3.3. Generation of ROS by PpIX and X-Ray Irradiation
Intracellular ROS production was assessed by pre-incubation of HeLa cells with cell-permeable fluorescent probes (APF or DHE) that show different sensitivities to specific ROS species (∙OH, O2− and 1O2). HeLa cells were incubated with PpIX for 6 h prior to X-ray irradiation. Cells without PpIX treatment were irradiated under the same conditions as those used for controls. ROS levels are expressed in terms of fluorescence intensity. Figure 4a shows the effects of PpIX on intracellular ROS levels at different X-ray doses in the presence of APF. X-ray irradiation in the absence of PpIX increased ROS levels in HeLa cells. After pre-incubation with PpIX, ROS levels increased with the X-ray dose and PpIX concentration. Figure 4b shows the effects of PpIX on the intracellular ROS levels for various X-ray doses in the presence of DHE. No change was observed in the intracellular ROS by increasing X-ray irradiation in the absence of PpIX. After pre-incubation with PpIX, the ROS levels increased with PpIX concentration and X-ray dose.
Figure 4  Detection of intracellular ROS levels in HeLa cells exposed to increasing PpIX concentrations and X-ray doses, using aminophenyl fluorescein (APF) (a) or dihydroethidium (DHE) (b). PpIX was added 6 h prior to X-ray irradiation. Before X-ray irradiation, APF or DHE was added to HeLa cells at a final concentration of 10 μM and incubated in the dark for 30 min at 37 °C. After X-ray irradiation, the medium was removed, and the cells were washed with PBS. The resulting fluorescence was measured using a microplate reader. The data correspond to the mean ± SD (n = 4). Statistical significance (p < 0.01) relative to the experiment performed without PpIX at the same irradiation dose is indicated by (TT). Statistical significance (p < 0.01) relative to the experiment performed without irradiation at the same PpIX concentration is indicated by (+).   In our previous study, using standard reactions, we showed that APF had a higher sensitivity for detecting ∙OH, unlike DHE, which showed higher sensitivity for detecting O2− and s1O2 [3]. Therefore, these results suggest that X-rays increase ∙OH generation and that PpIX enhances the production of ∙OH, O2− and 1O2.