14 Proc. Estonian Acad. Sci. Chem., 2007, 56, 1, 14–37 On the mechanism of reactive oxygen species generation in tumour cells subjected to the phototoxic action of haematoporphyrin derivative: effect of heating Lyudmila Chekulayeva, Igor Shevchuk * , Vladimir Chekulayev, and Elena Oginskaya Institute of Chemistry, Tallinn University of Technology, Akadeemia tee 15, 12618 Tallinn, Estonia Received 1 December 2006, in revised form 23 January 2007 Abstract. The main aims of the study were: (1) to enrich the existing knowledge on the mechanism of H 2 O 2 , superoxide (O 2 –• ), 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 O 2 –• (a precursor of H 2 O 2 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 H 2 O 2 and oxygen radicals, and that in tumour cells subjected to PDT with HPD the generation of H 2 O 2 and O 2 –• 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 H 2 O 2 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 O 2 –• 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 O 2 –• in EAC cells, which correlated well with an increase in the rate of their HPD-photosensitized killing. Studies showed that the intensification of O 2 –• 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. Key words: photodynamic therapy, tumour, reactive oxygen species, xanthine oxidase, hyper- thermia. * Corresponding author, igor@chemnet.ee