High-throughput Assays for Superoxide and Hydrogen Peroxide

Abstract

Background: NADPH oxidases (Nox) are involved in pathogenesis of inflammatory and fibrotic diseases. Results: High-throughput screening methodology is developed for discovery and determination of Nox inhibitors and mechanism. Conclusion: Rapid and specific monitoring of superoxide and hydrogen peroxide is possible, enabling high-throughput screening of Nox isoform-specific inhibitors. Significance: The proposed strategy will enable more rigorous research on the chemical biology of Nox enzymes.

Signalling mechanisms underlying doxorubicin and Nox2 NADPH oxidase‐induced cardiomyopathy: involvement of mitofusin‐2

“…Furthermore, the cell death profile induced by DOX in this setting is consistent with increased apoptosis leading to secondary necrosis, whilst the concentrations of DOX used in this study (0.5–5 μM) are relevant to plasma levels found clinically up to 1 h post‐treatment (0.1–10 μM) (Anderson et al, 1999). In a pharmacological approach, the observed effects of the pan‐NADPH oxidase inhibitor, VAS2870, on DOX (5 μM)‐induced cell death add weight to the involvement of Nox2 signalling in this model, although it should be noted that VAS2870 can exert Nox2‐independent actions (Gatto et al, 2013) and is cytotoxic at high concentrations (Zielonka et al, 2014), which we observed at 100 μM. However, at a concentration of 50 μM, which maintained cell viability, VAS2870 was shown to reduce both cytotoxicity and caspase 3/7 activity.…”

Recent Developments in the Probes and Assays for Measurement of the Activity of NADPH Oxidases

“…A DEPMPO spin trap was used to detect O 2 •− produced by NADPH oxidase in intact HL60 cells differentiated into neutrophil-like cells ( d HL60) and stimulated with PMA (Fig. 7) (79). The EPR signal was inhibited by SOD but not by catalase, indicating that superoxide was responsible for the adduct formed, consistent with the spectral features observed, which are characteristic of a superoxide spin adduct.…”

“…Thus, by profiling the products formed, in addition to the use of specific inhibitors and scavengers, the identity of the oxidant(s) can be determined (98–100). In specific instances, where the involvement of other oxidants can be excluded, boronate-based probes can be used to selectively monitor the activity of NADPH oxidases (24, 79, 91, 101). Several boronate-based fluorogenic probes have been applied to monitor the activity of NADPH oxidases, including coumarin boronic acid (CBA), which upon reaction with H 2 O 2 undergoes oxidation to 7-hydroxycoumarin (COH, also known as umbelliferone) (79, 102, 103).…”

“…In specific instances, where the involvement of other oxidants can be excluded, boronate-based probes can be used to selectively monitor the activity of NADPH oxidases (24, 79, 91, 101). Several boronate-based fluorogenic probes have been applied to monitor the activity of NADPH oxidases, including coumarin boronic acid (CBA), which upon reaction with H 2 O 2 undergoes oxidation to 7-hydroxycoumarin (COH, also known as umbelliferone) (79, 102, 103). The major limitation of boronic probes is their relatively low reactivity toward H 2 O 2 , reducing the possibility of quantitative analyses of H 2 O 2 but, on the other hand, allowing the bioorthogonal mode of detection (90).…”

“…For example, the HPLC-based analysis of HE and its oxidation products initially took 60–75 min per sample, was later shortened to 10 min, and most recently has been shortened to less than 90 s per sample (69, 79, 95). With such rapid assays, multiple probes and their products can be separated and detected simultaneously, providing an opportunity for real-time, simultaneous monitoring of NADPH-oxidase-derived O 2 •− and H 2 O 2 .…”