Quantifying redox signals: a tool to investigate adaptive responses to oxidative stress.

Abstract

In the context of redox biology's oxidative eustress/distress model, the contrasting roles of ROS, such as hydrogen peroxide, under normoxic and toxic oxidative stress conditions has been more clearly elucidated. However, a fundamental question in the field has been understanding how dynamic redox signalling processes contribute to balancing the cellular response to oxidative eustress/distress.We employed a quantitative approach, evaluating redox signals based on area under the curve (AUC), signal amplitude, time, and duration. These parameters allowed us to investigate how dynamic profiles changed in response to various oxidants across three distinct experimental contexts. First, quantifying the dynamic response of the Tpx1/Pap1 redox-regulated pathway in Schizosaccharoymyces pombe revealed a graded signal and transcriptional response to input peroxide concentrations. Secondly, we applied this method to establish the upper and lower limits of detection for the redox probes HyPer7 and roGFP-TSA2/Tpx1 in Saccharomyces cerevisiae and S. pombe, providing a set of criteria for improved selection and comparison of probes. Thirdly, we quantitatively evaluated the dynamic response of the mammalian NRF2/Keap1 pathway following pre-exposure to DEM and tBHQ. Our findings revealed that, unlike the Tpx1/Pap1 pathway, this pathway showed that the timing of oxidant exposure influenced the NRF2 response. These metrics have therefore provided useful insights to several different pathways in both experimental and analytical contexts. Importantly, these measures allow for further exploratory questions to be asked and provide a fundamental framework for assessing dynamic responses of redoxregulated pathways associated with cell physiology and disease.