Oxygen-consumption based quantification of chemogenetic H₂O₂ production in live human cells

Reactive Oxygen Species (ROS) in the form of H₂O₂ can act both as physiological signaling molecules as well as damaging agents, depending on their concentration and localization. The downstream biological effects of H₂O₂ were often studied making use of exogenously added H₂O₂, generally as a bolus and at supraphysiological levels. But this does not mimic the continuous, low levels of intracellular H₂O₂ production by for instance mitochondrial respiration. The enzyme d-Amino Acid Oxidase (DAAO) catalyzes H₂O₂ formation using d-amino acids, which are absent from culture media, as a substrate. Ectopic expression of DAAO has recently been used in several studies to produce inducible and titratable intracellular H₂O₂. However, a method to directly quantify the amount of H₂O₂ produced by DAAO has been lacking, making it difficult to assess whether observed phenotypes are the result of physiological or artificially high levels of H₂O₂. Here we describe a simple assay to directly quantify DAAO activity by measuring the oxygen consumed during H₂O₂ production. The oxygen consumption rate (OCR) of DAAO can directly be compared to the basal mitochondrial respiration in the same assay, to estimate whether the ensuing level of H₂O₂ production is within the range of physiological mitochondrial ROS production. In the tested monoclonal RPE1-hTERT cells, addition of 5 mM d-Ala to the culture media amounts to a DAAO-dependent OCR that surpasses ∼5% of the OCR that stems from basal mitochondrial respiration and hence produces supra-physiological levels of H₂O₂. We show that the assay can also be used to select clones that express differentially localized DAAO with the same absolute level of H₂O₂ production to be able to discriminate the effects of H₂O₂ production at different subcellular locations from differences in total oxidative burden. This method therefore greatly improves the interpretation and applicability of DAAO-based models, thereby moving the redox biology field forward.