L N Bezdetnaia's research while affiliated with Institute of Biomedical Chemistry, Moscow and other places

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.

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).

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.

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.

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.

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.

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.

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.

Irradiation with near ultra-violet light is shown to lead to a decrease in the electric stability of liposome membranes. The presence of 8-methoxypsoralen during irradiation enhanced the effect. Vacuumization of the sample or presence of alpha-tocopherol inhibited the decrease of the electric stability. 8-methoxypsoralen is supposed to decrease the electric stability of liposome membranes by photosensitizing lipid peroxidation.