Versatile Histochemical Approach to Detection of Hydrogen Peroxide in Cells and Tissues Based on Puromycin Staining

Hydrogen peroxide (H2O2) is a central reactive oxygen species (ROS) that contributes to diseases from obesity to cancer to neurodegeneration but is also emerging as an important signaling molecule. We now report a versatile histochemical approach for detection of H2O2 that can be employed across a broad range of cell and tissue specimens in both healthy and disease states. We have developed a first-generation H2O2-responsive analogue named Peroxymycin-1, which is based on the classic cell-staining molecule puromycin and enables covalent staining of biological samples and retains its signal after fixation. H2O2-mediated boronate cleavage uncages the puromycin aminonucleoside, which leaves a permanent and dose-dependent mark on treated biological specimens that can be detected with high sensitivity and precision through a standard immunofluorescence assay. Peroxymyem-1 is selective and sensitive enough to image both exogenous and endogenous changes in cellular H2O2 levels and can be exploited to profile resting H2O2 levels across a panel of cell lines to distinguish metastatic, invasive cancer cells from less invasive cancer and nontumorigenic counterparts, based on correlations with ROS status. Moreover, we establish that Peroxymycm-1 is an effective histochemical probe for in vivo H2O2 analysis, as shown through identification of aberrant elevations in H2O2 levels in liver tissues in a murine model of nonalcoholic fatty liver disease, thus demonstrating the potential of this approach for studying disease states and progression associated with H2O2. This work provides design principles that should enable development of a broader range of histochemical probes for biological use that operate via activity-based sensing.