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.     

Photophysical and Redox Properties of a Series of Phthalocyanines: Relation with Their Photodynamic Activities on TF-1 and Daudi Leukemic Cells

Abstract— The photodynamic therapy (PDT) efficiency of five phthalocyanines, chloroaluminum phthalocyanine (AlPc), dichlorosilicon phthalocyanine (SiPc), bis (tri- n -hexylsi-loxy)silicon phthalocyanine (PcHEX), bis (triphenyl-siloxy)silicon phthalocyanine (PcPHE) and nickel phthalocyanine (NiPc), was assessed on two leukemic cell lines TF-1 and erythroieukemic and B lymphoblastic cell lines, Daudi, respectively. AlPc showed the best photocytotox-icity leading to 0.008 surviving fraction at 2 × 10⁻⁹ M for TF-1 and 4 × 10⁻⁹ M for Daudi. At 5 × 10⁻⁷ M , SiPc and PcHEX induced a significant photokilling, whereas NiPc and PcPHE were inactive. Laser flash photolysis and photoredox properties of the phthalocyanines were investigated to try to relate these parameters with the biological effects. AlPc showed the longest triplet lifetime: 484 fis in dimethyl sulfoxide/H₂O. This value was increased up to 820 u.s when AlPc was complexed with human serum albumin used as a membrane model. Such an enhancement was not observed with the silicon phthalocyanines. Upon irradiation, all the phthalocyanines generated singlet oxygen with 0.29–0.37 quantum yield values. The reduction potentials of the excited states obtained from measurement in the ground state and energy of the excited triplets show that AlPc is the best electron acceptor. The in vitro photocytotoxicity observed and the measured parameters are in agreement with a key role of electron transfer in PDT assays involving these phthalocyanines.     

INACTIVATION OF Trypanosoma cruzi TRYPOMASTIGOTE FORMS IN BLOOD COMPONENTS BY PHOTODYNAMIC TREATMENT WITH PHTHALOCYANINES

Abstract— -Three phthalocyanine dyes HOSiPcOSi(CH₃)₂(CH₂)₃N(CH₃)₂ (Pc 4), HOSiPc-OSi(CH₃)₂(CH₂)₃N+(CH₃)3I- (Pc 5) and aluminum tetrasulfophthalocyanine hydroxide (AlOHPcS₄) were evaluated for their ability to inactivate the trypomastigote form of Trypanosoma cruzi in fresh frozen plasma (FFP) and red blood cell concentrates (RBCC). The compound Pc 4 was found to be highly effective in killing T. cruzi, Pc 5 less effective and AlOHPcS₄ ineffective. With FFP as the medium, a complete loss of parasite infectivity in vitro (≥5 log₁₀) was found to occur with 2 μ M Pc 4 after irradiation with red light (>600 nm) at a fiuence of 7.5 J/cm², while with RBCC as the medium, a complete loss was found to occur at a fiuence of 15 J/cm². Even without illumination, Pc 4 at 2 μ M also killed about 3.7-4.1 log₁₀ of T. cruzi in FFP during 30 min. Observed differences in T. cruzi killing by the various phthalocyanines may relate to differences in binding; Pc 4 binds to the parasites about twice as much as Pc 5. Ultrastructural analysis of treated parasites suggests that mitochondria are a primary target of this photodynamic treatment. The data indicate that Pc 4 combined with exposure to red light could be used to eliminate bloodborne T. cruzi parasites from blood components intended for transfusion. The inactivation of T. cruzi by Pc 4 in the dark suggests a possible therapeutic application.     

THE EFFECT OF IRRADIANCE ON VIRUS STERILIZATION AND PHOTODYNAMIC DAMAGE IN RED BLOOD CELLS SENSITIZED BY PHTHALOCYANINES

Abstract— Phthalocyanines are being studied as photosensitizers for virus sterilization of red blood cells (RBC). During optimization of the reaction conditions, we observed a marked effect of the irradiance on production of RBC damage. Using a broad-band light source (600–700 nm) between 5 and 80 mW/ cm², there was an inverse relationship between irradiance and rate of photohemolysis. This effect was observed with aluminum sulfonated phthalocyanine (AlPcS_n) and cationic silicon (HOSiPc-OSi[CH₃]₂ [CH₂]₃N⁺[CH₃]₃I^- phthalocyanine (Pc5) photosensitizers. The same effect occurred when the reduction of RBC negative surface charges was used as an endpoint. Under the same treatment conditions, vesicular stomatitis virus inactivation rate was unaffected by changes in the irradiance. Reduction in oxygen availability for the photochemical reaction at high irradiance could explain the effect. However, theoretical estimates suggest that oxygen depletion is minimal under our conditions. In addition, because the rate of photohemolysis at 80 mW/cm² was not increased when irradiations were carried out under an oxygen atmosphere this seems unlikely. Likewise, formation of singlet oxygen dimoles at high irradiances does not appear to be involved because the effect was unchanged when light exposure was in D₂O. While there is no ready explanation for this irradiance effect, it could be used to increase the safety margin of RBC virucidal treatment by employing exposure at high irradiance, thus minimizing the damage to RBC.     

The Relationship of Phthalocyanine 4 (Pc 4) Concentrations Measured Noninvasively to Outcome of Pc 4 Photodynamic Therapy in Mice

The ability to noninvasively measure photosensitizer concentration at target tissues will allow optimization of photodynamic therapy (PDT) and could improve outcome. In this study, we evaluated whether preirradiation tumor phthalocyanine 4 (Pc 4) concentrations, measured noninvasively by the optical pharmacokinetic system (OPS), correlated with tumor response to PDT. Mice bearing human breast cancer xenografts were treated with 2 mg kg⁻¹ Pc 4 iv only, laser irradiation (150 J cm⁻²) only, Pc 4 followed by fractionated irradiation or Pc 4 followed by continuous irradiation. Laser irradiation treatment was initiated when the tumor to skin ratio of Pc 4 concentration reached a maximum of 2.1 at 48 h after administration. Pc 4 concentrations in tumor, as well as in Intralipid in vitro , decreased monoexponentially with laser fluence. Pc 4-PDT resulted in significant tumor regression, and tumor response was similar in the groups receiving either fractionated or continuous irradiation treatment after Pc 4. Tumor growth delay following Pc 4-PDT correlated with OPS-measured tumor Pc 4 concentrations at 24 h prior to PDT ( R ² = 0.86). In excised tumors, OPS-measured Pc 4 concentrations were similar to the HPLC-measured concentrations. Thus, OPS measurements of photosensitizer concentrations can be used to assist in the scheduling of Pc 4-PDT.     

SULFOPHTHALOCYANINES FOR PHOTODYNAMIC INACTIVATION OF VIRUSES IN BLOOD PRODUCTS: EFFECT OF STRUCTURAL MODIFICATIONS

Abstract— Transmission of infectious diseases through blood transfusions is well known. Ultraviolet irradiation, solvents and detergents provide a means of sterilizing noncellular blood components. However these harsh methods are not applicable to cellular blood products. Recently, attempts have been made to sterilize biological fluids using photodynamic treatment and phthalocyanine (Pc) dyes have been advanced as photosensitizers for this purpose. We have evaluated a series of water-soluble Pc, chelated with different central metal ions, substituted to different degrees with sulfonato and r-butyl groups, for their effectiveness to reduce virus infectivity in red blood cell suspensions. Vaccinia virus cytopathogenicity was determined by endpoint serial dilutions in the CV-1 cell line. Anti-viral activity increased with the central metal ion in the following order: Ga(III) < Al(III) < Zn(II), and varied inversely with the degree of sulfonation. Furthermore, addition of a t -butyl group onto the trisulfonated dyes (PcS₃[ t -Bul) resulted in a 5–40-fold increase in anti-viral potency, suggesting that amphiphilicity enhances the photodynamic activity of the dye. Strong anti-viral photosensitizing properties cannot be the sole selection criterion. Of equal importance is the preservation of blood component integrity. Accordingly, the photohemolytic activity of the dyes was evaluated using the rate of hemolysis as a parameter and a toxicity index was defined. Among the most active dyes, the AlPcS₃( t -Bu) complex exhibited the most favorable anti-viral properties combined with a low toxicity index. Our results suggest that trisulfophthalocyanines, bearing an additional t -butyl group to enhance amphiphilicity, are particularly promising dyes for photodynamic blood sterilization.     

PHOTODYNAMIC EFFECTS OF NEW SILICON PHTHALOCYANINES: In vitro STUDIES UTILIZING RAT HEPATIC MICROSOMES AND HUMAN ERYTHROCYTE GHOSTS AS MODEL MEMBRANE SOURCES

Abstract— Photodynamic therapy (PDT) of cancer is a modality that relies upon the irradiation of tumors with visible light following selective uptake of a photosensitizer by the tumor tissue. There is considerable emphasis to define new photosensitizers suitable for PDT of cancer. In this study we evaluated six phthalocyanines (Pc) for their photodynamic effects utilizing rat hepatic microsomes and human erythrocyte ghosts as model membrane sources. Of the newly synthesized Pc, two showed significant destruction of cytochrome P-450 and monooxygenase activities, and enhancement of lipid peroxidation, when added to microsomal suspension followed by irradiation with ∼ 675 nm light. These two Pc named SiPc IV (HOSiPcOSi[CH₃]₂[CH₂]₃N[CH₃]₂) and SiPc V (HOSiPcOSi[CH₃]₂[CH₂]₃N[CH₃]₃¹ I) showed dose-dependent photodestruction of cytochrome P-450 and monooxygenase activities in liver microsomes, and photoenhancement of lipid peroxidation, lipid hydroperoxide formation and lipid fluorescence in rnicrosomes and erythrocyte ghosts. Compared to chloroaluminum phthalocyanine tetrasulfonate, SiPc IV and SiPc V produced far more pronounced photodynamic effects. Sodium azide, histidine, and 2,5-dimethylfuran, the quenchers of singlet oxygen, afforded highly significant protection against SiPc IV- and SiPc V-mediated photodynamic effects. However, to a lesser extent, the quenchers of superoxide anion, hydrogen peroxide and hydroxyl radical also showed some protective effects. These results suggest that SiPc IV and SiPc V may be promising photosensitizers for the PDT of cancer.     

ON THE ACRIDINE AND THIAZINE DYE SENSITIZED PHOTODYNAMIC INACTIVATION OF LYSOZYME—SINGLET OXYGEN SELF-QUENCHING BY THE SENSITIZERS

Abstract— The quantum yield of the photodynamic inactivation of lysozyme increases in the sequence acridine orange, methylene blue, proflavine and acriflavine (1:5:6:12). At least up to protein concentrations of 0.1 m M , singlet oxygen is exclusively responsible for the inactivation of the enzyme. For methylene blue, acriflavine and proflavine the quantum yields decrease considerably with increasing dye concentrations. From measurements in H₂O and D₂O buffer solutions it was concluded that in the case of methylene blue the effect is mainly caused by the quenching of singlet oxygen [rate constant (3–4) × 10⁸ M ⁻¹ s⁻¹]. For the acridine sensitizers both singlet oxygen and dye triplet quenching processes have to be taken into consideration. It has been found that all sensitizers act as competitive inhibitors of the enzymatic reaction of lysozyme. However, the dye-protein interaction near the active center cannot be responsible for the observed dye self-quenching effect.     

FURTHER IN VIVO STUDIES ON THE PARTICIPATION OF SINGLET OXYGEN IN THE PHOTODYNAMIC INACTIVATION AND INDUCTION OF GENETIC CHANGES IN SACCHAROMYCES CEREVISIAE

Abstract —In vivo participation of singlet excited oxygen (¹O₂, ¹Δ9) in the photodynamic inactivation and induction of genetic changes (gene conversion) in acridine orange-sensitized yeast cells was investigated by using N₃^-, an efficient ¹O₂ quencher, and D²O, a known agent for the enhancement of the lifetime of ¹O₂. The addition of N₃^- protected the cells from both photodynamic actions. From an analysis of the concentration-dependent protection, about 80% of the induction of the genetic change is explainable on the basis of ¹O₂ mechanism. The quantitative estimation of the N₃^- protection in the inactivation was not possible because of the sigmoidal nature of the inactivation curve. The replacement of H₂O with D₂O during illumination was effective in enhancing the photodynamic inactivation but almost completely ineffective for the gene conversion induction. The deuterium effect with the cell system was clearly not as large as would be expected from in vitro experiments. This, however, could be explained from the kinetic consideration that natural quenchers of ^lO₂ in the cell would mask the deuterium effect. By experiments with different cell stages it was demonstrated that these two modifying effects were dependent on the intracellular reaction environment. The conclusion is that ¹O₂ must be the major intermediate responsible for the photodynamic actions in acridine orangesensitized yeast cells.     

HEMATOPORPHYRIN DERIVATIVE (PHOTOFRIN®) PHOTODYNAMIC ACTION ON Ca2+ TRANSPORT IN MONKEY KIDNEY CELLS (CV-1)

Abstract After 24 h incubation with Photofrinm^® (PF), photodynamic action has been studied on Ca²⁺ transport in CV-1 cells. A moderate increase of the cytosolic free Ca²⁺ concentration [Ca²⁺] is observed immediately after a dose of irradiation which yields a survival rate of less than 5% at 48 h. In parallel, studies on digitonin-permeabilized cells indicate that such a treatment inhibits endoplasmic reticulum Ca²⁺ uptake with few alterations of this process in mitochondria. In contrast, ADP-stimulated respiration is impeded and intracellular ATP level decreases. It is suggested that direct damage to endoplasmic reticulum as well as mitochondrial disturbance are the primary mechanisms responsible for a nontransient elevation of [Ca^2+]i preceding cell death.     

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⁻.     

MIGRATION OF EXCITATION ENERGY FROM THIONINE TO METHYLENE BLUE IN MICELLES

Abstract— The main absorption bands of thionine (Th⁺) and methylene blue (MB⁺) in aqueous solution lie at 598 nm and 664 nm, respectively. This position permits excitation energy transfer from Th⁺ to MB⁺, but not vice versa. We describe here studies of such transfer between these molecules adsorbed on micelles of sodium lauryl sulfate (SLS), imitating, at least to some extent, the state of pigments in chloroplasts.
The SLS concentration was varied from 3.0 to 11 × 10^-3 M. In the presence of dye, aggregation to micelles, each containing 70–100 detergent molecules, begins at about 3.0 × 10^-3 M SLS. Practically all dye ions are adsorbed on these micelles as soon as their formation begins.
Energy transfer from adsorbed Th⁺ ions to adsorbed MB⁺ ions can be demonstrated by observing the quenching of the fluorescence of thionine and the sensitization of that of methylene blue.
At [Th⁺] = [MB⁺] = 1 × 10^-5 M , the most efficient energy transfer (82 per cent efficiency, as derived from measurements of the quenching of Th⁺ fluorescence, or 90 per cent, as derived from sensitization of MB⁺ fluorescence) is observed at the lowest SLS-concentration (3.0 × 10^-3 M ), when the only micelles present are those formed by aggregation of dye-carrying low molecular complexes of SLS with dye cations. Each micelle carries, under these conditions, 10–14 molecules of the two dyes, and the distance between two closest dye ions is about 16 A. Transfer becomes less efficient as the SLS-concentration increases, causing pigment molecules to distribute themselves among a greater number of micelles.     

Distribution of Chlorophyll- and Bacteriochlorophyll-derived Photosensitizers in Human Blood Plasma

Chlorophyll a and, in particular, bacteriochlorophyll a derivatives are promising candidates for photosensitizers in photodynamic therapy. The distribution of 21 (bacterio)chlorophyll derivatives among human blood plasma fractions was studied by iodixanol gradient ultracentrifugation and in situ absorption spectroscopy. Modifications of the natural pigments involved the central metal (Mg²⁺, Zn²⁺, Pd²⁺, none), the isocyclic ring (closed, open and taurinated), substituents at C-3 (vinyl, acetyl, 1-hydroxyethyl) and C-17³ (phytyl ester, free acid). Cellular blood components bound only a small fraction of the pigments. Distribution among low-density lipoproteins (LDL), high-density lipoproteins (HDL) and high-density proteins (HDP) of the plasma was influenced as follows: (1) application in Cremophor^® EL slightly altered pigment distribution by lipoprotein modification, (2) only very polar pigments with multiple hydrophilic substituents showed substantial HDP binding, (3) the presence of the esterifying alcohol at C-17³ caused enrichment in LDL, this was more pronounced with bacteriochlorophylls than with chlorophylls, (4) substituents at C-3 had only little influence on the distribution, (5) Zn²⁺-complexes were enriched in HDL compared to Mg²⁺ and Pd²⁺ complexes, indicating specific binding of the former. Equilibration of pigments among the different fractions was largely complete within 3 h.     

Ca2+ INFLUX INDUCED BY PHOTODYNAMIC ACTION IN HUMAN CEREBRAL GLIOMA (U-87 MG) CELLS: POSSIBLE INVOLVEMENT OF A CALCIUM CHANNEL

Abstract The plasma membrane has been implicated as a critical target of photodynamic action on cells. We have observed that the photosensitization of human cerebral glioma (U-87 MG) cells by hematoporphyrin derivative (HpD) causes a large increase in intracellular calcium [Ca²⁺]. This increase in [Ca²⁺]_i was solely due to the influx of extracellular Ca²⁺ through the plasma membrane and showed a dependence on HpD concentration, light dose and concentration of calcium in the extracellular medium. The magnitude of the Ca²⁺ influx decreased with increasing postirradiation time, which suggests that the cell membrane partially recovers from the photodynamic injury. The photoinduced Ca²⁺ influx was inhibited by the Ca²⁺ channel blocker diltiazem and the reducing agent dithioerythritol. These findings are discussed in terms of possible activation of a Ca²⁺ channel as a result of photosensitization.     

HEMATOPORPHYRIN DERIVATIVE-INDUCED PHOTODYNAMIC INHIBITION OF Na+/K+-ATPase IN L929 FIBROBLASTS, CHINESE HAMSTER OVARY CELLS AND T24 HUMAN BLADDER TRANSITIONAL CARCINOMA CELLS

The photodynamically induced inhibition of Na⁺/K⁺-ATPase with hematoporphyrin derivative as photosensitizer was studied in murine L929 fibroblasts, Chinese hamster ovary (CHO) cells and T24 human bladder transitional carcinoma cells. In T24 cells the inhibition of the enzyme activity appeared to be caused by ATP depletion rather than by direct damage from the enzyme itself. In L929 and CHO cells, on the other hand, the inhibition was caused by photodynamic damage from the enzyme molecule. For all three cell lines it was shown that a causal relationship between photodynamically induced reduction in Na⁺/K⁺-ATPase activity and the loss of clonogenicity is highly unlikely.     

Variation in Photodynamic Efficacy during the Cellular Uptake of Two Phthalocyanine Photosensitizers

A decrease in the efficacy of photodynamic therapy (PDT) with phthalocyanine photosensitizers was observed for lymphoblastic murine and human cell lines as the time between the addition of the photosensitizer, aluminum phthalocyanine (AlPc), to the culture medium and exposure to light was increased from 4 h to 18 h. The total intracellular concentration of photosensitizer did not decrease significantly during this 18 h interval. For the murine cell lines, the maximum cytotoxic and mutagenic effects were observed when the time between addition of the photosensitizer and irradiation was between 1 and 4 h. The time course of the variations in efficacy did not vary greatly from one murine cell line to another, even though the cell lines differ markedly in the extent of their cytotoxic and mutagenic response. The time course of the variation was similar for cytotoxicity and mutagenicity, as well as for the induction of DNA fragmentation. The human lymphoblastic cell line, WTK1, showed less variation in survival and mutability with time than did the murine cell lines. With Pc 4 (HOSiPcOSi[CH3]2[CH2]3N[CH3]2) as the photosensitizer, the photocytotoxicity for murine L5178Y (LY)-Sl cells did not change significantly as the time between addition of Pc 4 and irradiation was increased from 2 to 18 h. However, the mutagenicity decreased by a factor of three during this interval. The mutagenicity of PDT with Pc 4 was much less in LY-Sl cells than that with AlPc. The results suggest that the variation in the efficacy observed for AIPc-induced photocytotoxicity is caused by changes in the intracellular distribution and/or the aggregation of the photosensitizer with time after its addition.     

Cationic Hypericin Derivatives as Novel Agents with Photobactericidal Activity: Synthesis and Photodynamic Inactivation of Propionibacterium acnes

The present communication describes for the first time the synthesis and preliminary testing of two cationic hypericin derivatives. Uncharged hypericin derivatives with ω , ω '-attached C₂-linkers leading to a pyridyl or a 4-dimethylaminophenyl residue were prepared and subsequently quaternized by means of iodomethane. Photobactericidal activity was assessed using Propionibacterium acnes . The quaternary N , N , N -trimethyl-anilinium derivative displayed a pronounced photodynamic inactivation of the bacteria at low incubation concentrations (<100 n m ) and a short incubation time (1 h) after illumination with yellow light (590 nm, 20 J cm⁻²), whereas the photobactericidal efficacy of the N -methyl-pyridinium derivative was negligible under identical experimental conditions.     

PHOTOELECTRIC EFFECTS FROM CHLOROPHYLL a IN BILAYER MEMBRANES

Abstract— The photophysical and photochemical properties of thiophene derivatives have been studied by fluorescence and by 353 nm laser flash spectroscopy. α-Terthienyl and its derivatives show a moderate fluorescence quantum yield (less than 0.1) in cyclohexane, ethanol, or TritonX–100 micelles. An additional thiophene ring increases this value to 0.2 in ethanol or micelles. The transient triplet state of the six thiophenes is characterized by strong absorptions (ε⋍ 50000 M ^-1 cm^-1) in the visible region. These triplet states are very long lived. They react with oxygen, producing singlet oxygen very efficiently because of their high quantum yield of triplet formation (0.1 to 0.3). They do not react with excellent hydrogen or electron donors such as indole, N-acetyl tryptophanamide or cysteine. The hydrophobic thiophenes investigated are, therefore, Type II photodynamic agents almost exclusively.     

TOLUIDINE BLUE: THE MODE OF PHOTODYNAMIC ACTION IN YEAST CELLS

Abstract— Toluidine blue, a thiazine dye, was shown to have in vivo photodynamic activity through singlet oxygen (O₂¹Δ _g ) production. This was based mainly on the effective protection by N^-₃ and the marked enhancement in D₂O for the sensitized inactivation of yeast cells. The mode of the in vivo activity was, however, quite different from that of acridine orange, for which the singlet oxygen mechanism has also been proposed. The most characteristic feature in the toluidine blue-sensitization was the total lack of the induction of gene conversion (at trp 5), while the survival went down below 10%. The non-induction of genetic changes was confirmed at several pH's in the neutral region, whereas the inactivation was seen in parallel to the reported pH dependence of singlet oxygen production in vitro . Direct measurements by microspectrophotometry showed none of the toluidine blue was accumulated in the cell. It was also ascertained from acridine-sensitized induction of gene conversion that toluidine blue never interfered with the binding of acridine orange to cellular DNA. These findings suggested that the unique mode of photodynamic activity of toluidine blue is attributable to its action from outside of the cell. Furthermore, comparisons between the photodynamically treated cells (with toluidine blue) and non-treated cells with respect to the response to UV irradiation excluded certain cell functions relating to the expression of gene conversion from the possible damage sites. The photo-reactivation process of UV induced gene conversion was not disturbed by the pre-toluidine blue sensitition. In view of the foregoing results, the plasma membrane was tentatively suggested as the most likely site of damage.     

PHOTOIONIZATION OF CRYSTAL VIOLET IN AQUEOUS SOLUTION

Abstract— Laser flash photolysis studies were carried out on a triphenylmethane dye, crystal violet (CV⁺), at 248 nm in aqueous solutions. The results show that CV⁺ undergoes photoionization and the resulting transients CV^-2+, hydrated electrons (e^-_aq) and CV⁺ radical formed by the reaction of e^-_aq with CV⁺ have been characterized. Studies using suitable scavengers were done to support the characterization of the transient species. Laser intensity effects show that the ionization is biphotonic. Two mechanisms are proposed to explain the observed photoionization involving higher excited singlet state and/ or another long-lived excited state of the dye.