On the mechanism of erythrocyte hemolysis induced by photooxidized psoralen.

Contemporary concepts on a possible mechanism of erythrocyte hemolysis induced by photooxidized psoralen - the medicinal photosensitizing furocoumarin - are reviewed. The hypothesis on the mechanochemical mechanism of hemolysis is considered in view of recent data on photoinduced aggregation in photooxidized psoralen solutions. Appropriate chemical structures of photoproduct hemolysins and aggregating photoproducts are discussed.

Effects of glutathione peroxidase and catalase on hemolysis and methemoglobin modifications induced by photooxidized psoralen.

Psoralen photooxidation products (POP products) were obtained by UVA irradiation (365 nm, 180-640 W/m2) of an aqueous psoralen solution with fluences of 0-800 kJ/m2. Preincubation of POP products with glutathione peroxidase (GSHPer) or catalase, as well as presence of catalase during UVA irradiation of the aqueous psoralen solution did not influence their hemolytic activity. However, both GSHPer and catalase inhibited POP-induced conversion of methemoglobin. This indicates that hydrogen peroxide and psoralen peroxides destructible by GSHPer, which are being produced during psoralen photooxidation, do not possess hemolytic activity. Furthermore, hydrogen peroxide does not appear to serve as an intermediate in the process of hemolysin formation. Hydrogen peroxide generated during psoralen photooxidation is apparently the main POP product responsible for MetHb conversion.

Photohemolysis sensitized by the furocoumarin imperatorin and its oxyfunctionalized derivatives.

The dark and photosensitized (366 nm) hemolytic effects of imperatorin and its photooxidation products, the hydroperoxides I and II as well as the corresponding alcohol of the hydroperoxide I (imperatorin alcohol), were studied on human erythrocytes. Imperatorin was shown to photosensitize hemolysis, its fluence (D) dependence of the rate of photohemolysis (V) followed the equation V = V0 + aD2 + bD1/2, in which V0 is the dark hemolysis rate and a and b are constants. At fluences below 200 kJ/m2, the main hemolytic contribution derives from the bD1/2 component, which is due to the in situ formation of the imperatorin hydroperoxides, while at fluences higher than 200 kJ/m2, the main contribution corresponds to the aD2 component due to the two-photon damage of cell membranes. Hydroperoxides I and II induce oxyhemoglobin cross-linking, as well as its conversion to methemoglobin and hemichrome. These reactions involve hydroxyl and alkoxy radicals, as the hemolysis and oxyhemoglobin conversion could be inhibited by t-butanol and butylated hydrotoluene. For comparison, the dark hemolytic effect of the imperatorin alcohol was approximately 10-fold less than of the hydroperoxides.

[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.

Suppression of delayed-type hypersensitivity and hemolysis induced by previously photooxidized psoralen: effect of fluence rate and psoralen concentration.

The kinetics of the formation of biologically active psoralen photooxidation (POP) products were analyzed by the biological effects produced. Effects of the UV light fluence rate and psoralen concentration during the preirradiation were investigated to assess the yield of POP products, which were active in vivo (inducing suppression of delayed-type hypersensitivity [DTH] reaction to sheep red blood cells) and in vitro (altering the human erythrocyte membrane permeability). It was shown that the reciprocity law of the irradiation fluence rate and time was not valid in the case of POP-induced hemolysis and DTH suppression. Immunosuppressive POP products were more efficiently formed at low fluence rate (20.8 W/m2), whereas POP hemolysins were more efficiently produced at a high fluence rate (180 W/m2) of UV light. The yield of immunosuppressive POP products was enhanced in dilute psoralen solutions, while the POP hemolysins yield increased with increasing psoralen concentration. A kinetic scheme for psoralen photoproduct formation was proposed. Kinetic analysis showed that a labile intermediate was produced as the result of excitation of psoralen. This intermediate was either converted to a stable immunosuppressive POP product, or two intermediates combined to form a POP hemolysin. It is proposed that PUVA therapy conditions are more favorable for the formation of immunosuppressive rather than membrane-damaging psoralen photooxidation products.

[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.

Products of psoralen photooxidation possess immunomodulative and antileukemic effects.

It has been proposed that the therapeutic effect of PUVA (psoralens+UVA radiation) is connected to its immunomodulative properties, and that the molecular basis of such properties is the oxygen-independent photoaddition of psoralens to DNA. We have investigated effects of preliminary photooxidized psoralens (POP) on the delayed-type hypersensitivity reaction (DTH) to sheep red blood cells and on growth of grafted T-cell lymphoma EL-4 in mice. We have shown that intravenous injection of POP at low concentrations activated, and at high concentrations suppressed, DTH. The POP products are thermolabile. They preserved their immunosuppressive activity for 3 days at room temperature and lost it in several min at 58 degrees C. Incubation of POP in the presence of Fe2+ during 2 h before intravenous injection leads to complete loss of its immunomodulative activity, suggesting a peroxidic nature of POP products. The POP-inhibited growth of grafted T-cell lymphoma independent of the mode of POP application in mice (intravenous or subcutaneous injections, oral or nasal administration). Our data suggest that photooxidative reactions of psoralens, in addition to oxygen-independent photoaddition to DNA, form the basis for biological activity of these drugs.

Degradation of psoralen photo-oxidation products induced by ferrous ions.

Psoralen was irradiated at 366 nm in aerated aqueous or ethanol solutions. Fe2+ ions reduced photo-oxidized psoralen (POP) with the formation of free radicals and electronically excited states. Free radicals were detected by the electron spin resonance (ESR) method using the spin trap C-phenyl-N-tert-butyl-nitrone (PBN), and electronically excited states were registered by chemiluminescence (ChL) accompanying the destruction of POP by Fe2+ ions. PBN could not scavenge directly free radicals generated by the reduction of POP with Fe2+ and required the presence of ethanol during the reaction. Analysis of ESR spectra indicated that PBN trapped hydroxyethyl free radicals which were produced as a byproduct in the reaction of POP and Fe2+. The dependence of the yield of PBN adducts on the fluence of psoralen irradiation and the concentration of Fe2+ ions was measured. Although both ESR and ChL estimated the POP products destructible by Fe2+ (POPFe), they gave information about different POPFe products. A kinetic analysis showed that ChL-estimated POPFe products were produced with the participation of two molecules of psoralen (one in the electronically excited state and one in the ground state), whereas ESR-estimated POPFe products were produced with the participation of one molecule of psoralen in the excited state. ESR-estimated products were stable in both water and ethanol solutions and could be stored for 20 h without significant decay; pre-incubation of POP solutions with catalase or glutathione-peroxidase decreased the yield of PBN adducts by 50%. ChL-estimated products were essentially less stable, about 30% being spontaneously destroyed during storage in ethanol solution at room temperature; pre-incubation of these products with catalase decreased the ChL by 90%. The possible biological role of POPFe products is discussed.

Fe2+ ions and reduced glutathione--chemical activators of psoralen-sensitized photohaemolysis.

The influence of Fe2+ ions and reduced glutathione (GSH) on the haemolysis of erythrocytes photosensitized (366 nm) by psoralen (PUVA haemolysis) was investigated. PUVA haemolysis was induced by the low fluence rate (24 W m-2) and high fluence (greater than 150 W m-2) of UV-A irradiation. It has been shown that Fe2+ ions and GSH activated PUVA haemolysis at both fluence rates of irradiation. PUVA haemolysis activation by Fe2+ ions was more pronounced than that by GSH. It is supposed that activation caused by Fe2+ ions and GSH is connected with their ability to reduce lipid peroxides or psoralen peroxides with the subsequent formation of free radicals. The regeneration of endogenous Fe2+ by reduced glutathione is also possible.

Photohemolysis sensitized by psoralen: reciprocity law is not fulfilled.

The effect of the intensity of ultraviolet-A (UV-A) radiation (366 nm) on delayed photohemolysis sensitized by psoralen (PUV-A hemolysis) was studied. It was shown that PUV-A hemolysis induced by UV-A radiation at low fluence rate (20 W m-2) develops according to the well-known colloid-osmotic mechanism: there was no threshold dose of PUV-A treatment. After irradiation all the cells were hemolysed. The rate of PUV-A hemolysis was proportional to the square of the fluence. Hemolysis was delayed in the presence of sucrose. When the fluence rate of UV-A radiation was increased to 150 W m-2, the character of PUV-A hemolysis changed drastically. A threshold fluence appeared, below which PUV-A hemolysis was not induced. At fluences slightly exceeding the threshold, only part of the cells in the suspension were lysed. The dependence of the portion of hemolysing cells on fluence was S-shaped. Increasing the fluence resulted in complete (100%) hemolysis. The rate of complete hemolysis decreased at higher fluences, but was many-fold higher than the rate of low-intensity PUV-A hemolysis at equal fluences. The main features of high intensity PUV-A hemolysis (dependences on fluence and temperature, effect of sucrose) were the same for the hemolysis induced by the addition of previously photooxidized psoralen. We suggest that high intensity PUV-A hemolysis is induced with participation of photooxidized psoralen as an intermediate.

Psoralen-photosensitized damage of rat peritoneal exudate cells.

Psoralen-sensitized photodamage (PUVA) of rat peritoneal exudate cells was investigated. Quartz-activated luminol-dependent chemiluminescence (ChL) was registered and the amount of trypan-positive cells was determined. Irradiation of peritoneal exudate cells in the presence of psoralen resulted in a dose-dependent monotonous inhibition of ChL. The reciprocity law of irradiation intensity and duration of irradiation was not valid for the observed inhibition of ChL: the inhibition increased with higher intensity. When psoralen previously photooxidized in ethanol (POP) was added to peritoneal exudate cell suspension, a double-phase response depending on psoralen irradiation dose was obtained: ChL activation was observed at low doses of UVA, ChL inhibition at high doses. Chemiluminescence inhibition correlated well with the increase in the number of trypan-positive cells. It may be supposed that the observed effects of PUVA or POP treatment are caused by cell cytoplasmic membrane damage.

[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.

[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.