[Dual role of photosensibilizator merocyanine 540 as a bactericidal agent and immune reaction regulator].

AIM: Develop conditions for inactivation of staphylococcus by using photosensibilizator merocyanine 540 (MC540) for the production of antigenic preparation (AP). Study some of immune reactions to AP and the possibility of regulation of DTH reaction to AP under the effect of MC540. MATERIALS AND METHODS: Merocyanine 540 (MC540, Sigma-Aldrich, Switzerland) is used in the study. MC540 and Staphylococcus aureus, strain 78 (Sa78) were irradiated by light of a mercury-quartz lamp DRSH-250 (Zelenograd). C56BL/6 line mice were immunized once by subcutaneous administration of AP. DTH reaction was tested 7 days after the immunization. Functional activity of peritoneal exudate macrophages was determined 1 and 9 days after the immunization. Immune modulating effect of MC540 in DTH was determined after its per os administration to mice 1 hour after AP sensibilization. RESULTS: In order to obtain AP, S. aureus suspension at the concentration of 2.5 x 10(7) CFU/ml in 25 microM MC540 solution and 0.25 M NaCl solution were exposed to irradiation for 5 minutes. During DTH reaction induction its intensity dependence on AP dose was revealed. A persistent increase of a lysosomatic enzyme cathepsin D in macrophages of peritoneal exudate after a single administration of AP was noted. During MC540 irradiation an accumulation of photoproducts that have a pronounced immune suppression effect in DTH reaction had a dose-dependent character. CONCLUSION: Use of saline allows to increase bactericidal potential of a photosensibilizator (PS). However during therapy of localized forms of infection a possible immune modulating effect of PS on macro organism should be considered. By varying PS dose and irradiation time not only maximum bactericidal effect can be achieved but also regulation of inflammatory reactions in the area of PS effect can be ensured.

[Investigation of aggregates of dyes by the method of resonance light scattering: correction of spectra].

Resonance light scattering, a phenomenon of an abrupt enhancement of Rayleigh light scattering in close proximity to an absorption band, is easily detectable in solutions of strongly absorbing chromophores, which form large aggregates with strong pi-pi-electronic coupling among the chromophores. Resonance light scattering spectra need to be corrected for the sensitivity of spectrofluorimeter as well as for the effects of internal light filter. A method for correcting the measured resonance light scattering is described. It was shown by the method that the addition of KCl induces the formation of extended supramolecular aggregates (probably of H-type) of the anionic dye merocyanine 540 in water. The spectra of the resonance light scattering of the photosensitizer meta-tetra(hydroxyphenyl)chlorin (mTHPC, foscan ) indicate the formation of J-aggregates of this dye in aqueous medium.

[Photohemolysis sensitized by psoralen: dependence on pH].

The effect of pH on the hemolysis of erythrocytes photosensitized (366 nm, 23 Wt/m2) by psoralen has been studied. The dependence of the photohemolysis rate (V) on irradiation dose (D) was described by the equation V = Vo + kD, where Vo is the rate of hemolysis without irradiation (dark), and k is the constant. The index of the power at dose x was approximately equal to 2, and its value did not change as the pH of the erythrocyte suspension was changed. It was found that changes in pH led to a sharp change in the value of coefficient k and correspondingly V. The lowest rate of photohemolysis was observed in the pH range from 8.0 to 8.4. As pH was changed from 3.4 to 9.0 or from 8.0 to 7.4, the V value increased approximately twofold. At pH below 7.4, an abrupt increase (approximately fourfold) in V was observed, with the pK value being equal to 7.3. The psoralen molecule lacks titratable acidic and basic groups; therefore, the effects of pH can hardly be assigned to changes in the photophysical properties of the sensitizer. The increase in V in the alkaline region is prohably related to the acceleration of photooxidation of reduced glutathione, whereas the jump of V at pH of about 7.3 may be due to the titration of the product of psoralen photooxidation. The latter assumption is confirmed by the data of hign performance liquid chromatography. In these experiments, psoralen was oxidized in ethanol and mixed with the phosphate buffer at different pH values followed by a qualitative and quantitative analysis by high performance liquid chromatography of photoproducts. Several photoproducts of psoralen have been identified whose content depended on pH. The curve of titration of one photoproduct was similar in shape to the pH dependence of psoralen-photosensitized hemolysis.

[Photobiophysics of furanocoumarins]. / Fotobiofizika furokumarinov.

Furocoumarins (psoralens) are photosensitizers of plant origin, which increase the sensitivity of biological objects to near ultraviolet (UV-A, 320-400 nm). In combination with UV-A, they are successfully used for treating many dermal and autoimmune diseases (PUVA therapy and photophoresis). Along with therapeutic effects, the furocoumarin photochemotherapy induces a number of side-effects (erythema, edema, hyperpigmentation, and premature aging of skin). All photobiological effects of furocoumarins result from their photochemical reactions. Therefore, in order to advance the therapy, it is necessary to know the photochemical mechanisms of induction of both side- and therapeutic effects. The types of photoreactions of furocoumarins classified with respect to reactive photoproducts interacting with substrate were considered. Primary emphasis was placed on reactions proceeding with the participation of photooxidation products of furocoumarins. Among these photoproducts, at least two types can be distinguished. Some of them possess membranotoxic properties, others produce the immunosuppressory action in vivo. The photochemical mechanisms of the formation of the photoproducts of furocoumarins are different. It was found that, by varying the illumination conditions (intensity of UV-A radiation or the concentration of the photosensitizer), it is possible to obtain the photoproducts of furocoumarins that have either membranotoxic or immunosuppressory properties. It was found that the mechanisms of the immunosuppressive action of the photooxidation products of furocoumarins have some features in common with those underlying the PUVA therapy and photophoresis. It is assumed that the photochemical basis of the therapeutic action of furocoumarins is the reactions with the involvement of the products of their photooxidation.

[Photohemolysis sensitized by deuteroporphyrin-IX derivatives: determination of binding strength of dyes to erythrocytes]. / Fotogemoliz, sensibilizirovannyi proizvodnymi deiteroporfirina-IX: opredelenie prochnosti sviazyvaniia krasitelei s éritrotsitami.

A comparative analysis of the ability of 4-(1-methyl-3-hydroxybutyl)-deuteroporphyrin-IX (I) and 2,4-di-(1-methyl-3-hydroxybutyl)-deuteroporphyrin-IX (II) to photosensitize hemolysis of human erythrocytes was performed. The photohemolytic efficiency of dye I was shown to be about 60 times higher than that of dye II. It was found that a part of each dye tightly binds to erythrocyte membranes and is not removed by washing. A method for estimating the share of the dye tightly bound to the membrane (beta) was proposed, which takes into account the shielding effect produced by the free dye and the photohemolytic efficiency of the bound dye. It was shown that the beta values for dyes I and II are 86 and 61% and correlate with the coefficients of distribution of the dyes in the octanol/water system (20.7 and 17.0, respectively).

[Photo oxidative reactions of psoralens and their role in therapy of dermatoses]. / Fotookislitel'nye reaktsii psoralenov i ikh rol' v terapii dermatozov.

Psoralenes (furocoumarins) in combination with ultraviolet (UV) A radiation (320-400 nm) are used in the treatment of vitiligo, psoriasis, cutaneous T-cell lymphoma and other skin and autoimmune diseases (PUFA therapy). The mechanism of psoralene-photosensitive modification of biologically significant molecules, such as DNA, protein unsaturated lipids, antioxidants, etc. is considered in the paper. Particular emphasis is laid on the mechanism and biomedical significance of photo reactions proceeding via the stage of formation of psoralene photo oxidation products (POP). POP causes oxidative damage to many molecules in in vitro experiments and mediates the T-cell immunity cell in vivo, as appeared as suppression of delayed hypersensitivity and contact sensitivity in mice. The use of randomized double blind control has indicated that POP has therapeutical effects on eczema. A new modality of psoralene photo chemotherapy that consists in the UV-A radiation of psoralene solution followed by its administration (POP therapy) to patients. Unlike conventional PUFA therapy, POP therapy does not require the patients' skin to be exposed to UF-A radiation, which allows adverse effects, such erythema, skin hyperpigmentation, etc. to be avoided.

[Effect of Fe2+ ions on the hemolysis induced by products of psoralen photo-oxidation]. / Vliianie ionov Fe2+ na gemoliz, indutsirovannyi produktami fotookisleniia psoralena.

The effects of Fe2+ ions on haemolysis induced by previously photooxidized psoralen (POP-haemolysis) were investigated. It was shown that POP-haemolysis was strongly activated by Fe2+ ions when ferrous ions were added to erythrocytes just after addition of POP. If POP was preincubated with Fe2+ before mixing with erythrocytes, then POP completely lost its ability to induce haemolysis. These data indicate the peroxidic nature of POP products responsible for haemolysis.

[Death of cells from mouse peritoneal exudate induced by products of reaction of Fe2+ ions with previously photo-oxidized psoralen]. / Gibel' kletok peritoneal'nogo ékssudata myshei, indutsirovannaia produktami reaktsii ionov Fe2+ s predvaritel'no fotookislennym psoralenom.

Toxic effect of previously photooxidized in water psoralen on survival of mouse peritoneal exudate cells was investigated. Cell survival was determined by trypan exclusion test. It was shown that photooxidized psoralen itself did not induce trypan-positive cells while decrease in cell survival was observed in the case of simultaneous addition of photooxidized psoralen and Fe2+ ions to cell suspension. To evaluate the quantity of psoralen photoproducts which react with Fe2+ photooxidized psoralen Fe2+ mixtures were titrated by different cell concentrations. Then parameter c50 (cell concentration such that 50% trypan-positive cells were observed) was determined. It was shown that c50 increased with the increase in preirradiation fluence of psoralen from 4.5 to 45 kJ/m2. But further increase in preirradiation fluence resulted in decrease of c50. The photodestruction of psoralen photoproducts which react with Fe2+ ions was proposed. Simultaneous addition of photooxidized psoralen, Fe2+ ions and antioxidant butylated hydroxytoluene significantly increased cell survival. That indicated free radical nature of reaction products of photooxidized psoralen with Fe2+ ions.

[Dark and photosensitized hemolysis of erythrocytes in the presence of dithranol]. / Temnovoi i fotosensibilizirovannyi gemoliz éritrotsitov v prisutstvii ditranola.

Ditranol (1,8-dihydroxy-9-antrone) induced dark lysis of erythrocytes. After irradiation of the cells with UV-light (366 nm UV-A light) in the presence of ditranol (DUVA-effect) the hemolytic effect increases. It has been found that antioxidant butylated hydroxytoluene BHT in the concentration 10(-7) M did not affect the dark lysis, while with increased BHT concentration up to 10(-5) M the hemolytic effect of ditranol was intensified. The presence of BHT in the above concentration under DUVA-effect did not change the velocity of cell lysis. Sodium aside did not affect the dark hemolysis of ditranol, but it inhibited photosensitized hemolysis.

[Inhibition of psoralen sensitized photohemolysis by a marigold (Bidentis tripartita) extract]. / Ingibirovanie fotogemoliza, sensibilizirovannogo psoralenom, ékstraktom cheredy (Bidentis tripartita).

Hemolysis induced by psoralen and UV-A radiation (PUVA-hemolysis) was significantly inhibited by the addition of Bidentia tripartita extract. The rate of hemolysis was reduced both when the extract was present during irradiation, or added after PUVA-treatment. The inhibition effect was more pronounced when the extract was present during irradiation.

[Psoralen sensitized thermoactivated photodamage to leukocyte membranes]. / Sensibilizirovannye psoralenom termoaktiviruemye fotopovrezhdeniia membran leikotsitov.

Psoralen sensitized photodamage of rat peritoneal exudate cells was investigated. Irradiation of cells induced latent lesions in membranes which during thermal activation at the post-irradiation stage were transformed into permeability channels for trypan blue. The effect linearly increased with fluence of irradiation which indicates one hit production of thermolabile psoralen photoproducts in the membranes.

[Phagocytic activity of granulocytes after UV-irradiation]. / Fagotsitarnaia aktivnost' granulotsitov posle UF-oblucheniia.

The effect of UV-irradiation on three functions of human isolated granulocytes (viability, phagocyte activity and capacity to reduce nitrotetrazolium blue) was investigated. The phagocytosis appeared to be the most sensitive function. The number of phagocytic cells was decreased under UV-doses as low as 0.63 kJ.m-2. Cell lethality was increased under UV-doses 4.32 kJ.m-2 and higher. The capacity to reduce nitrotetrazolium blue was uneffected even at the dose as high as 10.17 kJ.m-2.

[Antioxidant inhibition of hemolysis induced by photooxidized psoralen]. / Ingibirovanie antioksidantami gemoliza, indutsirovannogo fotookislennym psoralenom.

Antioxidants butylated hydroxytoluene++ (ionol) and 2,2,5,7,8-pentamethyl-chromanol-6-(alpha-T-0) were shown to inhibit hemolysis induced by the ethanolic solution of photooxidized psoralen (POP). This inhibition appeared to be the same whether antioxidants were present during irradiation or were added immediately after photooxidation. Therefore antioxidants do not influence POP formation, but inhibit the interaction of POP degradation products in water with biomolecules. Possibility of POP participation in phototoxic effects of furocoumarins is discussed.

[Relationship between absorption and fluorescence excitation spectra of psoralens and coumarins and their concentration]. / Zavisimost' spektrov pogloshcheniia i vozbuzhdeniia fluorestsentsii psoralenov i kumarinov ot kontsentratsii.

The shape of absorption spectra is changed and their maxima are red shifted with an increase of furocoumarin (psoralen, 8-methoxypsoralen and angelicin) concentration. Fluorescence excitation spectra of psoralen, 7-methoxycoumarin and 7-hydroxy-4-methylocumarin do not depend on the concentration in solutions. They are similar or coincident with absorption spectra of the most concentrated solutions. One may conclude the existence of different forms of furocoumarin and coumarin aggregates in solution. From the coincidence or similarity of fluorescence excitation spectra and absorption spectra in the most concentrated solutions it may be proposed that only the aggregated forms of psoralens and coumarins (the dimers or associates of higher order) are able to emit fluorescence.

[Effect of photo-oxidized psoralen on the permeability of erythrocyte and liposome membranes]. / Vliianie fotookislennogo psoralena na pronitsaemost' membran éritrotsitov i liposom.

Hemolysis of erythrocytes was induced after addition of ethanol solution of photooxidized psoralen, while the substance irradiated in absence of oxygen did not cause the cells hemolysis. The rate of hemolysis correlated with concentration of photooxidized psoralen in a sigmoid type. Similar concentration-dependent effect has been known for digitonin. Digitonin elevated the ionic permeability of lecithin-cholesterol liposomes but did not affect the permeability of lecithin liposomes. Photooxidized psoralen did not effect apparently on liposomes of both types. These data suggest that hemolytic effect of photooxidized psoralen did not relate to the substance interaction with cholesterol.

[Effect of antioxidants on the psoralen photo-oxidation process]. / Vliianie antioksidantov na protsess fotookisleniia psoralena.

Psoralens are capable of photosensitizing oxidation of unsaturated fatty acids due to the two-stage mechanism. During the first (light) stage psoralen solution in ethanol undergoes photooxidation under UV-irradiation (366 nm). At the second (dark) stage the addition of photooxidized psoralen (POP) to the aqueous solution of liposomes is followed by lipid oxidation. Antioxidants inhibited the UV-stage, but did not influence the dark one. Neither spectrophotometry, nor spectrofluorometry could detect photoproducts of psoralen involved in the two-stage oxidation of lipids. However, mixing of ethanol solution of POP with water resulted in the flash of chemiluminescence. The inhibition constants by antioxidants of photoproducts formation which are active in the two-stage oxidation of lipids were estimated by chemiluminescence. Stern--Volmer's constants for antioxidants: 2,6-dimethyl-3,5-diacetyl-1,4-dihydropyridine (DHP), 6-hydroxy-2,2,5,7,8-pentamethylchroman (chromanol--C1), water soluble sodium phenozan and butilated hydroxytoluen (ionol) appeared to be (7.4 +/- 2.2) X 10(3) M-1, (4.4 +/- 1.0) X 10(3) M-1, (3.3 +/- 0.7) X 10(3) M-1, (4.5 +/- 2.5) X 10(2) M-1, respectively. The biological importance of these two-stage oxidation photosensitized by furocoumarins is discussed.

[Photogeneration of singlet oxygen by psoralens]. / Fotogeneratsiia singletnogo kisloroda psoralenami.

Photosensitized luminescence of singlet (1 delta g) molecular oxygen with the maximum at 1272 nm has been found in solutions of psoralen, angelicin and 8-methoxypsoralen in CCl4. The luminescence excitation spectra, quantum yields of generation (gamma g) and rate constants of quenching (Kq) of 1 delta g by psoralens have been measured. The gamma g values 0,0055, 0,0026, 0,002 and Kq values 9 X 10(3), 11 X 10(3) and 12 X X 10(3) have been obtained for psoralen, angelicin and 8-methoxypsoralen, respectively. The role of 1 delta g in a photobiological action of psoralens is discussed.

[Effect of antioxidants on photoaddition of 8-methoxypsoralen to thymine, double-helix DNA and photosensitized inactivation of Escherichia coli]. / Vliianie antioksidantov na fotoprisoedinenie 8-metoksipsoralena k timinu, dvuspiral'noi DNK i fotosensibilizirovannuiu inaktivatsiiu Escherichia coli.

Effect of antioxidants--alpha-tocopherol and 2,6-di-tert-butyl-hydroxytoluene (BHT) on 2-methoxypsoralen (8-MOP) photoaddition was investigated. Spectrophotometric experiments have shown that under anaerobic conditions alpha-tocopherol inhibits photodimerization of 8-MOP and photoaddition of 8-MOP to thymine (in 80% ethanol solution). 50% inhibition is obtained at alpha-tocopherol concentrations of about 10(-5) M. The antioxidants (in concentrations from 10(-8) to 10(-4) M) displayed no effect on photoaddition of 8-MOP to double-stranded DNA in solution. The reactions of 8-MOP photoaddition to double-stranded DNA were registered by formation of fluorescent monoadducts and interstrand crosslinks. The antioxidants also did not inhibit 8-MOP-photosensitized inactivation of E. coli. Perhaps, 8-MOP addition to DNA cannot be inhibited by antioxidants due to steric hindrance. The role of 8-MOP photoaddition to DNA in the induction of PUVA-erythema is discussed.