A Critical Review of Methodologies to Detect Reactive Oxygen and Nitrogen Species Stimulated by NADPH Oxidase Enzymes: Implications in Pesticide Toxicity.

In this review, potential fluorescent probe applications for detecting reactive oxygen and nitrogen species (ROS/RNS) generated from NADPH oxidases (e.g., Nox2) and nitric oxide synthase enzymes are discussed in the context of pesticide toxicology. Identification of the specific marker products derived from the interaction between ROS/RNS and the fluorescent probes (e.g., hydroethidine and coumarin boronate) is critical. Due to the complex nature of reactions between the probes and ROS/RNS, we suggest avoiding the use of fluorescence microscopy for detecting oxidizing/nitrating species. We also critically examined the viability of using radiolabeling or positron emission tomography (PET) for ROS/RNS detection. Although these techniques differ in sensitivity and detection modalities, the chemical mechanism governing the reaction between these probes and ROS/RNS should remain the same. To unequivocally detect superoxide with these probes (i.e., radiolabeled and PET-labeled hydroethidine analogs), the products should be isolated and characterized by LC-MS/MS or HPLC using an appropriate standard.