Kahina Abbas

Nikola Babić

Fabienne Peyrot
email: fabienne.peyrot@parisdescartes.fr

Use of spin traps to detect superoxide production in living cells by electron paramagnetic resonance (EPR) spectroscopy

Keywords: EPR spectroscopy, ESR spectroscopy, Spin trapping, Macrophage, Free radical, Cyclic nitrone reactive oxygen species, SCE, saturated calomel electrode, SOD, superoxide dismutase, TAMs, triarylmethyl radicals

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Use of spin traps to detect superoxide production in living cells by electron paramagnetic resonance (EPR) spectroscopy

Introduction 1.1 Superoxide as a key intermediate in oxidative stress and a mediator of cell signaling

Superoxide radical anion (O2 •-) is a major by-product of aerobic metabolism as a result of monoelectronic reduction of molecular oxygen by enzymes in the mitochondrial respiratory chain [START_REF] D'autréaux | ROS as signalling molecules: mechanisms that generate specificity in ROS homeostasis[END_REF]. Other important sources, either endogenous or exogenous, include enzymes in the NADPH oxidase family (Nox), xanthine oxidase, microsomes, autoxidation reactions of reduced flavines, quinones, metal ions, metalloproteins, and exposition to ionizing radiation or photochemical irradiation. Characterized by both mild oxidizing and reducing properties, the reactivity of O2 •-results mainly from its interaction with radicals and cationic iron-sulfur clusters ([Fe-S] clusters). It is a precursor to more toxic species. Hydrogen peroxide (H2O2) arises from spontaneous and enzyme-catalyzed disproportionation or from reduction of O2 •-. The highly reactive hydroxyl radical (HO • ) quickly follows. Peroxynitrite (ONOO -) results from the direct reaction of O2 •-with nitric oxide (NO • ). In cells, O2 •-is efficiently removed by the antioxidant enzyme superoxide dismutase (SOD) [START_REF] Mccord | Superoxide Dismutase: an enzymic function for erythrocuprein (hemocuprein)[END_REF]. The instability of O2 •-and its poor ability to cross cell membranes due to its negative charge do not favor its role as a signaling molecule. However the SoxR transcription factor is an established example of specific O2 •-sensing through [Fe-S] clusters [START_REF] Hidalgo | An iron-sulfur center essential for transcriptional activation by the redox-sensing SoxR protein[END_REF]. Monitoring production of O2 •-in cells with ever increasing sensitivity is thus an important issue in biomedical research on inflammation, cancer, aging, or atherosclerosis among other fields. Several techniques have been developed to answer this question, some of which based on spectrophotometric or fluorescent probes or on HPLC detection [START_REF] Bartosz | Use of spectroscopic probes for detection of reactive oxygen species[END_REF][START_REF] Zielonka | Cytochrome c-mediated oxidation of hydroethidine and mito-hydroethidine in mitochondria: identification of homo-and heterodimers[END_REF]. Here we will concentrate on technical aspects of the spin trapping/EPR technique, delineating recent breakthroughs, inherent limitations and potential artifacts.

Principle of spin trapping

EPR spectroscopy is the most direct method for characterizing and measuring free radicals in chemistry and biology, despite its rather low sensitivity. Using conventional Xband (ν ≃ 9 GHz) spectrometers, the detection threshold is in the range of 2-3 µM for a 25 µL sample (however it highly depends on the experimental setup and the acquisition parameters). Most free radicals are highly reactive and have short half-life in biological environment, and thus their concentration does not reach measurable level under such conditions. Therefore, direct observation is not possible; free radicals are observed indirectly by using spin traps. A spin trap is a diamagnetic molecule that readily reacts with a reactive radical to form a more stable radical product called a spin adduct, the concentration of which can reach measurable level (Figure 1). This radical stabilization technique is called spin trapping. Spin trapping experiments with 5,5-dimethyl-1pyrrolidine N-oxide (DMPO, Figure 1) in chemical systems were first reported in 1967 by Iwamura and Inamoto [START_REF] Iwamura | Novel formation of nitroxide radicals by radical addition to nitrones[END_REF]. The method was further developed by Janzen and Blackburn [START_REF] Janzen | Detection and identification of short-lived free radicals by an electron spin resonance trapping technique[END_REF], who coined the terms spin trap and spin trapping, and its first application to cellular systems dates back 1979 with detection on suspensions of stimulated human neutrophils [START_REF] Green | The production of hydroxyl and superoxide radicals by stimulated human neutrophilsmeasurements by EPR spectroscopy[END_REF]. The best spin traps for O2 •-are compounds with a cyclic nitrone functional group that form persistent aminoxyl (nitroxide) radicals after addition of the reactive radical species. Though the spin traps are not selective of one radical, the resulting nitroxide spin adduct has a distinctive EPR spectrum which gives information about the trapped radical species. Indeed, both nitrogen and β-hydrogen hyperfine coupling constants in spin adducts derived from cyclic nitrones are sensitive to the nature of the trapped radical. A careful selection of the spin trap is however necessary to unequivocally distinguish oxygencentered and thiyl radical adducts [START_REF] Buettner | Spin trapping: ESR parameters of spin adducts[END_REF][START_REF] Karoui | Characterization of sulfur-centered radical intermediates formed during the oxidation of thiols and sulfite by peroxynitrite. ESR-spin trapping and oxygen uptake studies[END_REF][START_REF] Karoui | DEPMPO: an efficient tool for the coupled ESR-spin trapping of alkylperoxyl radicals in water[END_REF]. In the following, we will append -OOH and -OH to the spin trap name to refer to the O2 •-and the HO • adducts, respectively.

Spin traps developed for cell studies

DMPO was the first cyclic nitrone used in spin trapping studies in cells and is still most commonly used (average of 13 articles published each year since 1990 according to Web of Science TM ), despite well identified pitfalls. Indeed, detection of DMPO-OH following incubation with stimulated neutrophils was first considered as an evidence for HO • production in these cells [START_REF] Green | The production of hydroxyl and superoxide radicals by stimulated human neutrophilsmeasurements by EPR spectroscopy[END_REF][START_REF] Rosen | Hydroxyl radical generation by polymorphonuclear leukocytes measured by electron spin resonance spectroscopy[END_REF], before it was proved, both experimentally and by computation, that DMPO-OOH spontaneously (non-enzymatically) decomposes to several species including DMPO-OH with a half-life under 1 min [START_REF] Finkelstein | Production of hydroxyl radical by decomposition of superoxide spin-trapped adducts[END_REF][START_REF] Locigno | Nitric oxide release from the unimolecular decomposition of the superoxide radical anion adduct of cyclic nitrones in aqueous medium[END_REF][START_REF] Villamena | Superoxide radical anion adduct of 5,5-dimethyl-1-pyrroline N-oxide (DMPO). 2. The thermodynamics of decay and EPR spectral properties[END_REF], and that stimulated neutrophils do not produce HO • [START_REF] Britigan | Do human neutrophils make hydroxyl radical -Determination of free radicals generated by human neutrophils activated with a soluble or particulate stimulus using electron paramagnetic resonance spectrometry[END_REF]. Thanks to the very active research in the field of spin trap design, several derivatives of DMPO with tuned lipophilicity have been synthesized since, bearing electron-withdrawing groups at position 5 of the ring, either alkoxycarbonyl (e.g., EMPO [START_REF] Olive | 2-Ethoxycarbonyl-2methyl-3,4-dihydro-2H-pyrrole-1-oxide: evaluation of the spin trapping properties[END_REF], BMPO [START_REF] Zhao | Synthesis and biochemical applications of a solid cyclic nitrone spin trap: A relatively superior trap for detecting superoxide anions and glutathiyl radicals[END_REF], AMPO [START_REF] Villamena | Spin trapping by 5-carbamoyl-5-methyl-1-pyrroline N-oxide (AMPO): Theoretical and experimental studies[END_REF]) or dialkoxyphosphoryl substituents (DEPMPO [START_REF] Frejaville | 5-Diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) -a new phosphorylated nitrone for the efficient in-vitro and in-vivo spin-trapping of oxygen-centered radicals[END_REF], DIPPMPO [START_REF] Chalier | 5-Diisopropoxyphosphoryl-5-methyl-1-pyrroline N-oxide, DIPPMPO, a crystalline analog of the nitrone DEPMPO: synthesis and spin trapping properties[END_REF], CYPMPO [START_REF] Kamibayashi | Synthesis and characterization of a practically better DEPMPOtype spin trap, 5-(2,2-dimethyl-1,3-propoxy cyclophosphoryl)-5-methyl-1pyrroline N-oxide (CYPMPO)[END_REF]) (Figure 2). Not only do these spin traps offer a dramatically enhanced intrinsic stability of the corresponding O2 •-spin adducts in buffer, but no spontaneous decomposition to the HO • adduct is observed with these traps. The thermodynamically-preferred pathways for the spontaneous decomposition of O2 •-spin adducts and the decomposition of their one-electron oxidation and reduction products were explored by computational studies, showing the need of a "loose" bond acceptor in the structure but also of both a low electron affinity and a low ionization potential for improved adduct stability [START_REF] Villamena | Superoxide radical anion adduct of 5,5-dimethyl-1-pyrroline N-oxide (DMPO). 2. The thermodynamics of decay and EPR spectral properties[END_REF][START_REF] Han | Synthesis and spin-trapping properties of a new spirolactonyl nitrone[END_REF][START_REF] Villamena | Superoxide radical anion adduct of 5,5-dimethyl-1-pyrroline Noxide. 6. Redox properties[END_REF]. Most of these traps now are commercially available and demonstrate extended shelf-life compared to DMPO, although they remain expensive and can be difficult to obtain. Phosphorylated nitrones allow easier identification of the trapped species because the large hyperfine coupling to 31 P is highly dependent on the structure of the trapped radical. This can however be slightly detrimental to the sensitivity of detection due to a doubling of the number of lines in the EPR spectrum, which reduces the signal-to-noise ratio. The reaction of O2 •-with DEPMPO derivatives leads to a mixture of diastereoisomers with slightly different EPR spectra. Rapid equilibrium between conformers of the predominant trans adduct also impacts the EPR spectra [START_REF] Frejaville | 5-(Diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide: a new efficient phosphorylated nitrone for the in vitro and in vivo spin trapping of oxygenradicals[END_REF]. Further substitution at position 4 of the pyrroline ring in a cis position compared to the dialkoxyphosphoryl group in DEPMPO or DIPPMPO simplifies the EPR spectrum by favoring the formation of one diastereoisomer and stabilizing one conformation of the ring in the adduct [START_REF] Hardy | Mito-DEPMPO synthesized from a novel NH2-reactive DEPMPO spin trap: a new and improved trap for the detection of superoxide[END_REF]. Moreover, conjugation of the pyrroline Noxide ring with a permethylated -cyclodextrin (CD), as in CD-DEPMPO or CD-DIPPMPO [START_REF] Hardy | Improving the trapping of superoxide radical with a betacyclodextrin-5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) conjugate[END_REF][START_REF] Bézière | Metabolic stability of superoxide adducts derived from newly developed cyclic nitrone spin traps[END_REF], allows selective detection of O2 •-over HO • , probably due to competition for HO • between the methoxy groups in the CD and the nitrone function. Conjugation with a CD also guaranties extracellular localization of the spin trap, by contrast with DMPO and DEPMPO which are assumed to readily permeate cell membranes [START_REF] Abbas | Detection of superoxide production in stimulated and unstimulated living cells using new cyclic nitrone spin traps[END_REF][START_REF] Anzai | ESR measurement of rapid penetration of DMPO and DEPMPO spin traps through lipid bilayer membranes[END_REF]. These newly developed compounds are unfortunately not commercially available, which limits their application. A determinant characteristic of an efficient spin trap for O2 •-is the kinetic rate of the spin trapping reaction (kT). Kinetics studies of this reaction are however difficult to perform and lots of published absolute values of kT are still controversial due to biased methods of determination [START_REF] Keszler | Comparative investigation of superoxide trapping by cyclic nitrone spin traps: the use of singular value decomposition and multiple linear regression analysis[END_REF][START_REF] Lauricella | A new kinetic approach to the evaluation of rate constants for the spin trapping of superoxide/hydroperoxyl radical by nitrones in aqueous media[END_REF]. Comprehensive analysis of the spin trapping reaction by Villamena et al. using the density functional theory (DFT) approach greatly contributed to rationalize the design of improved spin traps [START_REF] Han | Synthesis and spin-trapping properties of a new spirolactonyl nitrone[END_REF][START_REF] Villamena | Reactivity of superoxide radical anion with cyclic nitrones: role of intramolecular H-bond and electrostatic effects[END_REF][START_REF] Han | Improved spin trapping properties by beta-cyclodextrin-cyclic nitrone conjugate[END_REF]. The nucleophilic nature of O2 •- addition to nitrones explains why increasing the positive charge at position 2 of the ring through electron-withdrawing groups at C-5 is beneficial to the spin trapping rate, provided excessive steric repulsion on position 5 is avoided. Also, initial H-bond interaction of O2 •-with an H-bond donor, such as an amide function, at position 5 further enhances its nucleophilicity towards the nitrone function, yielding higher kT. Whatever the spin trap, the reaction with O2 •-remains a slow process (kT < 100 M -1 s -1 ) [START_REF] Ouari | Recent developments and applications of the coupled EPR/spin trapping technique (EPR/ST)[END_REF] and large concentrations of spin traps (10-100 mM) are required to compete with O2 •- spontaneous disproportionation. Fortunately, most cyclic nitrones demonstrate low toxicity towards cellular systems at these concentrations, with the exception of BMPO which was moderately toxic in CHO cells and various cancer cell lines at 50 mM [START_REF] Abbas | Detection of superoxide production in stimulated and unstimulated living cells using new cyclic nitrone spin traps[END_REF][START_REF] Saito | Comparison of superoxide detection abilities of newly developed spin traps in the living cells[END_REF][START_REF] Khan | Spin traps: in vitro toxicity and stability of radical adducts[END_REF][START_REF] Rohr-Udilova | Cytotoxicity of novel derivatives of the spin trap EMPO[END_REF].

Spin adduct stability and metabolism

A major limitation to the application of spin trapping with cyclic nitrones in cell studies is the instability of the spin adducts in biological systems which leads to rapid loss of EPR signal. The rate of cell-induced spin adduct decay is highly dependent on the cell type and on the activation state of the cells [START_REF] Roubaud | Quantitative measurement of superoxide generation and oxygen consumption from leukocytes using electron paramagnetic resonance spectroscopy[END_REF][START_REF] Shi | Evaluation of spin trapping agents and trapping conditions for detection of cell-generated reactive oxygen species[END_REF]. Modification of the flux of O2 •-production can also influence spin adduct stability [START_REF] Samuni | Superoxide reaction with nitroxide spin-adducts[END_REF], since reduction of the adducts by O2 •-is thermodynamically favored and supposed to play an important role in spin adduct decay [START_REF] Villamena | Superoxide radical anion adduct of 5,5-dimethyl-1-pyrroline Noxide. 6. Redox properties[END_REF]. Investigations, using redox agents, cells, or cellular fractions, of the biochemical mechanisms responsible for signal decay and the structure of diamagnetic products enabled to distinguish between oxidative and reductive pathways involved on this phenomenon [START_REF] Bézière | Metabolic stability of superoxide adducts derived from newly developed cyclic nitrone spin traps[END_REF][START_REF] Abbas | Detection of superoxide production in stimulated and unstimulated living cells using new cyclic nitrone spin traps[END_REF][START_REF] Reinke | Degradation of DMPO adducts from hydroxyl and 1-hydroxyethyl radicals by rat liver microsomes[END_REF][START_REF] Rosen | Detection of superoxide generated by endothelial cells[END_REF][START_REF] Khramtsov | NMR spin trapping: Detection of free radical reactions using a phosphorus-containing nitrone spin trap[END_REF][START_REF] Zhao | Detection and characterization of the product of hydroethidine and intracellular superoxide by HPLC and limitations of fluorescence[END_REF][START_REF] Bézière | Metabolic stability of superoxide and hydroxyl radical adducts of a cyclic nitrone toward rat liver microsomes and cytosol: A stopped-flow ESR spectroscopy study[END_REF]. Ferric hemeproteins with sufficient access to the iron cofactor, such as cytochrome P450, hemoglobin, or horseradish peroxidase (HRP), but not cytochrome c, could induce homolytic or heterolytic cleavage of the hydroperoxyde function in the O2 •-adducts of small-size cyclic nitrones, yielding ring-opened diamagnetic compounds or the HO • adduct, which upon further oxidation of the aminoxyl (nitroxide) function would lead to a highly unstable oxoammonium cation as an intermediate (Figure 3A) [START_REF] Bézière | Metabolic stability of superoxide and hydroxyl radical adducts of a cyclic nitrone toward rat liver microsomes and cytosol: A stopped-flow ESR spectroscopy study[END_REF]. In fact, the reduction by intracellular reductants usually accounts for the largest contribution to EPR signal loss (Figure 3B). Reaction with cytosolic ascorbate forms EPR silent hydroxylamines, which are prone to fast elimination of small molecules, yielding the parent nitrone [START_REF] Khramtsov | NMR spin trapping: Detection of free radical reactions using a phosphorus-containing nitrone spin trap[END_REF][START_REF] Bézière | Metabolic stability of superoxide and hydroxyl radical adducts of a cyclic nitrone toward rat liver microsomes and cytosol: A stopped-flow ESR spectroscopy study[END_REF]. Glutathione peroxidase in the presence of glutathione (GPx/GSH) has been shown to catalyze the reduction of the O2 •-adduct to the HO • adduct [START_REF] Rosen | Detection of superoxide generated by endothelial cells[END_REF]. Analogues of DMPO and DEPMPO displayed similar behavior when submitted to these different pathways [START_REF] Bézière | Metabolic stability of superoxide adducts derived from newly developed cyclic nitrone spin traps[END_REF]. Only when the spin trap structure ensures extracellular localization, are the adducts stabilized against cell-induced decomposition, as observed with the permethylated -cyclodextrin conjugate CD-DIPPMPO [START_REF] Abbas | Detection of superoxide production in stimulated and unstimulated living cells using new cyclic nitrone spin traps[END_REF].

Comparison to other detection methods

Detailed reviews for comparing reactive oxygen species (ROS) detection methods have been published elsewhere and are beyond the scope of this work [START_REF] Bartosz | Use of spectroscopic probes for detection of reactive oxygen species[END_REF][START_REF] Wardman | Fluorescent and luminescent probes for measurement of oxidative and nitrosative species in cells and tissues: progress, pitfalls, and prospects[END_REF][START_REF] Debowska | Toward selective detection of reactive oxygen and nitrogen species with the use of fluorogenic probes--Limitations, progress, and perspectives[END_REF][START_REF] Dikalov | Methods for detection of mitochondrial and cellular Methods Accepted manuscript Published in final edited form as[END_REF]. We will thus focus only on general conclusions here to highlight differences in specificity and reliability of each method compared to spin trapping. Among the oldest means of detection, spectrophotometric probes, i.e. substances with optical properties that change upon reaction with O2 •-, are usually simpler to implement and are rather specific of O2 •-but they are less sensitive than spin traps [START_REF] Bartosz | Use of spectroscopic probes for detection of reactive oxygen species[END_REF]. Common approaches include monitoring of the reduction of cytochrome c by O2 •-(increase of light absorption at 550 nm) and of the reduction of various tetrazolium salts (among which nitro blue tetrazolium, NBT). Known limitations of NBT method are O2 •--independent reduction of the probe by cellular reductases and subsequent O2 •-production by interaction with oxygen. Fluorescent probes, i.e. non fluorescent (or weakly fluorescent) reagents that yield fluorescent products upon reaction with ROS, such as dihydrorhodamines or the diacetate and free form of dihydro-2',7'-dichlorofluorescein (DCFH2), have been most frequently used to measure oxidative stress in cells due to their good sensitivity and versatility. However they are not specific of O2 •-and actually have a very low reactivity with O2 •-in absence of peroxidase-like catalyst [START_REF] Wardman | Fluorescent and luminescent probes for measurement of oxidative and nitrosative species in cells and tissues: progress, pitfalls, and prospects[END_REF]. Besides, a major artifact has been described with 2',7'-dichlorofluorescein derivatives in that they generate ROS themselves within cells [START_REF] Marchesi | Photoreduction of the fluorescent dye 2 '-7 '-dichlorofluorescein: A spin trapping and direct electron spin resonance study with implications for oxidative stress measurements[END_REF][START_REF] Bonini | The oxidation of 2′,7′dichlorofluorescin to reactive oxygen species: A self-fulfilling prophesy?[END_REF]. Recently, a new series of fluorescent probes, that afford a specific non-redox detection of O2 •-and overcome interference with cellular reductants (glutathione and Fe 2+ ), has been proposed [START_REF] Hu | Fluorescent Probe HKSOX-1 for Imaging and Detection of Endogenous Superoxide in Live Cells and In Vivo[END_REF]. The lead compound HKSOX-1 is a trifluorosulfonate derivative of 5-carboxy-2',4',5',7'-tetrafluorofluorescein. Clearly, more work needs to be done to fully characterize these promising probes. Less frequently used, chemiluminescent probes for O2 •-based on lucigenin or luminol offer attractive high detection sensitivity. However, just as with NBT and DCFH2, redox cycling of the probes is known to generate O2 •-in cells and the detection is prone to many interferences with cellular reductants and oxidants, which are difficult to control [START_REF] Bartosz | Use of spectroscopic probes for detection of reactive oxygen species[END_REF][START_REF] Wardman | Fluorescent and luminescent probes for measurement of oxidative and nitrosative species in cells and tissues: progress, pitfalls, and prospects[END_REF]. As a consequence, there is a growing consensus against their use in living cells. Hydroethidine (HE) and analogues are much more reactive towards O2 •-than reduced fluoresceins and rhodamines, specifically yielding 2-hydroxyethidium (2-OH-E + ). Using HPLC but not fluorescence, 2-OH-E + can unequivocally be identified and separated from ethidium (E + ) and dimerization products arising from the reaction with various biologically relevant oxidants [START_REF] Zielonka | Cytochrome c-mediated oxidation of hydroethidine and mito-hydroethidine in mitochondria: identification of homo-and heterodimers[END_REF][START_REF] Zielonka | HPLC study of oxidation products of hydroethidine in chemical and biological systems: ramifications in superoxide measurements[END_REF]. This is thus considered a sound qualitative method of detection. The formation of 2-OH-E + is however a two-step process, the first oxidation step being non-specific, by opposition to spin trapping. The response of HE to O2 •-could thus be altered if another oxidant initially comes into play. Moreover, the rate of formation of 2-OH-E + within cells is dependent upon both HE cellular uptake and depletion by competing oxidation pathways. Quantitative monitoring of intracellular O2 •-could therefore require measurement of the ratio between 2-OH-E + and HE but this strategy needs further validation [START_REF] Maghzal | Improved analysis of hydroethidine and 2hydroxyethidium by HPLC and electrochemical detection[END_REF][START_REF] Zielonka | Hydroethidine-and MitoSOX-derived red fluorescence is not a reliable indicator of intracellular superoxide formation: another inconvenient truth[END_REF]. The low sensitivity of most commonly used spin traps is a major limitation of spin trapping. More sensitive EPR probes for ROS exist, such as diamagnetic hydroxylamines which yield nitroxide compounds upon oxidation by ROS [START_REF] Dikalov | Spin trapping of superoxide radicals and peroxynitrite by 1-hydroxy-3-carboxy-pyrrolidine and 1-hydroxy-2,2,6,6tetramethyl-4-oxo-piperidine and the stability of corresponding nitroxyl radicals towards biological reductants[END_REF][START_REF] Dikalov | Production of extracellular superoxide by human lymphoblast cell lines: comparison of electron spin resonance techniques and cytochrome C reduction assay[END_REF][START_REF] Dikalov | EPR detection of cellular and mitochondrial superoxide using cyclic hydroxylamines[END_REF]. They are designated as spin probes and should not be confused with spin traps where a covalent bond is formed with O2 •-. The rate constants of the reaction with O2 •-are at least a hundred times higher than with cyclic nitrones [START_REF] Bartosz | Use of spectroscopic probes for detection of reactive oxygen species[END_REF]. However, hydroxylamines do not afford unambiguous identification of the involved ROS, since they react with O2 •-, ONOO -, peroxyl radicals, and H2O2 in the presence of metal ions. Use of specific scavengers can help overcoming this drawback, provided it is proved that they access the site of detection. Hydroxylamines are also sensitive to autoxidation in air, which may lead to high background signals. Their reaction products, nitroxides, are sensitive to reduction in cellular systems (mainly by ascorbate or reductases) [START_REF] Bačić | In vivo evaluation of different alterations of redox status by studying pharmacokinetics of nitroxides using magnetic resonance techniques[END_REF], though to a lesser extent than nitrone spin adducts. Monitoring the decay of spin probes such as triarylmethyl radicals (TAMs) is another EPR method for ROS detection. However loss of EPR signal occurs upon reaction with both O2 and peroxyl radicals [START_REF] Decroos | Oxidation of tris-(p-carboxyltetrathiaaryl)methyl radical EPR probes: evidence for their oxidative decarboxylation and molecular origin of their specific ability to react with O2*[END_REF][START_REF] Decroos | Oxidative and reductive metabolism of tris(p-carboxyltetrathiaaryl)methyl radicals by liver microsomes[END_REF], and even if TAMs are not sensitive to reduction by ascorbate and NAD(P)H, reduction by reductases is quite efficient and can interfere with the detection.

In the end, it seems that the ideal, flawless detection method for O2 •-within cells is still out of reach. Yet, combination of well-characterized methods can help develop a more indepth analysis of ROS produced in cellular systems, spin trapping with cyclic nitrones coupled to EPR being one of them.

Material and methods

Spin trap stock solutions

It is essential that spin trap solutions are free of contamination by hydroxylamine and nitroxide impurities that can impair O2 •-detection and give artifacts. Contaminating hydroxylamines can be tested by treatment with ferricyanide which converts them to EPR-visible nitroxides [START_REF] Leinisch | Investigation of spin-Methods Accepted manuscript[END_REF]. Most commercially available DMPO contains paramagnetic impurities. More recently described spin traps are usually more stable upon long-term storage at -20°C or lower. However, DEPMPO and EMPO are colorless to slightly yellow oils that can also be contaminated. Distillation of the neat oils is difficult on a small scale. Purification methods of contaminated solutions of spin traps by sequential treatment by activated charcoal and ferricyanide have been proposed but can be rather tedious [START_REF] Keszler | Comparative investigation of superoxide trapping by cyclic nitrone spin traps: the use of singular value decomposition and multiple linear regression analysis[END_REF][START_REF] Oberley | The production of hydroxyl radical by bleomycin and iron (II)[END_REF][START_REF] Pietri | Real-time continuous-flow spin trapping of hydroxyl free radical in the ischemic and post-ischemic myocardium[END_REF][START_REF] Barbacanne | Detection of superoxide anion released extracellularly by endothelial cells using cytochrome c reduction, ESR, fluorescence and lucigeninenhanced chemiluminescence techniques[END_REF][START_REF] Jackson | Detection and removal of contaminating hydroxylamines from the spin trap DEPMPO, and re-evaluation of its use to indicate nitrone radical cation formation and S(N)1 reactions, Free Radic[END_REF]. By contrast, BMPO and CYPMPO are crystalline solids and are easily purified by recrystallization [START_REF] Zhao | Synthesis and biochemical applications of a solid cyclic nitrone spin trap: A relatively superior trap for detecting superoxide anions and glutathiyl radicals[END_REF][START_REF] Kamibayashi | Synthesis and characterization of a practically better DEPMPOtype spin trap, 5-(2,2-dimethyl-1,3-propoxy cyclophosphoryl)-5-methyl-1pyrroline N-oxide (CYPMPO)[END_REF]. Stock solutions of spin traps should be prepared in an appropriate buffered saline containing a metal chelator such as diethylene triamine pentaacetic acid (DTPA, 0.1 -1 mM) when metal ions are not required in the assay and should be stored at -20°C or below. A sufficient amount of the spin trap should be introduced in the assay before the start of O2 •-production to ensure satisfactory accumulation of the spin adduct above EPR detection limit. Enhancement of spin adduct decay by excessive amounts of spin trap has been described [START_REF] Lauricella | A new kinetic approach to the evaluation of rate constants for the spin trapping of superoxide/hydroperoxyl radical by nitrones in aqueous media[END_REF], however in the presence of cells this effect might be masked by cellinduced rapid spin adduct decay. Typical final concentrations used in the literature are 100 mM for DMPO, 50 mM for BMPO, and 5-20 mM for DEPMPO and CYPMPO. Use of phenol red-and serum-containing culture media in spin trapping experiments should be avoided since they contain many components susceptible to generate or scavenge free radicals or to reduce spin adducts [START_REF] Grzelak | Light-dependent generation of reactive oxygen species in cell culture media[END_REF][START_REF] Halliwell | Oxidative stress in cell culture: an under-appreciated problem?[END_REF]. If these are used for the culture, cells should be carefully washed with buffered saline before addition of the spin trap.

EPR experimental setups and sample preparation

In this section, we will only describe experimental setups requiring commercial X-band continuous wave (CW) spectrometers and resonators.

Detection on cell suspensions

Most spin trapping experiments in the literature have been performed on cell suspensions using either quartz flat cells or glass capillaries as sample holders. Phagocytic cells represent the most studied cell type with DMPO [START_REF] Green | The production of hydroxyl and superoxide radicals by stimulated human neutrophilsmeasurements by EPR spectroscopy[END_REF][START_REF] Rosen | Hydroxyl radical generation by polymorphonuclear leukocytes measured by electron spin resonance spectroscopy[END_REF][START_REF] Britigan | Do human neutrophils make hydroxyl radical -Determination of free radicals generated by human neutrophils activated with a soluble or particulate stimulus using electron paramagnetic resonance spectrometry[END_REF][START_REF] Hume | The production of oxygencentered radicals by bacillus-Calmette-Guerin-activated macrophages. An electron paramagnetic resonance study of the response to phorbol myristate acetate[END_REF][START_REF] Britigan | Stimulated human neutrophils limit iron-catalyzed hydroxyl radical formation as detected by spintrapping techniques[END_REF][START_REF] Pou | Spin-trapping and human neutrophils. Limits of detection of hydroxyl radical[END_REF][START_REF] Ramos | Spin trapping evidence for myeloperoxidase-dependent hydroxyl radical formation by human neutrophils and monocytes[END_REF], because high level of O2 •-production can be conveniently triggered by incubation with various stimulants. Besides, results have been obtained on menadione-treated endothelial cells or enterocytes [START_REF] Rosen | Detection of superoxide generated by endothelial cells[END_REF][START_REF] Mansbach | Detection of free radicals as a consequence of rat intestinal cellular drug metabolism[END_REF], endothelial cells exposed to anoxia-reoxygenation cycles [START_REF] Zweier | Measurement of endothelial cell free radical generation: evidence for a central mechanism of free radical injury in postischemic tissues[END_REF][START_REF] Zweier | Determination of the mechanism of free radical generation in human aortic endothelial cells exposed to anoxia and reoxygenation[END_REF] N. gonorrhoeae bacteria upon incubation with paraquat [START_REF] Hassett | Bacteria form intracellular free radicals in response to paraquat and streptonigrin. Demonstration of the potency of hydroxyl radical[END_REF]. More recent spin traps were also individually tested in the same phagocytic cell systems [START_REF] Saito | Comparison of superoxide detection abilities of newly developed spin traps in the living cells[END_REF][START_REF] Roubaud | Quantitative measurement of superoxide generation and oxygen consumption from leukocytes using electron paramagnetic resonance spectroscopy[END_REF][START_REF] Khramtsov | NMR spin trapping: Detection of free radical reactions using a phosphorus-containing nitrone spin trap[END_REF][START_REF] Dikalov | Production of extracellular superoxide by human lymphoblast cell lines: comparison of electron spin resonance techniques and cytochrome C reduction assay[END_REF]. The quartz flat cell is however a rather insensitive technique, poorly suited to low radical production (especially basal conditions), and requires large amounts of spin trap and cells. Artifacts due to cross-contamination between runs can occur if thorough cleaning of adherent proteins is not performed after analysis of each individual sample. More importantly, in such closed systems, oxygen supply is limited and hypoxia can impair detection of O2 •-, either because the production of oxygen-derived radicals themselves is lowered or because reductases become activated under these conditions enhancing spin adduct decay -a process that has been investigated using sterically-shielded nitroxides [START_REF] Swartz | Use of nitroxides to measure redox metabolism in cells and tissues[END_REF][START_REF] Rosen | Formation and Reduction of a Nitroxide Radical by Liver-Microsomes[END_REF]. Use of a disposable length of gas-permeable PTFE tubing (Extruded Sub-Lite-Wall, inside diameter 0.635 mm, wall thickness 0.051 mm; Zeus Industrial Products Ltd., Ireland) folded twice in a W shape and inserted in a 4-mm EPR quartz tube solves many of these problems. The small sample size (100 L) allows analysis of multiple conditions from the same stock of cells, comparison of spin trap efficiency of detection, and avoids artifacts of contamination. Shi et al. determined the optimal conditions for detection of O2 •-produced in human mammary epithelial cells, MCA-10A, upon treatment with a polycyclic aromatic hydrocarbon, 1,6-benzo[a]pyrene quinone (20 M) in phenol red-and serum-free cell growth medium containing 0.1 mM DTPA [START_REF] Shi | Evaluation of spin trapping agents and trapping conditions for detection of cell-generated reactive oxygen species[END_REF]. Only the HO • spin adducts derived from reduction of the O2 •-adducts in cells were detected in this case, as evidenced by inhibition with SOD. In this cellular system, the signal intensity was maximum using 2.5  10 6 cells/mL, 50 mM spin trap, normoxia conditions, and physiological temperature of 37°C. From the comparison of four spin traps (one of which did not give any signal), the authors concluded that the maximal intensities (measured as low-field peak height of EPR spectra) obtained with DMPO, BMPO, and DEPMPO were similar, but that detection was more transient with DMPO. The maximum intensity was reached later and lasted longer with DEPMPO. In a study using suspensions of RAW 264.7 macrophages (3.6  10 6 cells/ 100 L) in phosphate-buffered saline (PBS), we compared the spin trapping efficiencies of six spin traps (DMPO, BMPO, DEPMPO, DIPPMPO, Mito-DIPPMPO, and CD-DIPPMPO) at 5-10 mM final concentration and 21°C upon stimulation with phorbol 12-myristate 13-acetate (PMA, 5 M, 20 min) [START_REF] Abbas | Detection of superoxide production in stimulated and unstimulated living cells using new cyclic nitrone spin traps[END_REF] (Figure 4). Optimal detection was not obtained at such low concentrations with DMPO and BMPO, while 50 mM afforded satisfactory signal with both spin traps. Besides, with this cellular system, mixtures of O2 •-and HO • adducts were observed. The SOD-inhibitable signal of the HO • adduct was preponderant with DMPO, BMPO, DEPMPO, and DIPPMPO, while Mito-DIPPMPO and CD-DIPPMPO enabled a greater detection of the O2 •-adduct, with a low (if any) amount of HO • adduct in the case of CD-DIPPMPO (Figure 5). The sensitivity of detection was dramatically increased with CD-DIPPMPO compared to all other spin traps, making detection on unstimulated macrophages possible for the first time. The superiority of CD-DIPPMPO over other spin traps used was attributed to its extracellular localization which protects the spin adducts from intracellular reductants. It appears that the larger linewidth of CD-DIPPMPO-OOH, arising from a higher molecular tumbling correlation time, which could have reduced the signal-to-noise ratio compared to DEPMPO-OOH, is largely outbalanced by the increase in adduct stability. These last two examples show how dependent on the cell type the detection is, which means that optimal experimental conditions can vary from one study to another. Results obtained on cell suspensions may occasionally differ from physiological responses. Cell harvesting methods, such as scraping or trypsinization, can undermine cell integrity. Physical damage to cells induced by mechanical detachment may reduce viability [START_REF] Heng | Mechanical dissociation of human embryonic stem cell colonies by manual scraping after collagenase treatment is much more detrimental to cellular viability than is trypsinization with gentle pipetting[END_REF][START_REF] Mcneil | A method for incorporating macromolecules into adherent cells[END_REF] or alter cell metabolism [START_REF] Batista | Effects of different detachment procedures on viability, nitroxide reduction kinetics and plasma membrane heterogeneity of V-79 cells[END_REF], while partial enzymatic digestion of membrane-expressed proteins may impair cell function [START_REF] Revel | Adhesion of culture cells to their substratum[END_REF][START_REF] Wu | Versican protects cells from oxidative stress-induced apoptosis[END_REF][START_REF] Piercy | Effect of harvesting and sorting on β-1 integrin in canine microvascular cells[END_REF][START_REF] Mutin | Reevaluation of trypsin-EDTA for endothelial cell detachment before flow cytometry analysis[END_REF][START_REF] Hirai | Characterization of cellular motions through direct observation of individual cells at early stage in anchoragedependent culture[END_REF]. Therefore different alternative setups have been proposed to perform detection of O2 •-produced by adherent cells. In the next sections, we will review detection on the supernatant and detection directly performed on adherent cells.

Detection on supernatant and distant analysis

When adherent cells are incubated with spin traps, the spin adduct formed by trapping of extracellular O2 •-accumulates in the supernatant and can be detected by EPR, though this method has been used in few studies. Optimal incubation times and spin trap concentration depend both on the level of O2 •-production, on the spin trapping rate, and on the stability of the adduct in the presence of cells. Endothelium-derived O2 •-, a key player in atherosclerosis and vascular aging, is particularly difficult to measure because of its low level of production. Pre-confluent and post-confluent cultured bovine aortic endothelial cells (1.5-2.5  10 6 cells) in 25 cm 2 flasks were incubated with DMPO (150 mM in a mix containing 1g/L glucose, 0.2 g/L CaCl2, 0.0059 g/L DTPA, 0.15 g/L NaCl, 0.37 g/L KCl in sodium phosphate buffer at pH 7.4) for 15 min. The supernatant transferred to a quartz cell was used to assess production of O2 •-under stimulation by the calcium ionophore A23187 in a dosedependent manner (3-10 M) in PBS [START_REF] Barbacanne | Detection of superoxide anion released extracellularly by endothelial cells using cytochrome c reduction, ESR, fluorescence and lucigeninenhanced chemiluminescence techniques[END_REF]. The signal, identified as resulting from the DMPO-OH adduct, was completely inhibited by SOD (30 U/mL), whereas it was unaltered by addition of catalase (2000 U/mL), confirming it originated from the trapping of extracellular O2 •-. Production by unstimulated cells was barely detectable under these conditions. Use of a spin trap that leads to more stable spin adducts, such as BMPO, allows distant analysis of the supernatant after freezing in liquid nitrogen when an EPR spectrometer is not available at the site of experiment [START_REF] Khan | Distant spin trapping": a method for expanding the availability of spin trapping measurements[END_REF]. Dysfunctional porcine aortic endothelial cells obtained by successive passages were thus shown to produce significantly more O2 •-than functional cells using this method [START_REF] Perrier | Effect of uncoupling endothelial nitric oxide synthase on calcium homeostasis in aged porcine endothelial cells[END_REF]. There, one-hour incubation of the cells in the presence of 50 mM BMPO in phosphate buffer under agitation at 37°C, with or without addition of SOD (300 U/mL), was performed and the supernatant was rapidly frozen in liquid nitrogen. The samples were allowed to thaw just before analysis and were rapidly transferred to a 4-bore AquaX (Bruker, Wissembourg). This specific sample cell, compatible with most standard EPR cavities, is made of closely spaced quartz capillaries with fittings that make it easy to fill and rinse with a syringe while installed within the spectrometer. Series of samples can be analyzed in a short period of time using this system. Adherent proteins can be removed by flushing the system with sodium hydroxide solution followed by buffer between runs since the cell is chemically-resistant. Detection on the supernatant with BMPO was also successfully applied to the detection of O2 •-produced extracellularly by Nox family enzymes overexpressed at the plasma membrane of human embryonic kidney HEK 293 cells (Figure 6). NADPH oxidase 5 (Nox5) and dual oxidase 2 (Duox2) are large homologues sharing a plasma membrane associated flavoprotein and an N-terminal extension comprising four and two EF-hand motifs, respectively, implicated in their Ca 2+ activation. Duox2 has an additional transmembrane helix and a peroxidase homology ectodomain with several glycosylated sites. While Nox5 and immature Duox2 catalyze the production of O2 •- [START_REF] Ameziane-El-Hassani | Dual oxidase-2 has an intrinsic Ca2+-dependent H2O2-generating activity[END_REF], mature Duox2 is involved in thyroid hormones biosynthesis as the H2O2 generator associated with thyroperoxidase. Mutations in Duox2 produce congenital hypothyroidism in humans. SOD-inhibitable and Ca 2+ -dependent accumulation of BMPO-OH in the supernatant following release of O2 •-by Nox5 but not by mature Duox2 at the plasmic membrane was observed (Figure 7). Besides, it confirmed the selectivity of detection of O2 •-versus H2O2 with BMPO since extracellular H2O2 productions by both cell lines in the presence of ionomycin were similar (as assayed by the increase in homovanilic acid fluorescence in the presence of HRP). On the contrary, BMPO did not allow detection of O2 •-produced by immature Duox2 at the endoplasmic reticulum in cell lines lacking the human Duox2 maturation factor hDuoxA2 (i.e. in HEK-hDuox2/pcDNA5FRT cells).

Damage to the cell layer and presence of cells in the supernatant upon freezing should be avoided because leakage of intracellular components will reduce the stability of the spin adducts in the sample, a problem which can be identified when fast signal decay occurs during EPR acquisition. Despite its simple implementation, a significant drawback of the detection on supernatant is the large volume of solution used to cover the cell layer in culture wells and the consequent large amount of spin trap required for the experiments.

Detection on undetached cells

As an alternative to detection on the supernatant, measurement can be performed directly on adherent cells introduced inside the EPR cavity by different tricks. Reinke et al. allowed polymorphonuclear leukocytes (PMNs) to adhere to the walls of a quartz flat EPR cell which was placed horizontally for 3 min before recording [START_REF] Tanigawa | Spin trapping of superoxide from glass adherent polymorphonuclear leukocytes induced by N-formylmethionyl-leucylphenylalanine[END_REF]. They could monitor a large and long-lasting N-formylmethionyl-leucyl-phenylalanine (fMLP)induced O2 •-release by adherent PMNs using DMPO, while no signal was detected when spin trapping was performed on PMNs in suspension. Unlike fMLP-, PMA-induced O2 •- release by PMNs was not influenced by cell adhesion. Britigan et al. performed spin trapping of O2 •-with DMPO (100 mM), in Hank's balanced salt solution without phenol red and containing 0.1 mM DTPA, using endothelial cells cultured on microcarrier beads (Cytodex 3, Pharmacia, Piscataway, NJ) [START_REF] Britigan | Insight into the nature and site of oxygencentered free radical generation by endothelial cell monolayers using a novel spin trapping technique[END_REF]. The final cell concentration in the quartz flat EPR cell with 25 vol% microcarrier beads reached 0.5-110 7 cells/mL. Upon exposure to paraquat (1 mM), DMPO-OH signal was observed. It was inhibited by SOD (30 U/mL), while it remained unchanged in the presence of catalase (400 U/mL) or DMSO, which is consistent with O2 •-trapping. However, the problems of oxygen supply inside flat quartz cells and of potential crosscontamination between runs already mentioned in previous sections remain with these techniques. To overcome these issues, we recently proposed to perform detection on cells attached to coverslip glasses initially designed for microscope studies [START_REF] Abbas | Mediumthroughput ESR detection of superoxide production in undetached adherent cells using cyclic nitrone spin traps[END_REF]. RAW 264.7 macrophages were incubated for 20 min with four spin traps (DEPMPO, DIPPMPO, Mito-DIPPMPO, or CD-DIPPMPO, 5-10 mM) and PBS containing DTPA (1 mM) in 24-well culture plates (150 L/well). Two coverslips were then mounted (cell layer facing outwards) on a quartz slide using EPR silent silicon vacuum grease (Bluestar silicones, Caronno Pertusella, Italy) and introduced inside the EPR TM cavity equipped with a flat quartz protection sleeve (Figure 8). All spin traps gave satisfactory detection upon macrophage stimulation with PMA and the results correlated those obtained with cell suspensions. Again, only the O2 •-adduct was observed with CD-DIPPMPO. This setup requires a reduced number of cells for one condition (500 000 cells on two coverslips) and a limited amount of spin trap. Moreover, sample preparation is straightforward which allows medium-throughput EPR detection of O2 •-production on adherent cells.

Choice of acquisition parameters

As mentioned in previous examples, the choice of EPR parameters for the experiments can be critical because the spin adducts usually form at low and transient concentrations.

It is good practice to pretune the cavity with some control sample (buffer or untreated cells) in order to save time when analyzing samples containing short-lived spin adducts. Microwave power between 10 and 20 mW is widely accepted as optimal value to obtain the maximal EPR intensity while avoiding saturation of the signal. However undesired heating of the sample can occur even at 20 mW, which can influence both cell metabolism, spin trapping rate, and spin adduct decay [START_REF] Ouari | Recent developments and applications of the coupled EPR/spin trapping technique (EPR/ST)[END_REF]. It is therefore important to keep this value constant and to be aware that results cannot be compared when different EPR sample cells are used (glass capillaries, flat cells, AquaX, gas-permeable tubing...). Modulation of the magnetic field is used in conventional EPR spectrometers in order to enhance the sensitivity, which results in the EPR spectrum being recorded as the first derivative of the absorption signal with respect to magnetic field. Recommended modulation amplitude is generally around 0.1 mT for nitroxides. However it can be increased up to 0.25 mT in order to fathom very weak signals of spin adducts from the baseline, with only negligible distortion of the EPR spectra. When looking at very weak signals, EPR spectroscopists usually increase signal-to-noise ratio by using large time constants to filter noise or by repeatedly acquiring the spectrum and subsequently adding each spectrum together. As a result, the signal increases proportionally with the number of scans N, random noise with √𝑁, and thus signal-tonoise ratio with √𝑁. However, modern spectrometers also allow recording of large series of spectra as a function of time and this function can be used as an advantage to further increase the sensitivity of detection with spin traps. By keeping the time constant low and recording large series of spectra that are then treated with statistical data analysis methods such as singular value decomposition (SVD), one can extract kinetic information in addition to performing noise filtration [START_REF] Keszler | Comparative investigation of superoxide trapping by cyclic nitrone spin traps: the use of singular value decomposition and multiple linear regression analysis[END_REF][START_REF] Lauricella | A new kinetic approach to the evaluation of rate constants for the spin trapping of superoxide/hydroperoxyl radical by nitrones in aqueous media[END_REF]. We used this method to unravel unstimulated O2 •-production in RAW 264.7 macrophage suspensions with CD-DIPPMPO [START_REF] Abbas | Detection of superoxide production in stimulated and unstimulated living cells using new cyclic nitrone spin traps[END_REF].

Interpretation of the results

Identification of the spin-trapped species and of the site of radical production

According to the theory of spin trapping , the trapped radical can easily be identified by the characteristic EPR spectrum of the spin adduct. Hyperfine coupling constants of O2 •- and HO • adducts of common spin traps have been tabulated [START_REF] Buettner | Spin trapping: ESR parameters of spin adducts[END_REF][START_REF] Bézière | Metabolic stability of superoxide adducts derived from newly developed cyclic nitrone spin traps[END_REF]. In the case of phosphorylated spin traps, significant lines for O2 •-and HO • adducts do not overlap, which makes interpretation more straightforward. Computer-assisted simulation of the EPR spectra with different softwares (such as WinSim, Roki, or Easyspin) helps in the determination of the relative proportions of overlapping species [START_REF] Duling | Simulation of multiple isotropic spin-trap EPR spectra[END_REF][START_REF] Rockenbauer | Automatic computer simulations of ESR spectra[END_REF][START_REF] Stoll | EasySpin, a comprehensive software package for spectral simulation and analysis in EPR[END_REF]. Interpretation of spin trapping results can sometimes be difficult due to the instability of the spin adducts in biological systems and the conversion of O2 •--to HO • -adduct (see section 1.4). However, relatively straightforward procedures exist to determine whether O2 •-or HO • radicals have been trapped [START_REF] Finkelstein | Spin trapping of superoxide and hydroxyl radical: Practical aspects[END_REF]. Most importantly, to prove extracellular O2 •- radical formation, incubation with SOD or polyethylene glycol-conjugated SOD (SOD-PEG) (30 U/mL) should prevent the formation of the O2 •-adduct, while catalase (200 U/mL) should have no effect. (Heat-inactivated SOD can be used to rule out interference of preservatives in commercial preparations of the enzymes.) Neither SOD nor SOD-PEG can cross the cell membrane at short incubation times and thus these controls cannot be used to track down intracellular production of O2 •-. The origin of HO • adduct can be identified by further controls (Figure 9). In the circumstances of HO • formation, addition of small amounts (1 vol%) of ethanol or DMSO will lead to an inhibition of the HO • adduct and will result in appearance of a new species due to the trapping of secondary -hydroxyethyl or methyl radicals, respectively. HO • adduct resistant to both SOD-and ethanol/DMSO-inhibition likely originates from the reduction of O2 •-adduct by intracellular components and can thus be regarded as a sign of intracellular O2 •-production, provided nucleophilic addition of water under the experimental conditions has been excluded (see section 4.1). Besides, intracellular versus extracellular localization of spin adducts can be probed by using membrane-impermeant paramagnetic salts, such as tri(oxalato) chromiate(III) (30 mM) [START_REF] Samuni | On the spin trapping and ESR detection of oxygen-derived radicals generated inside cells[END_REF] or tris(ethylenediamine)nickel(II) chloride (100 mM) [START_REF] Abbas | Detection of superoxide production in stimulated and unstimulated living cells using new cyclic nitrone spin traps[END_REF]. Concentration-and distance-dependent magnetic relaxation enhancement mechanisms (Heisenberg exchange) between the paramagnetic metal ions and the organic radicals lead to linebroadening and diminution of intensity of the EPR signal of spin adducts present in the extracellular space, while intracellular species are unaffected. Passive diffusion of the spin adducts away from their sites of production is however a possibility that must be taken into account for long-lived adducts.

Quantification

The EPR spectrum, being the first derivative of the absorption signal, contains information about the number of spins in the sample. Assuming the recording parameters have been properly chosen to avoid saturation of the signal, the intensity of the EPR spectrum -defined as the area under the absorption curve -is proportional to the number of spins and the concentration of the spin adduct easily follows from the comparison with an external standard of known concentration (usually a stable nitroxide). Can this quantity be used to derive a quantitative estimation of O2 •-production from cells? Roubaud et al. suggested that DEPMPO has ideal properties for such quantitative measurements [START_REF] Roubaud | Quantitative measurement of superoxide generation and oxygen consumption from leukocytes using electron paramagnetic resonance spectroscopy[END_REF]. Considering the poor spin trapping rate and the low spin adduct stability in the presence of biological systems, any attempt to quantify O2 •-production by cells using spin trapping is however extremely difficult and should be considered with caution, especially when the concentration of oxygen varies during the experiment, altering both the O2 •-production rate and the reducing potency of cells. In our opinion, only CD-DIPPMPO, with its high intrinsic adduct stability and its extracellular localization that prevents cell-induced decay, when used in combination with gas-permeable tubing, would make quantitative estimates more reliable.

Troubleshooting

False positives

Nitrones are very reactive organic compounds and various reactions that do not involve radical trapping may lead to nitroxides and undesired EPR signals. Specific warnings against misinterpretations appeared in the literature [START_REF] Finkelstein | Spin trapping of superoxide and hydroxyl radical: Practical aspects[END_REF][START_REF] Ranguelova | The fidelity of spin trapping with DMPO in biological systems[END_REF] oxidation of DMPO and related nitrones to the corresponding hydroxamic acid by metal ions or other oxidizing systems, followed by oxidation to a nitroxide characterized by a 7line EPR spectrum [START_REF] Rosen | Spin trapping of the primary radical involved in the activation of the carcinogen N-hydroxy-2-acetylaminofluorene by cumene hydroperoxide-hematin[END_REF]; ii) inverted spin trapping [START_REF] Chandra | Hydration of spin-trap cations as a source of hydroxyl adducts[END_REF][START_REF] Eberson | Inverted spin trapping. Part III. Further studies on the chemical and photochemical oxidation of spin traps in the presence of nucleophiles[END_REF], and iii) the Forrester-Hepburn mechanism [START_REF] Forrester | Spin traps -Cautionary note[END_REF] (Figure 10). These last two mechanisms lead to radicals that are identical to spin adducts but through combinations of mono-electronic oxidation of the spin trap and nucleophilic addition, in either order. Inverted spin trapping, where the oxidation precedes the nucleophilic addition, is a rather limited source of artifacts with cyclic nitrones in biological systems since their oxidation potentials are very high (EDMPO•+/DMPO  EDEPMPO•+/DEPMPO  1.9 V vs. SCE in acetonitrile [START_REF] Eberson | Inverted spin trapping. Part III. Further studies on the chemical and photochemical oxidation of spin traps in the presence of nucleophiles[END_REF][START_REF] Tuccio | Decay of the hydroperoxyl spin adduct of 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide -an EPR kinetic-study[END_REF]) compared to biologically-relevant oxidation potentials, but it should be considered in the presence of high valency metal ions (Fe 3+ , Cu 2+ , Mn 4+ ). These species can oxidize the nitrone to the radical cation and make it susceptible to hydration to form the HO • adduct (water being the most abundant nucleophile in biological systems). The Forrester-Hepburn mechanism, where the first step involves nucleophilic attack of the β-carbon of the nitrone by nucleophiles such as water, sulfite, or thiols and formation of a readily-oxidizable hydroxylamine product, is potentially a greater source of artifacts, especially with nitrones bearing a higher positive charge than DMPO on this position like in DEPMPO, and in the presence of trace metal ions that further activate the electrophilic character of the nitrone function by complexation [START_REF] Potapenko | Nonradical mechanism of (bi)sulfite reaction with DEPMPO: cautionary note for SO3*-radical spin trapping[END_REF][START_REF] Potapenko | Reversible reactions of thiols and thiyl radicals with nitrone spin traps[END_REF]. Nucleophilic addition of water leading to the HO • adduct is the main concern in the absence of metal chelators. Timmins et al. proposed a technique involving pre-incubation with isotopically labeled spin traps to identify such artifacts [START_REF] Timmins | Use of isotopically labelled spin traps to determine definitely the presence or absence of Methods Accepted manuscript Published in final edited form as[END_REF]. This was further refined by Leinisch et al. [START_REF] Leinisch | Investigation of spin-Methods Accepted manuscript[END_REF][START_REF] Leinisch | Evaluation of the Forrester-Hepburn mechanism as an artifact source in ESR spin-trapping[END_REF] and completed by auxiliary experiments by Ranguelova et al. [START_REF] Ranguelova | The fidelity of spin trapping with DMPO in biological systems[END_REF].

False negatives

Unsuccessful EPR detection using spin traps should not be considered as a proof of absence of radical production by the cellular system. The actual spin trap concentration at the site of O2 •-production inside cells may be limiting if the rate of cell membrane crossing by DMPO and DEPMPO is slow [START_REF] Anzai | ESR measurement of rapid penetration of DMPO and DEPMPO spin traps through lipid bilayer membranes[END_REF]. The low spin trapping rate and fast cellinduced decay of spin adducts can prevent accumulation of the spin adduct above the detection limit of EPR when low fluxes of O2 •-are produced. Incubation with preformed adducts or stable nitroxides can be used to probe the metabolic potential of the cells and test this hypothesis [START_REF] Abbas | Detection of superoxide production in stimulated and unstimulated living cells using new cyclic nitrone spin traps[END_REF]. Simultaneous production of NO • may also prevent trapping of O2 •-with cyclic nitrone spin traps [START_REF] Pignitter | Inefficient spin trapping of superoxide in the presence of nitric-oxide: implications for studies on nitric-oxide synthase uncoupling, Free Radic[END_REF].

Conclusions and perspectives

Since the first application of the technique of spin trapping in cellular systems in the 1970s, many improvements were achieved regarding the structures and properties of the spin traps designed for O2 •-detection. The specificity of detection remains the greatest strength of this methodology, together with the absence of redox cycling of the probes within cells. Nowadays extracellular detection of O2 •-production can be performed with excellent sensitivity using standard spectrometers and CD-DIPPMPO, however commercial unavailability of the new-generation spin traps is a serious brake to further spreading. On the other hand, low sensitivity for the detection of intracellular O2 •- production is still a major shortcoming of the technique, which has up-to-now prevented detection of mitochondrial O2 •-within cells for example, despite progresses in CW EPR spectrometer sensitivity and in chemical design of cyclic nitrone spin traps. Future ) and wild-type HEK 293 cells (HEK-WT) were cultured as previously described [START_REF] Ameziane-El-Hassani | Dual oxidase-2 has an intrinsic Ca2+-dependent H2O2-generating activity[END_REF]. Human Duox2-transfected HEK 293 cells were transiently transfected with human Duox2 maturation factor (hDuoxA2) that allows expression of the mature enzyme at the plasma membrane [START_REF] Grasberger | Identification of the maturation factor for dual oxidase. Evolution of an eukaryotic operon equivalent[END_REF]. The Duox2 cell lines transfected with the empty expression vector (pcDNA5FRT) were used as controls in this case. Cells (~1x10 6 cells) in 6-well culture plates were washed with Hepes buffered Earle's solution (pH 7.2) and incubated for 30 min at 37°C in the same buffer containing EDTA, CaCl2, ionomycin, and BMPO. The Ca 2+ dependence of the activity was evaluated with 30-min pre-incubations with BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester), an intracellular calcium chelator), followed by incubations with the ionomycine-free mixture.

Methods

  Accepted manuscript Published in final edited form as: Methods. 2016 May 6; 109: 31-43. doi: 10.1016/j.ymeth.2016.05.001 © 2020. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ 10/35

Methods

  Accepted manuscript Published in final edited form as: Methods. 2016 May 6; 109: 31-43. doi: 10.1016/j.ymeth.2016.05.001 © 2020. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ 11/35

Methods

  Accepted manuscript Published in final edited form as: Methods. 2016 May 6; 109: 31-43. doi: 10.1016/j.ymeth.2016.05.001 © 2020. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ 12/35

Figure legends : Figure 1 :

 :1 Figure legends:

Figure 2 :

 2 Figure 2: Structure of cited spin traps. The seven-glucopyranose unit of the permethylated -cyclodextrin in CD-DEPMPO and CD-DIPPMPO is shown as a cylinder and drawing is not to scale.

Figure 3 :Figure 4 :

 34 Figure 3: Proposed mechanisms for the disappearance of O2 •-and HO • spin adducts of BMPO: A) in the presence of ferric hemeproteins (Hp), B) in the presence of intracellular reductants. (Adapted from reference [46])

Figure 5 :

 5 Figure 5: Representative EPR spectra obtained by Abbas et al. [29] after 3-min incubation of BMPO (10 mM), DEPMPO (10 mM), or CD-DIPPMPO (5 mM) in the presence of PMAstimulated RAW 264.7 macrophages (3.6±0.3  10 6 cells/100 L) in PBS (pH 7.1-7.4) and 1 mM DTPA. The y-axis was expanded 5-fold for BMPO and 2-fold for DEPMPO for the sake of clarity. Only BMPO-OH is observed in the upper spectrum. The middle spectrum shows superimposition of DEPMPO-OOH () and DEPMPO-OH (•). Only CD-DIPPMPO-OOH is seen in the bottom spectrum. EPR parameters: microwave frequency, 9.85 GHz; microwave power, 10 mW; modulation frequency, 100 kHz; modulation amplitude, 0.2 mT; receiver gain, 60 dB; time constant, 40.96 ms; conversion time, 41.04 ms; data points, 1024; sweep width, 15 mT; sweep time, 42.02 s. Noise was filtered using the SVD method (see section 2.3).

Figure 6 :

 6 Figure6: Procedure for spin trapping experiments with HEK 293 cell lines, when EPR detection was performed on the supernatant. Stable human Nox5b-transfected HEK 293 cells (HEK-hNox5) and wild-type HEK 293 cells (HEK-WT) were cultured as previously described[START_REF] Ameziane-El-Hassani | Dual oxidase-2 has an intrinsic Ca2+-dependent H2O2-generating activity[END_REF]. Human Duox2-transfected HEK 293 cells were transiently transfected with human Duox2 maturation factor (hDuoxA2) that allows expression of the mature enzyme at the plasma membrane[START_REF] Grasberger | Identification of the maturation factor for dual oxidase. Evolution of an eukaryotic operon equivalent[END_REF]. The Duox2 cell lines transfected with the empty expression vector (pcDNA5FRT) were used as controls in this case. Cells (~1x10 6 cells) in 6-well culture plates were washed with Hepes buffered Earle's solution (pH 7.2) and incubated for 30 min at 37°C in the same buffer containing EDTA, CaCl2, ionomycin, and BMPO. The Ca 2+ dependence of the activity was evaluated with 30-min pre-incubations with BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester), an intracellular calcium chelator), followed by incubations with the ionomycine-free mixture.

Figure 7 :Figure 8 :

 78 Figure 7:Representative EPR spectra obtained with BMPO on the supernatant of HEK-hNox5 cells, HEK-hDuox2/hDuoxA2 cells, and respective control cells, or without cells, upon stimulation by ionomycin or after incubation with BAPTA-AM. 27 EPR spectra were sequential recorded over 18 min, the sum of which is shown. Same EPR parameters as in Figure5except: microwave frequency, 9.82 GHz; microwave power, 5.1 mW; sweep

Figure 9 :

 9 Figure 9: Flow chart for determining the origin of HO • adduct of cell-permeant spin traps such as DMPO, DEPMPO, or BMPO.

Figure 10 :

 10 Figure 10: Different pathways leading to artifactual detection of nitroxides in biological systems. DMPO is used as a model spin trap in this figure but similar mechanisms exist for other cyclic nitrones. The EPR spectrum of 5,5-dimethyl-2-pyrrolidone-N-oxyl (DMPOX) is characterized by 7 lines and hyperfine coupling constants AN = 0.72 mT and AH = AH' = 0.41 mT in water [99]. Nu: stands for nucleophiles such as H2O, sulfite or thiols.

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  Methods Accepted manuscript Published in final edited form as: Methods. 2016 May 6; 109: 31-43. doi: 10.1016/j.ymeth.2016.05.001

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  , or Methods Accepted manuscript Published in final edited form as: Methods. 2016 May 6; 109: 31-43. doi: 10.1016/j.ymeth.2016.05.001

© 2020. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ 9/35

  . Possible artifacts include: i) Methods. 2016 May 6; 109: 31-43. doi: 10.1016/j.ymeth.2016.05.001 © 2020. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/

	Methods Accepted manuscript
	Published in final edited form as:
	14/35

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© 2020. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ 13/35

© 2020. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ 16/35

© 2020. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/

© 2020. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ 23/35

Acknowledgments

The results in Figures 6 and7 were obtained through a collaboration between F. Peyrot, R. Ameziane-El-Hassani, Y.-M. Frapart, J.-L. Boucher, D. Mansuy, M. El Mzibri, A. Virion, and C. Dupuy, and were presented as a poster during SFRR Europe 2007 meeting in Vilamoura, Portugal. HEK-hNox5 cells used in this study were kindly provided by Dr K. H. Krause, Biology of Aging Laboratories, University of Geneva, Switzerland.


Takanori Fukuoka
email: tfukuoka@landscape.kobe-u.ac.jp

Sadahisa Kato

Green Infrastructure Implementation Case Study in Asia Monsoon Climate-In case of ABC Water Design Guideline in Singapore with Sustainable Stormwater Management Concept Développement d'une infrastructure verte dans un climat de moussons en Asie. Recommandations pour la conception d'ouvrages à Singapour intégrant le concept de gestion durable des eaux pluviales

Keywords:

L'objectif de cette étude est d'analyser des stratégies pour une gestion durable des eaux pluviales et d'analyser les lignes directrices en matière de conception d'ouvrages d'eau ABC (ABC-WDG) à Singapour. Depuis très longtemps, Singapour est confrontée à de sérieux problèmes hydrologiques, tels que des pénuries d'eau, des inondations et des questions liées à la qualité de l'eau. Les lignes directrices nationales en matière de conception des ouvrages d'eau, soit ABC-WDG, donnent un aperçu global pour promouvoir l'aménagement de l'eau urbaine et la mise en oeuvre d'infrastructures vertes. De plus, ABC-WDG a créé un système de certification pour améliorer la gestion durable des eaux pluviales. Le système professionnel ABC est unique, et sert aussi de système éducationnel qui promeut des concepts d'aménagement des eaux urbaines. Par le biais de cette étude de cas de l'ABC-WDG, et avec la présentation de la conjoncture actuelle, nous explorons les problèmes à Singapour et les stratégies pour la mise en oeuvre d'infrastructures vertes dans les climats de mousson en Asie, et de la gestion durable des eaux pluviales en milieu urbain.

INTRODUCTION

Research Background and Purpose

This research aims to analyze Water Design guideline, which challenges to promote implementation of Green Infrastructure (GI) projects with sustainable stormwater management concept in order to reduce water disaster risk and to seek more sustainable stormwater management. Specifically, how site-scale GI projects can contribute to larger, city scale GI implementation by setting up active design guidelines.

The effects of rapid urbanization on global climate change have resulted in the mutation of natural water cycle such as infiltration and evapotranspiration. Dense urban environment set with high rises and multi complex buildings have increased levels of solar radiation which in turn exacerbates the urban heat island effect. Combined with large areas of monolithic impervious surfaces and a conventional, underground piped linear stormwater management system face potential risk of flood. From now on, as part of climate change adaptation strategies in highly urbanized area, Water Sensitive Urban Design(WSUD) may become essential approaches to reduce further water related disaster. In other words, GI implementation with sustainable stormwater management potentially have very important role. Sustainable stormwater management in this article can be defined as an alternative way of managing stormwater runoff by using combination of various stormwater tools such as green roof, pervious surfaces, vegetated swale in order to reduce stormwater runoff and speed, promote onsite retention and infiltration, and increase evapotranspiration which all contribute to recover fragmented natural water cycle in urban area. This research targets GI implementation in Asia monsoon climate areas with significant precipitation. The purpose of this research is to analyze methods and to obtain knowledge of promoting GI implementation at broader city scale through studying Water Design Guideline in Singapore. Current trend in stormwater management is limited to site scale stormwater harvesting and infiltration methods, and more holistic and diverse GI implementation methods need to be applied in much broader scale. GI implementation methods are technically available, but there are disconnection how to implement such methods into actual projects. To breakthrough this limited GI with sustainable stormwater management, communication media with citizen or architects are essential to explain how GI can be implemented and what the impacts are. As one of GI promoting communication media, water design guideline can function as effective tools. Towards applying broader scale GI implementation with sustainable stormwater management, water design guideline needs to be studied in order to reveal what required elements are as well as how GI concept is communicated with diverse readers.

GI need be mainly studied in two categories, which are policy (planning, incentives) and implementation (planning and design through project implementation). The purpose of this is research is to study Water Design Guidelines in Singapore as case study for GI implementation in Asia monsoon climate, and to analyze important structure and methods towards promotion of GI implementation with sustainable stormwater management in broader scale.

Previous Research Review

Previous sustainable stormwater management related research in Japan mainly focus in the area of stormwater harvesting and infiltration methods and concept, methods and techniques as hard facilities, and described in various book and manuals such as Rainwater Architecture (AIJ ed. 2005), Stormwater harvesting manuals (AIJ ed.2011, SHASEJ ed.2011) GI related research in Japan are relatively new field. Stormwater Infiltration Road Model Study through GIS (Inoue 2011), Green open scape and stormwater management relationship (Kato 2012), several research case studies in GI policy in Philadelphia (Endo 2011), Green Street implementation in Portland (Hanai 2011), and GI implementation in Portland(Fukuoka 2014). As listed, previous research can be categorized into quantitative based stormwater management and techniques, and GI policy or case studies. However, methodologies for GI implementation at broader scale, and guideline case study have novelty and original approach to seek GI implementation for diverse target.

1-3. Methods of Research

Author conducted multiple interviews with Public Utility Board (PUB) and Urban Redevelopment Agency (URA) government officers regarding innovative ABC Water Design Guidelines (ABC-WDG) from March 25 through 26 in 2015. This research is based on interviews above. Firstly, at URA, author conducted an interview with Mr.Teck Leong Lim(director), Ms.Dixi Mengnote, Ms. You Ling Lim. URA explained ABC-WDG from city planning point of view, gave presentation and conducted questions and answers session. Secondly, author conducted another interview with Ms. Geok Ling Chen (sustainability team director), Ms. Suat Ong, and Ms. Enid Chen at PUB. At an interview, PUB provided ABC-WDG overview, framework, and project implementation and its problems, and conducted questions and answers session. After interviews above conducted in Singapore, ABC-WDG framework, promotion methods of GI implementation and certification system analyzed.

ABC-WATER DESIGN GUIDELINES IN SINGAPORE 2.1 Background of ABC-WDG

Singapore is located in the center of Southeast Asia, and consists of 63 islands. Population is 5.3 million, and population density is the second in the world. Singapore island is 42km long to east to west, and 23km to south to north. Island's highest point is 163m, and several major rivers run through on relatively flat land. Historically, Singapore has been relying on 40% of drinking water resource from Malaysia through Pipe Lines. However, due to uncertain future with water issues, government made decision to be self sufficient for water by applying watershed based management system. Especially, 2,400mm annual rainfall was targeted as major water resource to be wisely used together with other gray water and dam solution. In this paper, overall structure of ABC Water Design Guidelines (ABC-WDG) by Public Utility Board (PUB) and how WBC-WDG function to change nation wide water resource management to create Garden Cities with water.

Summary of ABC-WDG

First edition of ABC-WDG was published in 2009 as holistic water design guidelines. In 2011, second edition was published with additional implemented case studies and regional characters. Then third edition is published in 2014. ABC stands for Active (Places for recreation), Beautiful (Holistic water management and amenity), Clean (enhance water quality), and designed to provide water for all. ABC-WDG has three strategic approaches, which are 1) Creation of ABC Water Master Plan and Green Infrastructure project implementation 2) Promotion of ABC water concept and application 3) 3P (People, Public, Private) partnership. First, ABC Water Master Plan created which are based on water shed based, and plotted important existing water resources such as rivers, canals, reservoirs and wetlands as well as potential project locations for future GI project implementation. Towards 2030, over 100 projects to be implemented based on this master plan. So far, 23 projects completed by June in 2014. Second, PUB functions as main agency to promote ABC water concept to all related governmental agencies as well as private developer. Third, ABC-WDG targets 3P. For diverse users various methods such as water education program and ABC water related activities held to promote at all different levels. ABC-WDG promotes watershed base, holistic water management. As shown in figure 2 and3, surface flow of all stormwater from roof and buildings, roads and green open spaces, and water bodies are designed to apply various GI projects in order to reduce stormwater runoff volume, and its speed. It is central to integrate sustainable stormwater management with GI projects. In other words, ABC-WDG seeks to enhance Blue and Green Infrastructure by maximizing performance of land. Treatment elements and c)Conveyance and storage elements. Firstly, catchment elements aim to collect water based on land use typologies such as road, canal, water bodies, pedestrian walkways, public open spaces, plazas and buildings. For different surface conditions, appropriate stormwater planning and design methodologies are shown with clear illustration. Project planning stage research and concept based on ABC water, building location and volume to meet ABC water goals, and applicable GI implementation methodologies at catchment stage. For instance, building is divided into various catchment elements such as green roofs, terraced green balconies at multi levels, and ground level elements such as planted areas and water features. ABC-WDG provides engineering and design procedures so that basic knowledge and methodologies can be easily integrated into projects. Secondly, Treatment elements such as swales, bio retention pond, detention pond, stormwater planter, rain garden and cleaning biotope methodologies and how to implement explained through visual information. Water treatment part covers wide range of methods and this provides concept, benefits, design methodologies to management issues. Thirdly, Conveyance and storage focus on large water bodies and provide methodologies such as bio engineering, erosion control and water quality control.

ABC-WDG DEVELOPMENT AND PROMOTION OF NAITON WIDE WSUD

ABC-WDG related Water law change and influence

Since 2009 when ABC-WDG published for the first time, there was no mandatory water regulation to force sustainable stormwater management. However, in 2013, there was revisions to COP on surface water drainage 7.1.5 to set surface runoff rate must be 0.55 for all new development sites over 0.2ha.

Developer is now required to submit documents to show GI implementation model and calculation(how sustainable stormwater management is applied on site-In other words, combination of multiple stormwater design tools for various surfaces at the project). Also, there is requirement that all documents need to be reviewed by ABC professionals who are trained to give advises on ABD-WDG related issues.

ABC

CONCLUSIONS

In conclusion of this case study research, following four points revealed. Firstly, ABC-WDG provides city to nation scale sustainable stormwater management vision through GI project implementation. Though stormwater design guideline is often limited to site to region scale, it is very unique to provide large scale water design guidelines with clear visions for diverse stakeholders. Secondly, Singapore finally regulated surface water runoff ration to all new development over 0.2ha. Nation wide water design guidelines and policy can provide good balance between voluntary and regulated approaches in order to create dynamic project implementation over time to meet nation wide ABC water goal. Thirdly, ABC-WDG is not limited to provide sustainable stomwater management and design methods, but also providing ABC Water Certificate and Professional system which help to develop collection of good ABC case studies and network of human resources to promote ABC further. Lastly, ABC-WDG indicate possibility of Green Infrastructure implementation strategies in Asia Monsoon climate.

LIST OF REFERENCES

Figure

  Figure-1:Gray Infrastructure (left) and Green Infrastructure with sustainable stormwater management (right) (LID Low Impact Development Design Manual by University of Arkansas, edited by author)

Figure- 2

 2 Figure-2(left) ABC-WDG function as holistic, watershed base management 3(right)ABC-WDG illustrates how stormwater is managed from roof to water river mouth、ABC WDG(2014): PUB Singapore2.3 Structure of ABC-WDGABC-WDG provides actual stormwater design tools for three different stages which are applicable to

Certificate and ABC Water Professional Program ABC

  certificate system started in 2010, July by PUB in order to recognize and further promote ABC water program. By 2015 April, 49 projects (29 public, 20 private) are certified as ABC water project.

	Certification system consists of 3 criteria which are Active(30points), Beautiful (30points), Clean (30
	points), Innovative(20 points) and total of 110npoints evaluated in detail. It is not only limited to
	sustainable stormwater treatment through GI, but also management, participation and original
	approaches. To support this Certificate system, ABC Professional program started which started in
	2011.Currently over 200 registered ABC professionals, and they help to consult how to implement GI
	through ABC-WDG.

  1) Communication from the Commission to the European Parliament, The Council, The European Economic and Social Committee and The Committee of The Regions Green Infrastructure (GI)(2013) : < http://eur-

lex.europa.eu/legal-content/EN/TXT/?uri=CELEX:52013DC0249> 2)EPA(2014):What is Green Infrastructure?<http://water.epa.gov/infrastructure/greeninfrastructure/gi_what.cfm 3) Kato, S. (2012) :An Overview of Green Infrastructure's Contribution to Climate Change Adaptation:13 th Japan-China-Korea Landscape Conference Proceedings, 224-228 4) ABC Water Design Guidelines(2009、2011、2014), Public Utility Board, Singapore