The main aims of the study were: (1) to enrich the existing knowledge on the mechanism of H2O2, superoxide (O2–•), and protein peroxides (PPO) formation in tumour cells subjected to photodynamic therapy (PDT) with haematoporphyrin derivative (HPD) and (2) to explain the stimulatory effect of heat stress on the generation of O2–• (a precursor of H2O2 and a very reactive hydroxyl radical) in HPD–PDT-treated cells. Experiments were performed on Ehrlich ascites carcinoma (EAC) cells, which were loaded with HPD in phosphate-buffered saline and then irradiated with red light at 630 nm in the same buffer. Studies showed that photoexcited HPD itself, i.e. in the absence of photooxidizable biomolecules, is a poor source of H2O2 and oxygen radicals, and that in tumour cells subjected to PDT with HPD the generation of H2O2 and O2–• could be largely explained by (i) photooxidation of certain cellular constituents (NAD(P)H), (ii) an increase in the activity of xanthine oxidase (XOD), and (iii) a photodamage to mitochondria. Besides, it was found that in cellular proteins the HPD-photosensitized oxidation of aromatic amino acids is responsible for the generation of H2O2 and PPO. Our data suggest that upon HPD–PDT the mild hyperthermia (ca 44 °C) produced by photoirradiation may enhance its tumouricidal effect via the stimulation of O2–• formation; it was found that a rise in the temperature from 30 to 44 °C strongly (by ca 2.5-fold) enhanced the generation of O2–• in EAC cells, which correlated well with an increase in the rate of their HPD-photosensitized killing. Studies showed that the intensification of O2–• formation is mediated by the stimulatory effects of heating on the activity of XOD as well as the production of this radical by the respiratory chain of mitochondria. Nevertheless, the obtained results indicate that severe hyperthermia (at temperatures > 45 °C) could induce, contrary to mild hyperthermia, a reduction in the efficiency of HPD–PDT due to suppression of the activity of XOD in tumour cells.
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