A. Van de Vorst

QUANTUM YIELD OF SINGLET OXYGEN PRODUCTION BY XANTHENE DERIVATIVES

The singlet oxygen quantum yield (ϕ1o2) of 11 purified fluorescein derivatives was determined by reaction with singlet oxygen acceptors in aqueous and ethanolic solutions; in both solvents ϕ1o2 was enhanced with increasing halogenation. Tryptophan and 2,2,6,6‐tetramethylpiperidone were found to be unadapted for the determination of ϕ1o2, in our systems; however, the use of 9.10‐dipropionic anthracene acid andp‐nitrosodimethylaniline in conjunction with imidazole derivatives was suitable for 1O2 detection in water. Both methods lead to results in excellent agreement. As in ethanol. ϕ1o2, was equal to the triplet state quantum yield (ϕT), the comparison between the two solvents showed that ϕT in water was greater than in ethanol. The comparison between our values obtained with polychromatic light with published data obtained with monochromatic light suggests that the triplet quantum yield of fluorescein derivatives is wavelength independent.

MECHANISMS FOR DYE‐MEDIATED PHOTODYNAMIC ACTION: SINGLET OXYGEN PRODUCTION, DEOXYGUANOSINE OXIDATION AND PHAGE INACTIVATING EFFICIENCIES

Abstract The production of singlet oxygen (1O2) upon irradiation of several dyes in aqueous solution at pH 9.0, was quantitatively analyzed on the basis of the appearance of stable nitroxide radicals using the amine 2,2,6,6‐tetramethyl‐4‐piperidone as 1O2 acceptor. The dyes were checked for purity, their concentrations uniformized in terms of absorbance values and a correction factor was introduced which took into account the amount of photons absorbed. The rates of 1O2 production (in arbitrary units per min) were: 71 with rose Bengal, 70 with methylene blue, 61 with eosin Y, 18 with thiopyronine, 10 with proflavine and 9 with acridine yellow. Production of 1O2 was not observed with 9‐aminoacridine, acridine orange, quinacrine and ethidium bromide. Irradiated lumichrome initiated, with the same amine, another type of reaction. The rates of two other photoreactions were also determined under similar experimental conditions by following (i) the deoxyguanosine decomposition in which case the reaction was found to be less sensitive but largely parallel to the 1O2 production and (ii) the bacteriophage ØX174 inactivation in which case the dyes showed differences in their relative efficiencies. The proteinic capsid of the phage appeared as an effective impermeability barrier towards externally generated 1O2. Moreover, some of the dyes studied intercalated into the phage DNA, a process known to favor radicalar reactions.

Electron Spin Resonance Evidence of the Generation of Superoxide Anion, Hydroxyl Radical and Singlet Oxygen During the Photohemolysis of Human Erythrocytes with Bacteriochlorin A

Abstract— Photodynamic therapy with bacteriochlorin a (BCA) as sensitizer induces damage to red blood cells in vivo. To assess the extent of the contributuion of reactive oxygen species (ROS) and to determine a possible reaction mechanism, competition experiments with assorted ROS quenching or/and enhancing agents were performed in human erythrocytes as model system and in phosphate buffer. In the erythrocyte experiments, a 2% suspension was incubated with BCA for 1 h, washed with phosphate‐buffered saline, resuspended and subsequently illuminated with a diode laser using a fluence rate of 2.65 mW/cm2. Potassium leakage and hemolysis were light and BCA dose dependent. Adding tryptophan (3.3 mM), azide (1 mM) or histidine (10 mM) to the erythrocyte suspension before illumination delayed the onset of K‐leakage and hemolysis suggesting a type II mechanism. The D2O did not affect K‐leakage nor photohemolysis. Adding mannitol (13.3 mM) or glycerol (300 nM) also caused a delay in the onset of K‐leakage and hemolysis, suggesting the involvement of radicals. In phosphate buffer experiments, it was shown using electron spin resonance (ESR) associated with spin‐trapping techniques that BCA is able to generate 02∼* and OH* radicals without production of aqueous electron. Visible or UV irradiation of the dye in the presence of the spin trap 5,5‐dimethyl‐1‐pyrroline‐iV‐oxide (DMPO) gave an ESR spectrum characteristic of the DMPO‐hydroxyl radical spin adduct DMPO‐OH. Addition of ethanol or sodium formate produced supplementary hyperfine splittings due to the respective CH3CHOH * and CO2‐1 radical adducts, indicating the presence of free OH*. Production of DMPO‐OH was partly inhibited by superoxide dismutase (SOD), catalase and desferoxamine, suggesting that the iron‐catalyzed decomposition of H2O2 was partly involved in the formation of one part of the observed OH *. The complementary inhibition of DMPO‐OH production by azide and 9,10‐anthracenedipropionic acid (ADPA) was consistent with 1O2 production by BCA followed by reaction of 1O2 with DMPO and decay of the intermediate complex to form DMPO‐OH and free OH*. All our results seem to indicate that BCA is a 50%/50% type 1/type 2 sensitizer in buffered aqueous solutions and confirmed that the dye‐induced hemolysis of erythrocytes was well caused by a mixed type 1/type 2 mechanism.

Singlet oxygen production and photoisomerization: Two competitive processes for merocyanine 540 irradiated with visible light

The quantum yields of singlet oxygen production by merocyanine 540 have been measured during visible light irradiation performed in methanol and ethanol. These appear to be one hundred times smaller than the quantum yield for rose bengal measured under the same conditions. Flash photolysis experiments demonstrate the ability of merocyanine 540 molecules to isomerize under visible light irradiation: the isomerization quantum yields are about 0.65 in both ethanol and methanol. This information combined with the fluorescence quantum yield data account for the low values for singlet oxygen production.

Photosensitized production of singlet oxygen by merocyanine 540 bound to liposomes

The production of singlet oxygen by merocyanine 540 was studied in dimyristoyl-phosphatidylcholine liposomes using two singlet oxygen probes: 9,10-anthracenedipropionic acid (water soluble) and 9,10-dimethylanthracene (liposoluble). Upper and lower limits of singlet oxygen quantum yield for bound merocyanine 540 were determined to be 0.055 and 0.015 respectively. The diffusion characteristics of singlet oxygen were examined using the isotropic enhancement effect of D2O and the inhibitory effect of sodium azide. It was shown that 1O2 spent more than 87% of its lifetime in a vesicle environment. When the singlet-reacting substrate and the dye were both located in the bilayer, approximately 40% of the singlet oxygen remained in the liposomes where it was originally generated.

Phototoxicity of phenothiazine derivatives. II. Photosensitized cross-linking of erythrocyte membrane proteins

Irradiation in the presence of O2, with near-UV light of five promazine (PZ) derivatives added to erythrocyte ghost membranes, causes covalent cross-linking between proteins as revealed by a progressive decrease in the amounts of proteins separable by electrophoresis after denaturation. The induction of cross-links in the two spectrin subunits is a single-hit process as a function of the irradiation time; relatively the rate constants (in min-1) of the photoreactions were 0.060 with chlorpromazine (CPZ), 0.039 with methoxypromazine (MTPZ), 0.031 with PZ, 0.029 with triflupromazine (TFPZ) and 0.006 with acepromazine (ACPZ). A main photochemical intermediate implicated in the spectrin aggregation seems to be the cation radical of the PZ derivatives. Indeed, (i) the chemically generated cation radicals can induce the reaction in the dark; (ii) the photoaggregation is regularly reduced upon addition of increasing concentrations of NaN3; (iii) NaN3 similarly affects the amount of cross-links induced by the isolated cation radicals. Hydroxyl radicals are also involved in the photocross-linking when the reaction is initiated only by MTPZ and not by the other sensitizers. In the absence of oxygen during irradiation, PZ, MTPZ and ACPZ completely loose their cross-linking activities whereas CPZ and TFPZ remain as efficient as in the presence of oxygen.

ALTERATION OF GUANINE RESIDUES DURING PROFLAVINE MEDIATED PHOTOSENSITIZATION OF DNA

Abstract— Mild photodynamic treatments of proflavine‐calf thymus DNA complexes induce a unique and quantitatively important alteration of the guanine residues which can be related to the lethal lesions due to the combined action of proflavine and light on phages. The ‘altered guanine’ is destroyed by HClO4 but is recovered after partial DNA depurination under the form of two photoproducts. The first product, Gox, elutes as guanine on a Sephadex column but has a modified UV absorbance spectrum. It gives rise by further irradiation to another product, X, which elutes at pH 9.7 as a pyrimidine compound and presented a maximal UV absorbance at 246 nm. Product X is also selectively released by piperidine fixation onto the photo‐damaged DNA. The guanine degradation process is markedly decreased in the presence of the singlet oxygen quencher, NaN3. The photodynamic lesion inhibits the enzymatic degradation of the DNA but generates locally denatured regions that are sensitive to S1 endonuclease.

Mechanism for Strand-break Induction in DNA–proflavine Complexes Exposed to Visible Light

Proflavine bound-superhelical phi XRFI DNA Molecules undergo single-strand scission upon irradiation with visible light at high fluence rate. As shown by agarose gel electrophoresis analyses, the nicking reaction is (i) oxygen-dependent, (ii) strongly inhibited by catalase and an electron scavenger such as cystamine, and (iii) totally suppressed by ceruloplasmin and radical scavengers such as t-butanol sodium benzoate. This indicates that H2O2, e-, O2 and OH, respectively, are involved in the cleavage process. NaN3, a singlet-oxygen quencher, has very little effect on strand-breakage but it prevents almost completely the alteration of guanine residues (a lesion already observed after irradiation at low fluence rate). Since, in the presence of NaN3, strand scission can occur and guanine (as the other bases) recovered intact, it follows that the radical intermediates produced during breakages are probably not involved in any permanent modification of the DNA bases.

Photodynamic potentialities of some phenothiazine derivatives

SummaryThe quantum yields of fluorescence and phosphorescence and the triplet lifetimes were determined for 29 phenothiazine derivatives; theφp values are varying from 0.2 to 1. The irradiation of phenothiazine derivatives in aerated solutions yields from the triplet state to the formation of singlet oxygen and more especially of superoxide ions and cation-radicals characteristic of the dye. Relative values of the formation rate were determined for these two mechanisms. Production of cation-radicals was correlated with phototoxicity in vivo. Chlorination enhances the two phenomena and acetylation reduces to nothing. A maximum value was estimated for the quantum yield of photoionization.

Photodynamic effect of proflavine on ϕX174 bacteriophage, its DNA replicative form and its isolated single-stranded DNA: Inactivation, mutagenesis and repair

SummaryIn contrast to that what is observed with most inactivating agents, proflavine-mediated photoinactivation is about 10 times more efficient on double-stranded ϕX 174 replicative form DNA (RFI) than on isolated single-stranded ϕX 174 DNA. Both ϕX RFI DNA and encapsidated DNA have similar sensitivities to proflavine and light treatment.With the three substrates studied, reactivation can occur through high multiplicity of infection and depends upon the cellular rec A gene product. No effect of the pol A, uvr A or lex A gene mutations has been found on either phage or DNA inactivation rates.The photodynamically induced lesions can be repaired at least in part, by the SOS repair system induced in the host-cells by a 100 J ·m-2 UV irradiation. SOS repair does not occur with bacteria (or spheroplasts) irradiated in the presence of chloramphenicol.Reversion frequency of the ϕX 174 amber mutations indicates that 1) photodynamically induced lesions are mutagenic whether the rec A gene product is present or not in the indicator bacteria; 2) induction of the SOS repair system is accompanied by a mutagenic process which almost results in a two fold increase of the reversion frequency; and 3) multiplicity reactivation occurs through a recombinational process and is not mutagenic per se.