[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.

[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.

The Attenuation of Effectors and Induction of Suppressors of Delayed Type Hypersensitivity Reaction under the Treatment with Psoralen Photooxidation Products.

Psoralens, together with ultraviolet light A (PUVA), are used for the treatment of the series of T cell mediated diseases. The role of photooxidative reactions in psoralen phototherapy is not entirely clear. Using model of delayed type hypersensitivity (DTH) reaction to sheep red blood cells and evaluating the antibody production in mice, we investigated the influence of produced in vitro psoralen photooxidation products (POP) on the functions of T and B effectors. The research showed POP to induce inhibition of the DTH reaction independently on the phase of immune response it was injected. It was revealed that in vitro POP treatment of splenocytes, containing mature effectors, partially impaired their capacity to transfer the DTH reaction. The POP effect was not related to the direct cytolysis. In vivo POP injection to sensitized mice-donors resulted in much stronger alteration of DTH effectors than in the case of in vitro POP treatment. We elaborated the model for evaluation of functional activity of DTH suppressors. Using the model, we found that, being intravenously injected, POP induced DTH suppressors other then T cells. Furthermore, it was shown that intravenously injected POP did not affect antibody production. Thus, the POP selectively influenced T cells and had no effect on B cells. Probably, psoralen photooxidation products may be used for the therapy of some hyperproliferative T cell mediated diseases.