Active oxygen intermediates in the degradation of hematoporphyrin derivative in tumor cells subjected to photodynamic therapy

Hematoporphyrin derivative (HPD), a sensitizer used in photodynamic therapy (PDT) of malignancies, is progressively destroyed during the treatment. Prior studies suggested that upon PDT the photobleaching of HPD in tumor tissues is largely mediated by self-sensitized singlet oxygen. However, little is known about the role of other reactive oxygen species (ROS). The main aim of this work was to clarify the significance of H2O2, superoxide ( O 2 - ) and hydroxyl (OH) radicals in bleaching of HPD in tumor cells subjected to PDT. Experiments were performed on Ehrlich ascites carcinoma (EAC) cells, which were loaded with HPD in PBS and then irradiated with red light at 630 nm in the same buffer. Studies showed that photosensitization of EAC cells by HPD led to the formation of significant amounts of H2O2, O 2 - and OH, and that these ROS could be involved in the photobleaching of HPD during PDT. In fact, we found that addition of catalase (CAT, a scavenger H2O2), Cu/Zn-superoxide dismutase (Cu/Zn–SOD) and Tiron (scavengers of O 2 - ), Na-benzoate, mannitol and deferoxamine (scavengers of OH) caused a substantial decrease in the rate of HPD photobleaching in EAC cells. In these cells, the inhibitory effects of Na-benzoate, mannitol and deferoxamine on the photodegradation of HPD correlated well with suppression of the OH generation, a highly active oxidizer. In EAC cells, the glutathione redox cycle and CAT (scavengers of H2O2) as well as Cu/Zn–SOD was found to suppress the photoinduced degradation of HPD. It was also established that HPD can directly scavenge H2O2 and oxygen free radicals; in a phosphate buffer its second-order rate constants were measured as 5.51 ± 0.32 × 103 M−1 s−1 (for the reaction with O 2 - ), 5.08 ± 0.31 × 104 M−1 s−1 (for H2O2), and 3.44 ± 0.08 × 1010 M−1 s−1 (for OH). Thus, our data suggest that OH could be one of the main oxidants mediating the photobleaching behavior of HPD in malignancies. Studies showed that photoexcited moieties of HPD can oxidize cell proteins with the formation of protein peroxides (PPO), which currently are regarded as a new form of ROS. Model experiments suggest that PPO could also participate in bleaching of HPD in tumors treated with PDT. It was found that HPD may destroy in tumor cells after cessation of photoirradiation and that this event is largely mediated by the presence of H2O2, a precursor of OH.