Objectives: Our previous work has shown that certain blue-light photosensitive compounds can generate reactive oxygen species (ROS) at levels that may be bactericidal. In the current study, we determined how the dose of light affects ROS production in mammalian cells exposed to these photosensitive chemicals.
Methods: Four blue-light sensitive chemicals (chlorin E6, riboflavin, pheophorbide A, pheophorbide A-poly-L-lysine, all 10 mM) were exposed to human THP1 monocytes for 30 min (n = 3). The cells were then exposed to blue light at different doses (6.8-108 J/cm2) using a standard quartz-tungsten halogen source (450 mW/cm2, 380-500 nm). Controls received no light and no photosensitive compounds. ROS levels in cells were measured using the difluorodihydrofluroescein diacetate assay over 2 h post-light exposure. Diamide (10 mM, 30 min) was used as a positive normalization control for ROS generation in cells. ROS levels were compared using ANOVA and Tukey post-hoc analysis (a = 0.05).
Results: Riboflavin itself generated no ROS, but generated the highest levels (8000% diamide, p < 0.01) of intracellular ROS when exposed to light in a dose-dependent manner. Pheophorbide A and pheophorbide A-PLL did not generate ROS by themselves, but blue light induced ROS levels ranging from 70-140% of diamide controls, depending on the light doses applied. Chlorin E6 generated the lowest levels of ROS (10-30%) with light exposure, but generated some ROS (10%) by itself. For all compounds, the light doses affected intracellular ROS level, but not proportionally, suggesting complex or overlapping mechanisms of generation.
Conclusion: We conclude that all these compounds are capable of generating ROS in cells upon exposure to blue light although each responded uniquely and in complex fashion to various doses of light. These chemicals show promise as sources of ROS for photo-activated disinfection in endodontics. (Swiss National Sciences foundation, grant # 310000-119938 for financial support).