Dose-dependent Intracellular Reactive Oxygen Species from Blue Light Chromophores

Objectives: Studies suggest that 30-50% of current endodontic treatments fail from residual bacterial infection. Photodynamic therapy using red-light chromophores (630 nm) to induce antimicrobial death via generated reactive oxygen species (ROS) have been reported, but red-light may thermally damage resident tissues. In the current study, we investigated a hypothesis that blue light chromophores (380-500 nm) generate intracellular reactive oxygen species in proportion to blue light dose. Methods: THP1 human monocytes (n = 3/condition) were exposed to 10 µM of four chromophores (chlorin e6, pheophorbide-a, pheophorbide-a-PLL, and riboflavin) for 30 min before activation with blue light (QTH, 7-108 J/cm², 15 s to 4 min). After activation, intracellular ROS were measured for 2 h using a dihydrofluorescein diacetate fluorescence assay. Controls received light or chromophores alone. Positive controls received diamide (5 mM, 30 min). To assess ROS lifetime, we compared ROS production for 2 h post irradiation with ROS for the last 15 min of the 2 h interval. Results: Chromophores and light alone generated low, transient ROS levels. All chromophores produced intracellular ROS levels higher than controls (p < 0.05) in a light-dose-dependent fashion. Riboflavin generated ROS levels that were the highest (20-80 fold over diamide, peak at 54 J/cm²), but these ROS decayed in less than 90 min. Pheophorbide-a-PLL generated ROS levels of 0.9-1.4 fold of diamide (peak at 14 J/cm²), with some persistence past 90 min (p > 0.05). Pheophorbide-a generated ROS at 0.7-1.1 fold of diamide with peak levels at 108 J/cm². Chlorin e6 generated the least ROS at 0.1-0.3 fold of diamide, with peak levels at 7 J/cm², but with significant (p < 0.05) persistence after 90 min. Conclusion: Blue light chromophores produce intracellular ROS in a dose-dependent fashion, with peak responses and ROS duration dependent on the chromophore used. (Funding: MCG Dental Research).