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. Peroxymycin-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 Peroxymycin-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.