Exposing cells to H2O2: A quantitative comparison between continuous low-dose and one-time high-dose treatments

Abstract Most studies investigating the influence of H 2 O 2 on cells in culture apply nonphysiological concentrations over nonphysiological time periods (i.e., a one-time bolus that is metabolized in minutes). As an alternative, the glucose oxidase/catalase (GOX/CAT) system allows application of physiologically relevant H 2 O 2 concentrations (300 nM–10 µM) over physiologically relevant time periods (up to 24 h). Recent findings suggest that bolus and GOX/CAT treatments can lead to opposing cellular responses, thus warranting a quantitative comparison between the two approaches. First, we established a reaction–diffusion model that can predict the behavior of the GOX/CAT system with spatiotemporal resolution, thus aiding selection of optimal experimental conditions for its application. Measurements of H 2 O 2 concentration in the cellular supernatant with the luminol/hypochlorite system were consistent with the predictions of the model. Second, we compared the impact of bolus and GOX/CAT treatments on cytosolic H 2 O 2 levels over time. Intracellular H 2 O 2 was monitored by the response of the thiol peroxidase Prx2 and the H 2 O 2 sensor roGFP2-Orp1. We found that Prx2 rapidly and reversibly responds to submicromolar H 2 O 2 levels and accurately reflects kinetic competition with cellular catalase. Our measurements reveal fundamental differences in the dynamic response of cellular H 2 O 2 concentrations following either bolus or GOX/CAT treatments. Thus, different, or even opposing, biological outcomes from differing means of H 2 O 2 delivery may be expected. Cellular responses induced by bolus treatment may not occur under GOX/CAT conditions, and vice versa.