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. 2017 Aug;12:35-49.
doi: 10.1016/j.redox.2017.02.001. Epub 2017 Feb 7.

Taking Up the Cudgels for the Traditional Reactive Oxygen and Nitrogen Species Detection Assays and Their Use in the Cardiovascular System

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Taking Up the Cudgels for the Traditional Reactive Oxygen and Nitrogen Species Detection Assays and Their Use in the Cardiovascular System

Andreas Daiber et al. Redox Biol. .
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Reactive oxygen and nitrogen species (RONS such as H2O2, nitric oxide) confer redox regulation of essential cellular functions (e.g. differentiation, proliferation, migration, apoptosis), initiate and catalyze adaptive stress responses. In contrast, excessive formation of RONS caused by impaired break-down by cellular antioxidant systems and/or insufficient repair of the resulting oxidative damage of biomolecules may lead to appreciable impairment of cellular function and in the worst case to cell death, organ dysfunction and severe disease phenotypes of the entire organism. Therefore, the knowledge of the severity of oxidative stress and tissue specific localization is of great biological and clinical importance. However, at this level of investigation quantitative information may be enough. For the development of specific drugs, the cellular and subcellular localization of the sources of RONS or even the nature of the reactive species may be of great importance, and accordingly, more qualitative information is required. These two different philosophies currently compete with each other and their different needs (also reflected by different detection assays) often lead to controversial discussions within the redox research community. With the present review we want to shed some light on these different philosophies and needs (based on our personal views), but also to defend some of the traditional assays for the detection of RONS that work very well in our hands and to provide some guidelines how to use and interpret the results of these assays. We will also provide an overview on the "new assays" with a brief discussion on their strengths but also weaknesses and limitations.

Keywords: Dihydroethidium oxidative fluorescence microtopography; Fluorescence and chemiluminescence-based assays; L-012-enhanced chemiluminescence; Lucigenin-enhanced chemiluminescence; Oxidative stress; Redox signaling.


Fig. 1.
Fig. 1
Detection of eNOS uncoupling in hypertensive animals (AT-II infusion model) by oxidative fluorescence microtopography. (A) To determine eNOS-dependent ROS formation, vessels were preincubated with the NOS inhibitor L-NAME (500 µM, lower panel), embedded in Tissue Tek resin, frozen, cryo-sectioned, and stained with DHE (1 µM) . It should be noted that DHE does not react with “accumulated” ROS (most likely superoxide) in the cryo-sections, which would have been decomposed during storage, but DHE is oxidized by de novo formed ROS coming from uncoupled eNOS or NADPH oxidase after freezing and thawing. For detailed methodology see , , , . (B) Densitometric data are presented as bar graphs. (C) eNOS uncoupling was assessed by densitometric quantification of DHE staining in the endothelial cell layer which was extracted from the whole microscope image. A fixed area was used for densitometric quantification and the procedure is shown for one representative endothelial cell layer of AT-II treatment group. The method of densitometric quantification of endothelial DHE staining was adopted from a published protocol (A-C) Stainings were selected from unused pictures and graphs were drafted de novo from original data published in Schuhmacher et al., Hypertension 2010 . Aortic endothelial DHE staining correlated well with endothelial dysfunction measured by ACh-dependent relaxation using isometric tension recording (D), impaired calcium ionophore-stimulated NO formation determined by EPR (E) and eNOS S-glutathionylation quantified by IP and Western blot analysis (F). (D-E) From Hausding et al., Basic Res. Cardiol. 2013 . With permission of Springer-Verlag Berlin Heidelberg. Copyright © 2013. (F) From Kröller-Schön et al., Antioxid. Redox Signal. 2014 . With permission of Mary Ann Liebert, Inc. Copyright © 2014. The scheme summarizes these positive correlates of eNOS uncoupling: endothelial dysfunction, impaired NO formation and eNOS S-glutathionylation as shown here, as well as increased asymmetric dimethyl-L-arginine (ADMA) levels, oxidative disruption of the zinc-sulfur-complex in the dimer binding interface of eNOS and oxidative depletion of tetrahydrobiopterin (BH4) as shown elsewhere (G). Adverse phosphorylation (Thr495, Tyr657) and S-nitros(yl)ation of eNOS were discussed to be involved in eNOS uncoupling but final evidence is still missing. All of these eNOS modifications and modulators of eNOS activity have been discussed in detail as potential redox switches leading to eNOS uncoupling or at least dysfunction , , .
Fig. 2.
Fig. 2
Detection of NADPH oxidase activity in hypertensive animals (AT-II infusion model) by lucigenin enhanced chemiluminescence (ECL) in cardiac membrane fractions. Lucigenin ECL was used for decades to detect NADPH oxidase-derived superoxide formation in cells and tissue membrane fractions , , , . We found that in vivo angiotensin-II (AT-II) infusion increased lucigenin (5 µM) ECL, which was suppressed by nebivolol (N) in vivo treatment (A). Importantly, increased NADPH (200 µM)-triggered lucigenin ECL showed nice correlation with membranous content of the regulatory cytosolic NADPH oxidase (isoform 2) subunits p67phox and Rac-1 (B). In order to investigate the underlying mechanism of this pleiotropic effect of nebivolol we incubated membrane fractions from Ang-II infused rats with different β-blockers (N, nebivolol; C, carvedilol; A, atenolol; M, metoprolol) and measured lucigenin ECL, which was decreased by nebivolol and carvedilol (C). These membrane fraction suspensions were subjected to another ultracentrifugation step after incubation with the β-blockers. The cytosolic fraction (supernatant) was discarded after the 100,000×g centrifugation step and the membrane pellet was used for Western blotting. It turned out that nebivolol and carvedilol that suppressed the lucigenin ECL signal had partially dislocated the regulatory subunits p67phox and Rac-1 (D). The detailed in vitro protocol is provided in (E). All details on the membrane NADPH oxidase assay are provided in . Graphs were drafted de novo from original data published in Oelze et al., Hypertension 2006 .
Fig. 3.
Fig. 3
Superoxide, nitric oxide and peroxynitrite formation in lipopolysaccharide (LPS)-induced endotoxic shock. L-012 enhanced chemiluminescence (ECL) was used for phorbol ester (PDBu)-triggered oxidative burst measurement in whole blood (A), which mainly originates from NADPH oxidase-derived superoxide in granulocytes or more correctly its degradation product hydrogen peroxide in the presence of peroxidases, as known for all other luminol derivatives . Since L-012 also generates appreciable chemiluminescence upon reaction with peroxynitrite, this species could contribute to the overall chemiluminescence signal as well . The L-012 signal nicely correlates with nitrosyl-iron hemoglobin (Hb-NO) levels (B), a surrogate parameter of inducible nitric oxide synthase (iNOS) activity in leukocytes, as measured by electron paramagnetic resonance (EPR) spectroscopy in whole blood of LPS-treated rats . Representative EPR spectra are shown below the quantification bar graph. Both together, Nox-derived superoxide burst and iNOS-derived nitric oxide, yield peroxynitrite, which in turn leads to protein tyrosine nitration as detected by dot blot analysis using a specific antibody against protein-bound 3-nitrotyrosine (C). Alternatively, nitrated proteins can also originate from a (myelo)peroxidase/H2O2/nitrite-dependent pathway, which largely depends on the nature of invading pathogens . Likewise, L-012 ECL can be also used for the detection of oxidative burst (and thereby inflamed tissues) in living animals using in vivo luminescence imaging devices (D, Steven et al. unpublished; L-012 dose in mice: 100 mg/kg injected i.p. in 200 µl DMSO 5 min before sacrifice, then 10 min illumination and signal recorded with an IVIS Spectrum Imager, PerkinElmer, Waltham, MA, USA). All of these parameters nicely reflected the severe inflammatory phenotype induced by LPS treatment and the beneficial anti-inflammatory and antioxidant effects of the dipeptidyl peptidase 4 inhibitors (DPP4i) linagliptin (Lina) and sitagliptin (Sita) as well as the glucagon-like peptide 1 analogue (GLP1a) liraglutide (Lira) . L-012 ECL also had prognostic value in LPS-triggered endotoxic shock as demonstrated by the survival curves (E) . From Steven et al., Basic Res. Cardiol. 2015 . With permission of Springer-Verlag Berlin Heidelberg. Copyright © 2015.
Fig. 4.
Fig. 4
Overview on the simplified model of redox biology in the cardiovascular system with the 3 different categories of assays for RONS detection: The “old”, traditional assays, the state-of-the-art techniques and the cutting-edge (future) assays, always in the same vertical line as the species they can be applied for. Superoxide was identified as an antagonist of the “endothelium-derived relaxing factor” (EDRF = nitric oxide) far before EDRF was widely accepted to be nitric oxide by the famous experiment of Gryglewski, Palmer and Moncada based on the transfer of the perfusate from badykinine-stimulated endothelial cell culture to an organ bath with denuded (endothelium-devoid) aortic ring segments . The vasodilatory potency of EDRF coming from the cell culture was increased by addition of superoxide dismutase (SOD) to the buffer on the cells, supporting the break-down of EDRF by superoxide. From previous work we know today that NO and superoxide react in a diffusion controlled reaction to form peroxynitrite (ONOO-) , . Without this reaction superoxide is dismutated either by SODs or undergoes spontaneous self-dismutation to form hydrogen peroxide, which is largely involved in redox signaling pathways via oxidation of specific thiol residues or inactivated by catalases (Cat), glutathione peroxidases (GPx) or peroxiredoxins. Peroxynitrite can cause widespread oxidative damage in proteins (tyrosine nitration [3-NT] and methionine sulfoxidation [oxMet]) but also lipids and DNA molecules . Abbreviations: IHC, immunohistochemistry; DHR, dihydrorhodamine; PR, plate reader; EPR, electron paramagnetic resonance; ECL, enhanced chemiluminescence; DHE, dihydroethidium; Pox, peroxidase; DCF-DA, dichlorofluorescein-diacetate; DAF, diaminofluorescein.

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