Significance: Since the discovery of the superoxide dismutase enzyme, the generation and fate of short-lived oxidizing, nitrosating, nitrating, and halogenating species in biological systems has been of great interest. Despite the significance of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in numerous diseases and intracellular signaling, the rigorous detection of ROS and RNS has remained a challenge. Recent Advances: Chemical characterization of the reactions of selected ROS and RNS with electron paramagnetic resonance (EPR) spin traps and fluorescent probes led to the establishment of species-specific products, which can be used for specific detection of several forms of ROS and RNS in cell-free systems and in cultured cells in vitro and in animals in vivo . Profiling oxidation products from the ROS and RNS probes provides a rigorous method for detection of those species in biological systems. Critical Issues: Formation and detection of species-specific products from the probes enables accurate characterization of the oxidative environment in cells. Measurement of the total signal (fluorescence, chemiluminescence, etc .) intensity does not allow for identification of the ROS/RNS formed. It is critical to identify the products formed by using chromatographic or other rigorous techniques. Product analyses should be accompanied by monitoring of the intracellular probe level, another factor controlling the yield of the product(s) formed. Future Directions: More work is required to characterize the chemical reactivity of the ROS/RNS probes, and to develop new probes/detection approaches enabling real-time, selective monitoring of the specific products formed from the probes. Antioxid. Redox Signal . 28, 1416–1432.

Using high throughput screening-compatible assays for superoxide and hydrogen peroxide, we identified potential inhibitors of the NADPH oxidase (Nox2) isoform from a small library of bioactive compounds. By using multiple probes (hydroethidine, hydropropidine, Amplex Red, and coumarin boronate) with well defined redox chemistry that form highly diagnostic marker products upon reaction with superoxide (O₂^̇̄), hydrogen peroxide (H₂O₂), and peroxynitrite (ONOO⁻), the number of false positives was greatly decreased. Selected hits for Nox2 were further screened for their ability to inhibit ONOO⁻ formation in activated macrophages. A new diagnostic marker product for ONOO⁻ is reported. We conclude that the newly developed high throughput screening/reactive oxygen species assays could also be used to identify potential inhibitors of ONOO⁻ formed from Nox2-derived O₂^̇̄ and nitric oxide synthase-derived nitric oxide.