D‐amino acid oxidase as a chemogenetic tool for spatiotemporally controlled hydrogen peroxide production: The oxygen connection

Background and PurposeD‐amino acid oxidase (DAO) has been developed as a chemogenetic tool, to precisely manipulate redox levels in tissues and cells by generating H$_{2}$O$_{2}$ in the presence of D‐amino acids. The enzyme's significant oxygen consumption during H$_{2}$O$_{2}$ production may influence cellular oxygen levels. We set out to explore if DAO activation results in hypoxia, which in turn alters the activity of cellular oxygen sensors such as prolyl‐4‐hydroxylase domain enzymes, and thus the activation of the hypoxia‐inducible factor (HIF) pathway.Experimental ApproachWe analysed oxygen the consumption rate (OCR), pericellular oxygen concentration and HIF‐1α stabilisation in the cytoplasm or mitochondrial matrix of HEK293 cells stably overexpressing DAO. The obtained experimental data were used to test whether a simple mathematical model can be used to predict oxygen levels.Key ResultsFor HEK293 cells expressing DAO in either the cytoplasm or mitochondrial matrix, we found a dose‐dependent increase in OCR upon stimulating the DAO reaction, which resulted in a decreased pericellular oxygen concentration. Correspondingly, HIF‐1α protein levels were stabilised over time, reflecting the biological cellular response to DAO‐induced hypoxia. Furthermore, we provide a simplified diffusion model that allows users to input experimental parameters and assess the risk of hypoxic conditions in cell cultures.Conclusions and ImplicationsHigh DAO activity can induce hypoxia because of increased oxygen consumption. Caution should be taken to prevent hypoxic conditions in cell culture experiments when using DAO. A provided spreadsheet is intended to help estimate oxygen levels in combination with OCR measurements.