[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.

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.

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.

[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.