2 system under aerobic conditions. However under anaerobic conditions we have determined that when a cell permeabilizer was added to the MB/H202 system, the photosensitized destruction of C. vulgaris proceeded via a combination of Type I and Type II mechanisms. The presence of PEI enforces MB/H2O2 to be active toward the destruction of C. vulgaris whether oxygen is present or absent. Under aerobic and anaerobic conditions the activity of NFR was suppressed in the presence of PEI as a result of electrostatic interactions between the photosensitizer and the cell permeabilizer. The decrease in fluorescence recorded is indicative of destruction of the chlorophyll a pigment.     

THE PHOTODYNAMIC IMMOBILIZATION OF ARTEMIA SALINA NAUPLII BY POLYCYCLIC AROMATIC HYDROCARBONS AND ITS RELATIONSHIP TO CARCINOGENIC ACTIVITY

Abstract— The rates of the photosensitized immobilization of the nauplii of the crustacean Artemia salina were measured as a function of irradiation time and the amount of light absorbed by the sensitizers. The nauplii were incubated in the dark in dilute solutions of the sensitizers for periods of 2 and 22 h prior to irradiation. Nineteen carcinogenic and 22 noncarcinogenic polycyclic aromatic hydrocarbons were used as sensitizers. Relative photodynamic activities (RPA) were determined from the rates of immobilization using benz[ c ]acridine as a standard (RPA = 1). High RPA was restricted to carcinogenic compounds with 4 and 5 fused rings, compounds with 6 or more fused rings had low RPA regardless of their carcinogenic activities. The correlation of RPA with carcinogenic activity was excellent ( P = 0.006 and 0.009 for the 2 and 22 h dark incubations, respectively). It is suggested that carcinogenesis by polycyclic aromatics may result from sublethal photodynamic effects.     

PHOTODYNAMIC DAMAGE AND ITS REPAIR IN Vibrio cholerae

Abstract— Repair of photodynamic damage induced by acriflavine and visible light has been examined in three strains of Vibrio cholerae differing in their capabilities to repair ultraviolet (UV) light induced DN A damage. Excision repair deficient wild type cells of strain 154 are more sensitive to photodynamic treatment compared to repair proficient cells of strain 569B. However, no difference in their capabilities to repair of damage following photodynamic treatment can be detected. No single-strand breaks in the irradiated cell DNA are observed when the cell survival is more than 10%. Single-strand breaks observed at cell survival less than 5% are not dark repairable even in excision repair proficient wild type cells. Repair of membrane damage can partially account for the recovery observed at low doses. In contrast, radiation-sensitive mutant 569B_s cells which lack both excision and medium-dependent dark repair for UV-lesions are most efficient in repairing damage induced by photodynamic treatment.     

Photodynamic inactivation of isolated crayfish mechanoreceptor neuron: different death modes under different photosensitizer concentrations

To study the mechanism of photodynamic nerve cell killing, isolated crayfish mechanoreceptor neurons were photosensitized by the sulfonated aluminum ophthalocyanine Photosens. Neuron activity was continuously recorded until irreversible abolition. Intense (10(-5) M Photosens) or weak (10(-7) M Photosens) photosensitization induced different bioelectric neuron responses: firing activation followed by irreversible depolarization block or gradual inhibition until firing abolition, respectively. These bioelectric responses were accompanied by different biochemical and morphological changes. In the case of intense photosensitization, neuron nuclei swelled and then shrank. Succinate dehydrogenase (SDH) was inhibited, and the plasma membrane was compromised just after firing cessation. Weak photosensitization did not induce these changes but caused swelling of the endoplasmic reticulum and destruction of the matrix, cristae and membranes in some of the mitochondria. Other mitochondria, however, retained the normal structure. Plasma membrane damage, SDH inhibition, nucleus shrinkage and impairment of the nuclear border occurred after 2-4 h. It is concluded that intense photosensitization induced necrotic processes during irradiation, whereas weaker impact caused delayed necrosis 2-4 h later. The observed electrophysiological neuron responses to photodynamic therapy may be considered as early hallmarks of different modes of forthcoming cell death.     

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.     

Effects of charged amphiphiles in depolarising solutions on potassium efflux and the osmotic fragility of human erythrocytes

The effect of the presence of charged amphiphiles during the incubation of human erythrocytes in a sucrose-substituted low-Cl(-) solution on the shift of the osmotic resistance profile and the net K+ efflux was investigated. Osmotic fragility was determined by fitting the complementary error function to the haemolysis resistance curve. K+ efflux was calculated from the increase in the K+ concentration in supernatant measured by inductively coupled plasma atomic emission spectrometry (ICP-AES). The cationic amphiphile hexadecyltrimethylammonium bromide (CTAB) at 14 microM decreases, whereas the anionic amphiphile sodium dodecyl sulfate (SDS) at 50 microM increases the shift of the haemolysis resistance curve of erythrocytes incubated in isotonic sucrose by 0.069 and 0.079 %NaCl, respectively. Both the positively and the negatively charged amphiphile caused a significant change in the K+ efflux into isotonic sucrose solution: CTAB decreased and SDS increased K+ efflux by about 40%. In view of the lack of effect of the investigated compounds on the haemolysis resistance curve and K+ efflux from human erythrocytes incubated in isotonic NaCl solution, these results suggest that the insertion of charged amphiphiles into the erythrocyte membrane modulates the properties of the K+ transport pathway which is activated under low ionic strength (LIS) conditions.     

HYDRODYNAMIC EFFECTS IN THE PHOTOSENSITIZED LYSIS OF LIPOSOMES*

Abstract— The photosensitized lysis of phosphatidylcholine liposomes incorporating methylene blue in the membrane or in the presence of external methylene blue is promoted by hydrodynamic agitation concurrent with or subsequent to irradiation with red light. The results implicate the attack of singlet oxygen on an unsaturated lipid component as the key photochemical step, which is followed by additional membrane damage induced by hydrodynamic action.     

INACTIVATION OF LIPID-CONTAINING VIRUSES BY HYDROPHOBIC PHOTOSENSITIZERS AND NEAR-ULTRAVIOLET RADIATION

Abstract—The hydrophobic photosensitizers acridine and phenothiazine inactivate the lipid-contnining viruses PM2,φ6, and herpes simplex when samples are illuminated with near-UV radiation. φ23–1- a . which is insensitive to organic solvents and presumably contains no lipids. is not inactivated under comparable conditions. For acridinc, the inactivation of virus requires that oxygen be present and is inhibited by sodium azide, implicating the involvement of singlet oxygen. For phenothiazine, oxygen is not required for photosensitized inactivation. Treatment of PM2 with acridine and near-UV light caused a complete disruption of the virion, as determined by sucrose gradient analysis of treated and untreated samples. These data and related observations suggest that lipid-containing viruses are inactivated through photosensitized membrane damage.     

Hypochlorous acid-induced oxidative damage of human red blood cells: effects of tert-butyl hydroperoxide and nitrite on the HOCl reaction with erythrocytes

Hypochlorous acid, one of the most powerful biological oxidants, is believed to be important in the pathogenesis of some diseases. The purpose of this study was to further characterise the membrane and intracellular events which resulted in HOCl-induced oxidative impairments and haemolysis of human erythrocytes and interaction of different oxidative agents, which accumulated during respiratory burst, in the process of RBS oxidation. The sequence of cellular events after red blood cell exposure to HOCl: cell morphological transformations, oxidation of cellular constituents, enzyme modifications, and haemolysis have been evaluated. It was shown that HOCl-treated cells underwent colloid-osmotic haemolysis, preceded by rapid morphological transformations and membrane structural transitions. The activation energy of the process of haemolysis (after removal of the excess of oxidative agent) was estimated to be 146+/-22 kJ/mol at temperatures above the break point of Arrhenius plot (31-32 degrees C). This value corresponds to the activation energy of the process of protein denaturation. Modification of erythrocytes by HOCl inhibited membrane acetylcholinesterase (uncompetitive type of inhibition), depleted intracellular glutathione, activated intracellular glutathione peroxidase, but did not induce membrane lipid peroxidation. The presence of other oxidants, nitrite or tert-butyl hydroperoxide (t-BHP), promoted the oxidative damage induced by HOCl and led to new oxidative reactions.     

IMINIUM SALT OF COPPER BENZOCHLORIN (CDS1), A NOVEL PHOTOSENSITIZER FOR PHOTODYNAMIC THERAPY: MECHANISM OF CELL KILLING

The mechanism of cell killing by CDS1, an iminium salt of octaethylbenzochlorin with copper in the aromatic ring, in combination with light from a noncoherent light source was investigated. Using a standard clonogenic assay and the AY-27 FANFT turnor line. photoactivation of CDS1 was shown to be cytotoxic. The photodynamic cell killing ability of CDS1 required the presence of molecular oxygen. The reactive species generated by light activation of CDS1 were effectively quenched by N.N' -diphenyl- p -phenylenediamine. Additionally, the photodynamic effect of CDS1 was not cnhanced by dcuterium oxide. To characterize the reactive oxygen species generated by the photoactivation of CDS1 the well-characterized erythrocyte ghost model was used. Superoxide dismutase and catalase were potcnt inhibitors of CDS1-induced lipid peroxidation of erythrocyte membranes. Sodium azide only partially inhibited lipid peroxidation. These findings differed from the known singlet oxygen generator, tin (II) etiopurpurin dichloride (SnET₂). Sodium azide was a potent inhibitor of SnET₂-induced lipid peroxidation, whereas superoxide dismutase and catalase were totally ineffective. Based on these results, we conclude that CDS1 requires the presence of molecular oxygen for cell killing to occur but appcars to act primarily through a non-singlet oxygen mechanism.     

MODE OF ACTION OF α-TERTHIENYL ON ESCHERICHIA COLI: EVIDENCE FOR A PHOTODYNAMIC EFFECT ON MEMBRANES

The photodynamic effects of α-terthienyl (αT) in near-UV light (UV-A) on Escherichia coli showed close agreement with the light absorption of αT at different wavelengths suggesting that αT is the primary absorbing molecule responsible for the photosensitized reaction. Studies with DNA repair deficient mutants of E. coli indicated that the bactericidal action of αT/UV-A was not mediated by DNA damage, in direct contrast to the well-known photosensitizer, 8-methoxypsoralen. By using a closed borosilicate glass reaction vessel and various gas mixtures, it was demonstrated that photosensitization of both E. coli and a more resistant bacterium, Pseudomonas aeruginosa , was absolutely dependent on the presence of oxygen. The rate of killing by αT/UV-A showed a rather small dependence on preincubation temperatures, with quite rapid killing at 5°C, suggesting that the movement of αT across the cytoplasmic membrane of E. coli is not the rate limiting step in killing and perhaps is not even necessary for killing. Sodium dodecyl sulphate-polyacrylamide gels of cell membrane proteins after 15 and 30min of treatment with αT/UV-A showed substantial random crosslinking of these proteins. The results taken overall suggest that αT is a photodynamic photosensitizer which exerts its primary effect at the level of the cytoplasmic membrane.     

Red blood cell ghosts and intact red blood cells as complementary models in photodynamic cell research

Recent research on erythrocytes as model cells for photodynamic therapy showed differing behaviour of certain photosensitisers in erythrocytes compared to other cells. Differences of dye accumulation in the cell membrane were proposed to be the reason for the distinct photodynamic effects. Using pheophorbide a as an example, the combination of erythrocyte ghosts as models to follow the dye accumulation in the cell membrane and intact erythrocytes as model cells to show the photodynamic damage is provided. Evidence for the correctness of the combination of erythrocyte ghosts and intact erythrocytes as a functioning model system in photodynamic cell research is provided using the confocal laser scanning microscopy on intact, pheophorbide a loaded erythrocytes.     

Cytotoxicity of All‐Trans‐Retinal Increases Upon Photodegradation†

All‐trans‐retinal (AtRal) can accumulate in the retina as a result of excessive exposure to light. The purpose of this study was to compare cytotoxicity of AtRal and photodegraded AtRal (dAtRal) on cultured human retinal pigment epithelial cells in dark and upon exposure to visible light. AtRal was degraded by exposure to visible light. Cytotoxicity was monitored by imaging of cell morphology, propidium iodide staining of cells with permeable plasma membrane and measurements of reductive activity of cells. Generation of singlet oxygen photosensitized by AtRal and dAtRal was monitored by time‐resolved measurements of characteristic singlet oxygen phosphorescence. Photodegradation of AtRal resulted in a decrease in absorption of visible light and accumulation of the degradation products with absorption maximum at ～330 nm. Toxicity of dAtRal was concentration‐dependent and was greater during irradiation with visible light than in dark. DAtRal was more cytotoxic than AtRal both in dark and during exposure to visible light. Photochemical properties of dAtRal indicate that it may be responsible for the maximum in the action spectra of retinal photodamage recorded in animals. In conclusion, photodegradation products of AtRal may impose a significant threat to the retina and therefore their roles in retinal pathology need to be explored.     

The effect of phenyltin chlorides on osmotically induced erythrocyte haemolysis

The toxicity of many amphiphilic compounds may result from their effect on the lipid phase of biological membranes. Upon incorporation such compounds may change the properties of membranes in general and in particular alter the organization of membrane lipids. These changes should affect, among other things, the mechanical properties of membranes. We selected two amphiphilic compounds, diphenyltin dichloride (Ph₂SnCl₂) and triphenyltin chloride (Ph₃SnCl), which are known to be located at different regions of the lipid bilayer and to be toxic. As a model biological membrane the erythrocyte plasma membrane was used. Analysis of the haemolysis kinetics showed differences between the effect of the compound studied on mechanical properties at so‐called non‐lytic concentrations. Diphenyltin dichloride showed a limited effect on erythrocyte haemolysis, whereas triphenyltin chloride affected all the parameters measured (extent of initial haemolysis, extent of final haemolysis and membrane mechanical strength). We correlated these effects with the location of the investigated compounds in liposomes. The presented data show that triphenyltin chloride reduces the erythrocyte plasma membrane mechanical strength and increases the extent of haemolysis under osmotic stress conditions. Copyright © 2005 John Wiley & Sons, Ltd.