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

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

  • Anal Biochem. 2022 Oct 4;114934. doi: 10.1016/j.ab.2022.114934.
Kaiwen Mu 1 David D Kitts 2
Affiliations

Affiliations

  • 1 Food Science, Food Nutrition and Health Program, Faculty of Land and Food Systems, The University of British Columbia, 2205 East Mall, Vancouver, B.C, V6T 1Z4, Canada. Electronic address: muk@mail.ubc.ca.
  • 2 Food Science, Food Nutrition and Health Program, Faculty of Land and Food Systems, The University of British Columbia, 2205 East Mall, Vancouver, B.C, V6T 1Z4, Canada. Electronic address: david.kitts@ubc.ca.
Abstract

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.

Keywords

Aquaporin; Caco-2 cells; H(2)O(2); HyPer-3; Phenolic acids; Redox.

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