Application of a HyPer-3 sensor to monitor intracellular H2O2 generation induced by phenolic acids in differentiated Caco-2 cells

Intestinal epithelial cells (IECs) are an important point of contact between dietary food components consumed and subsequent whole-body utilization for body maintenance and growth. Selective bioactive phenolic acids, widely present in fruits, vegetables and beverages can generate hydrogen peroxide (H₂O₂) and contribute to the cellular redox balance, hence influencing well-known cellular anti-oxidant and pro-oxidant mechanisms. Our findings have showed that increasing extracellular H₂O₂ resulted in associated changes in intracellular H₂O₂ levels in Caco-2 cells (p < 0.05) which was facilitated by activity of a family of water channel membrane proteins, termed aquaporins (AQPs). To demonstrate this, a HyPer-3 genetically encoded fluorescent H₂O₂ sensitive indicator was used to enable fluorescent real-time imaging of intracellular H₂O₂ levels as a measure of changes occurring in extracellular H₂O₂ in differentiated Caco-2 cells exposed to different phenolic acids. The use of confocal microscopy and flow cytometry, respectively, captured visualization and quantification of H₂O₂ uptake in differentiated Caco-2 cells. DFP00173, an Aquaporin 3 (AQP3) inhibitor was effective at inhibiting the intracellular uptake of H₂O₂ and was sensitive to varied levels of H₂O₂ generated when different phenolic acids were added to the culture media. In summary, HyPer-3 was shown to be an effective technique to demonstrate relative capabilities of structurally different dietary phenolic acids that have potential to alter intestinal redox balance by changing intracellular H₂O₂, and either antioxidant or pro-oxidant activity, respectively.