DNA damaging capability of hematoporphyrin towards DNAs of various accessibilities

In this work we wanted to verify that photoactivation of DNA-non-binding porphyrin derivative hematoporphyrin IX (Hp) is able to induce damages in DNAs of various accessibilities such as B-conformation and superhelical isolated DNA, nucleoprotein complex and intracellular DNAs. It was found that photodynamic reaction of Hp results significant changes in thermal stability of isolated T7 DNA and induces single strand breaks in supercoiled Bluescript plasmid isolated from Escherichia coli cells. As optical melting measurements revealed, the irradiation of photosensitized T7 nucleoprotein complex leads to a destabilization of the protein capsid. The photodynamic reaction affected both the protein structure and DNA-protein interaction, however, the parameters corresponding to the DNA denaturation are not influenced. The accumulation of Hp in HeLa cells was followed by laser scanning confocal microscopy. The picture received is typical for lipophilic dyes. When Hp loaded cells were irradiated, a reduction of viability could be observed in a concentration and a light dose dependent manner; 12microM porphyrin induced almost complete cell killing after 30min irradiation. After similar treatment, alkaline agarose gel electrophoresis of isolated nuclear DNA did not show the presence of single strand breaks. The alkaline comet assay also failed to demonstrate any DNA damage in HeLa cells. We also considered the possibility of the generation of damages in intracellular SV40 DNA. According to the electropherograms there was no difference between the patterns of DNAs from treated and control samples.