New Probes under Investigation 2.7.1 Common probes used
to detect ROS in cells
So far, there are few ‘gold standard’ probes universally employed and specific enough to measure certain free radicals. The reader who wishes to delve into this area can see some good reviews published recently that cover the advantages and limitations of various reagents for the detection of ROS (Freitas et al., 2009; Niki 2010a,b; Rhee et al., 2010). Thus, this sec-tion only summarizes some of the most widely used reagents for cellular and non-cellular systems.
Cytochrome c
The reduction of cytochrome c has been widely used to estimate specifically ROS release in neutrophils stimulated with dif-ferent agents. The reaction is inhibited almost 100% by SOD addition. The product of the reaction: cytochrome c [Fe (III)] + O2•− → O2 + cytochrome c [Fe (II)] can be analysed at 550 nm. Although this assay is even less sensitive than lucigenin for determining the extracellular production of O2•−, it basically provides the same information.
Tetrazolium salts: NBT; MTT, XTT, WST-1 All these compounds have in common their ability to be reduced by reactive species to form a highly coloured formazan that can be analysed using spectrophotometry and vis-ualized in cells. These salts are particularly sensitive to O2•− generating the radical tetra-zoinil, which dismute to form the water-insoluble blue formazan. Formazan must be solubilized for its quantitation and there-fore attempts have been carried out using MTT or the WST-1 for the same purpose because they produce derivatives with increased water solubility. WST-1 com-pounds have low cost, less probability of dismutation of O2•− to H2O2 and their reduc-tion is almost 100% inhibited by SOD, sug-gesting low cell permeability.
Lucigenin
As indicated earlier, lucigenin can be used for its higher selectivity and sensitivity to O2•− production compared with tetrazolium salts. Superoxide is able to reduce luci-genin to form dioxetane. The latter is unstable and breaks spontaneously to form N-methylacridone in an excited state, which leads to light emission. Because lucigenin is highly selective to O2•−, its detection does not depend on the presence of MPO as it does for luminol. Although lucigenin can promote O2•− generation by redox recycling, the amount produced is minimal. Lucigenin is not a permeable molecule and therefore when using SOD, the emission of light is practically inhibited by 100%.
Dihydroethidium (hydroethidine) This probe is cell permeable and can be oxi-dized by superoxide to form the ethidium cation, which has a strong fluorescence.
Because hydroethidine (HE) could be oxi-dized by other ROS, there is still some debate as to whether the ethidium cation is really a specific product for the presence of O2•−. An HPLC analysis of the HE oxidation products promoted by superoxide revealed a peak corresponding to a new substance, which was assigned to 2-hidroxietidium.
The latter has different fluorescence proper-ties to the ethidium cation and its specific recognition should be performed by HPLC-FLD (Zhao et al., 2003, 2005; Fernandes et al., 2007). This compound has a minor tendency to produce superoxide by redox recycling and is used for detection of intra-cellular ROS. Disadvantages are that it has high photolability and that in cells undergo-ing apoptosis by the intrinsic pathway the release of cytochrome c may lead to artefac-tual oxidation of hydroethidine (Zielonka et al., 2008).
Dihydrorhodamine 123 and 2,7-dichlorodihydrofluorescein Dihydrorhodamine (DHR) is a lipophilic probe sensitive to hydrogen peroxide. Once inside the cell DHR undergoes oxidation and one of its amino groups tautomerizes to the imino form (Rho123), preventing it from leaving the cell (Henderson and Chappell, 1993). 2,7-Dichlorodihydrofluorescein (DCFH) is a derivative of fluorescein that can be oxi-dized by ROS generating intense fluorescent dichlorofluorescein (DCF). The diacetate derivative of DCFH is apolar and non- fluorescent and thus can enter cells, where it can serve as substrates for esterases to release DCFH. This latter is polar and is therefore trapped inside the cell. The probes respond to both ROS and RNS (Crow, 1997).
The main disadvantage of both DHR and DCFH is high sensitivity to photo-oxidation and a certain tendency to leak away from cells in long-term assays. Although both probes could be used, some authors preferred DHR instead of DCFH for H2O2
Antioxidants from Vegetal Sources 25
detection in living cells (Qin et al., 2008;
Sakurada et al., 1992).
Amplex Red
N-Acetyl-3,7-dihydroxyphenoxazine (Amplex Red) is a highly specific probe for the detec-tion of hydrogen peroxide produced in neu-trophils. When this probe is oxidized by hydrogen peroxide it generates a stable fluor-escent product resofurin, which can be ana-lysed using excitation at 520–550 nm and an emission at 585–595 nm. Although this compound is not interfered with by the autofluorescence of biological samples, some vegetal constituents could interfere with Amplex Red oxidation (Serrano et al., 2009; Mishin et al., 2010).
Tetramethylbenzidine
Tetramethylbenzidine (TMB) is a probe used to detect HOCl, generating a blue product when it is oxidized. A disadvantage of this probe is that it can be a substrate of MPO, limiting its applications (Freitas, 2009).
Diaminofluoresceins
These compounds (DAF-2 and DAF-FM) have been used successfully for recognizing the extracellular production of NO. In an analogous way to DCHF-DA, their acetylated derivatives are used for the recognition of intracellular production of NO. These probes are highly sensitive and allow moni-toring of NO production in real time.
Ethanol/a-(4-pyridyl-1oxide)-N-tert-butylnitrone (4POBN)
This probe is one of the few existing mol-ecules able to detect specifically the pro-duction of the hydroxyl radical.
MitoSOX Red
This is a fluorescent probe specifically oxi-dized by mitochondrial superoxide. It can be used to investigate the production of ROS in living cells. It should be noted that their oxidation can be prevented by adding SOD-mimetic agents such as Tiron and
FeTCPP. Although much hope was placed on this probe, its specific use to detect intra-cellular superoxide production is contro-versial. So, Zielonka and Kalyanaraman critically reviewed the reliability of this probe, concluding that it must be used with care and HPLC profiles should be traced in order to observe all HE oxidation products (Zielonka and Kalyanaraman, 2010).
2.7.2 New probes for the detection of ROS
Recently, substantial progress has been made in the synthesis of new and more spe-cific probes to detect ROS.
New probes for the hydroxyl radical (OH•) Traditionally, the hydroxyl radical is detected by reaction with salicylate or phe-nylalanine (Althaus et al., 1993; Halliwell and Kaur, 1997; Luo and Lehotay, 1997).
However, both strategies do not allow a realistic estimate of the production of hydroxyl radical and some new probes have been introduced.
terephthalate. The product of terephtha-late hydroxylation (2-hydroxy-terephthaterephtha-late) has a higher fluorescence intensity than 2-hydroxy-benzoate (Saran and Summer, 1999). Because of that, this probe has gained popularity for detecting OH radicals pro-duced from diverse sources, including liv-ing systems, in the micromolar range (Freinbichler et al., 2008a,b; Page et al., 2010). Detection could be done using fluor-escence readers or, even better, trough HPLC-FLD (Li et al., 2004).
dppec. Similarly, the fluorescent probe 1,2- dipalmitoylglycerophosphorylethanolamine (DPPEC) has been developed for the detec-tion of the hydroxyl radical in lipid mem-branes (Soh et al., 2008). The probe has high selectivity for this radical.
tempo. Recently, Maki and colleagues developed a new probe from the union of
perylene-3,4,9,10-tetracarboxyl bisimide and 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) (Maki et al., 2009). This compound emits fluorescence in the visible region and is highly selective for the hydroxyl radical.
rhodamine nitroxide probes. Most recently, Yapici and coworkers developed a series of rhodhamine nitroxide probes (I, II and III) specific for OH that can be used for fluores-cence and ESR detection (Yapici et al., 2012). These molecules have been success-fully assayed for OH detection in cell-free systems (Fenton reagent), ARPE-19 cell stimulated with PMA and tumour lines such as HeLa, HepG2 and SW-620.
A new probe for superoxide
Circularly permuted yellow fluorescent protein (cpYFP), previously used as the core structure for the Ca2+ indicator pericam (Nagai et al., 2001), is a novel biosensor for O2•−, the primal ROS generated by the elec-tron transfer chain. The fluorescence emis-sion (515 nm) of purified cpYFP when excited at 488 nm is five times brighter under strong oxidizing conditions. Extensive in vitro experiments revealed the superox-ide selectivity of cpYFP over other physio-logically relevant oxidants and metabolites.
The O2•− associated increase in cpYFP fluor-escence is completely reversed by the sub-sequent addition of Cu/Zn-superoxide dismutase (SOD, 600 U/ml) or prevented by prior addition of SOD. By contrast, cpYFP emission is unchanged by H2O2 and peroxy-nitrite, and is decreased by HO• and NO (Wang et al., 2008).
New probes for hydrogen peroxide
peroxifluor-1. In order to improve the spe-cific detection of H2O2, Chang and coworkers developed the peroxifluor-1 probe (Chang et al., 2004). Indeed, their response to H2O2 is 500 times greater than for other ROS. A fam-ily of boronate probes has been synthesized, such as peroxyresofurin-1 (PR-1), greenfluo-rescent PF-1 and the blue-fluogreenfluo-rescent peroxyxanthone-1 (PX1). Interestingly, all these probes are permeable and can detect
micromolar concentrations of H2O2 in vivo.
Boronate-derived probe oxidation can be used for studying localization, trafficking and in vivo production of H2O2 in various liv-ing systems (Lippert et al., 2011).
organelle-specific detection of h2o2 using snap-tagproteinlabelling. A refining of bor-onate chemistry has been recently devel-oped. Using SNAP-tag technology, site-specific protein labelling can be done in practically any cell compartment (nucleus, mitochondria, plasma membrane and endoplasmic reticulum). Hence, local-ized H2O2 production has been detected with one of these fusion products named SNAP-peroxy-Green (SNAP-PG). The spe-cificity of this probe was evaluated using scanning confocal microscopy (Dickinson et al., 2010; Srikun et al., 2010).
hyper. OxyR is a sensor and transcriptional regulator that can detect H2O2 through domains that may sense this ROS. The sen-sor domain is called OxyR-RD, and by fusion with cpYFP gives rise to the Hyper.
This probe has two excitation peaks at 420 and 500 nm, with emission at 516 nm. When exposed to H2O2, the 420 nm peak decreases while the 500 nm increases. This is there-fore a ratiometric sensor probe. Hyper is highly selective for H2O2 and cell transfec-tion is required for its use. Hyper has been demonstrated as a valuable tool to monitor hydrogen peroxide generated in different cellular compartments (Malinouski et al., 2011). The fusion Hyper-PTS1 has recently been used for specific hydrogen peroxide detection in peroxisomes as well (Gehrmann and Elsner, 2011).
New probes for hypochlorous acid
sulfonaphthoaminophenyl fluorescein (snapf). SNAPF is a newly developed fluorescein-derived probe for the specific detection of intracellular levels of HOCl generated by MPO activity. In the presence of HOCl, the 4-aminophenyl function suffers oxidative cleavage to release fluorescein. This probe can be used for the in vivo non-invasive detection of HOCl (Freitas et al., 2009).
Antioxidants from Vegetal Sources 27
2.8 What Should We do to Evaluate