Global Profiling of Reactive Oxygen and Nitrogen Species in Biological Systems

Background: Recently, new “targeted” fluorescent probes that react selectively with reactive oxygen and nitrogen species to yield specific products have been discovered. Results: High-throughput fluorescence and HPLC-based methodology for global profiling of ROS/RNS is described. Conclusion: This methodology enables real-time monitoring of multiple oxidants in cellular systems. Significance: The global profiling approach using different ROS/RNS-specific fluorescent probes will help establish the identity of oxidants in redox regulation and signaling. Herein we describe a high-throughput fluorescence and HPLC-based methodology for global profiling of reactive oxygen and nitrogen species (ROS/RNS) in biological systems. The combined use of HPLC and fluorescence detection is key to successful implementation and validation of this methodology. Included here are methods to specifically detect and quantitate the products formed from interaction between the ROS/RNS species and the fluorogenic probes, as follows: superoxide using hydroethidine, peroxynitrite using boronate-based probes, nitric oxide-derived nitrosating species with 4,5-diaminofluorescein, and hydrogen peroxide and other oxidants using 10-acetyl-3,7-dihydroxyphenoxazine (Amplex® Red) with and without horseradish peroxidase, respectively. In this study, we demonstrate real-time monitoring of ROS/RNS in activated macrophages using high-throughput fluorescence and HPLC methods. This global profiling approach, simultaneous detection of multiple ROS/RNS products of fluorescent probes, developed in this study will be useful in unraveling the complex role of ROS/RNS in redox regulation, cell signaling, and cellular oxidative processes and in high-throughput screening of anti-inflammatory antioxidants.

Recent advances in oxy-radical research reveal multiple roles for reactive oxygen species (ROS) 3 (superoxide radical anion, O 2 . ; hydrogen peroxide, H 2 O 2 ; hypohalous acids such as HOCl and HOBr; lipid hydroperoxides, LOOH; and carbonyl products derived from them) and reactive nitrogen species (RNS) (peroxynitrite, ONOO Ϫ ; nitrogen dioxide, ⅐ NO 2 ; nitrated lipids, LNO 2 ). These species may be involved in redox regulation of cellular proteins, signaling, proliferation and differentiation, senescence, and cell death (1)(2)(3)(4). However, in most cases, the identity of the species responsible for the described biologic effects remains to be determined. This is partly due to a lack of suitable methodologies and chemical probes that allow unequivocal detection and distinction between different ROS and RNS and also to the complex intracellular chemistry of these species. Although a considerable amount of work has been done to elucidate reaction mechanisms of ROS/RNS with biological agents, antioxidants, and detection probes (5)(6)(7), only recently has real progress been made in uncovering reaction mechanisms (e.g. kinetics, stoichiometry, product analysis) of ROS and RNS with fluorescent probes (8 -10). The purpose of this study is to determine the specific products of oxidation of fluorescent probes by ROS and RNS and reveal the strategic use of these probes for real-time detection of these species in a more complex biological setting. In recent years, new "targeted" chemical probes that are more specific and selective in their reaction with O 2 . , H 2 O 2 , and ONOO Ϫ were discovered (10 -12). These and the other breakthroughs described below have enabled us to perform global profiling of ROS/RNS in biological systems (8 -10). These breakthroughs are highlighted as follows. (i) Hydroethidine (HE) or dihydroethidium and its mitochondria-targeted analog conjugated to a triphenylphosphonium moiety (MitoSOX TM Red) react rapidly with superoxide (k ϳ10 6 M Ϫ1 s Ϫ1 ), forming a specific hydroxylated marker product, 2-hydroxyethidium (2-OH-E ϩ ) or 2-hydroxymitoethidium (2-OH-Mito-E ϩ ).
Other oxidants (ONOO Ϫ -derived radicals, hydroxyl radical, perferryl iron) react with HE and MitoSOX TM to form the corresponding oxidation/dimeric products (E ϩ , Mito-E ϩ , and dimeric products) but do not yield the same O 2 . -mediated hydroxylated products (13). Understanding of the oxidation mechanisms of HE and MitoSOX TM has made it possible to use these probes with discretion for detecting intracellular O 2 . and other oxidants formed from H 2 O 2 and iron or heme or ONOO Ϫ . (ii) Boronate-containing fluorogenic probes react directly and stoichiometrically with H 2 O 2 , albeit slowly (k ϳ1 M Ϫ1 s Ϫ1 ), to form phenolic products that can be monitored in cells. (iii) Furthermore, several aromatic boronates react directly, rapidly, and stoichiometrically with ONOO Ϫ (k ϳ10 6 M Ϫ1 s Ϫ1 ), forming the corresponding phenols as major products. Other reactive nitrogen species ( ⅐ NO/O 2 and ⅐ NO 2 formed from myeloperoxidase/NO 2 Ϫ /H 2 O 2 ) do not react with boronates in a similar manner, thus enabling selective detection of ONOO Ϫ (14). Importantly, the chemistry and mechanism of reaction of H 2 O 2 and ONOO Ϫ with most boronates are very similar (14). This has prompted the development of several custom-synthesized and targeted boronic acid/ester-based fluorophores that can be used for specific and sensitive detection of ONOO Ϫ in chemical and biological systems (12).
The combined use of HPLC and fluorescence detection techniques now enables real-time monitoring and detection with unequivocal characterization of oxidants in biological systems. In this study, we have used a multiwell plate reader as a highthroughput approach to monitor the formation of O 2 . , H 2 O 2 , ⅐ NO-derived nitrosating species, and ONOO Ϫ in both cell-free and cellular systems. Both newly synthesized and commercially available fluorescent probes were used in the present study for detecting O 2 . , H 2 O 2 , ⅐ NO-derived nitrosating species, and ONOO Ϫ .
Determination of O 2 . and ⅐ NO Fluxes-⅐ NO and O 2 . fluxes were determined as described previously (9). Briefly, ⅐ NO fluxes were determined from the rate of decomposition of DPTA-NONOate measured by following the decrease of its characteristic absorbance at 248 nm (⑀ ϭ 8.1 ϫ 10 3 M Ϫ1 cm Ϫ1 ). The rate of decay of DPTA-NONOate was multiplied by a factor of two to obtain the rate of ⅐ NO release, assuming that two molecules of ⅐ NO are released during the decomposition of one molecule of DPTA-NONOate. The flux of O 2 . generated by XO-catalyzed oxidation of hypoxanthine was determined by monitoring the cytochrome c(Fe 3ϩ ) reduction and the increase in absorbance at 550 nm (using a difference in the values of the extinction coefficients between reduced and oxidized cytochrome of 2.1 ϫ 10 4 M Ϫ1 cm Ϫ1 ). Cell Culture-RAW 264.7 cells were grown in DMEM (Invitrogen) medium supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 100 units/ml penicillin, and 100 g/ml streptomycin. Before the experiment, cells were incubated overnight (16 -20 h) with LPS (0.5 g/ml) and IFN-␥ (50 units/ ml). For monitoring ROS/RNS, the cells were washed and cotreated with the probes and PMA (200 ng/ml) in DPBS supplemented with pyruvic acid and glucose (DPBS-GP). For HPLC analyses, the dishes with cells were incubated for 1 h at 37°C in a CO 2 -free incubator. After incubation, an aliquot of the medium was collected and immediately frozen in liquid nitrogen, while the cells were washed twice with ice-cold PBS, scraped in 1 ml of PBS, and centrifuged (1 min, 1000 ϫ g). The supernatant was discarded, and the cell pellet was immediately frozen by immersion of the tube in liquid nitrogen. For measurements using the fluorescence plate reader, the plate with cells was placed immediately after the addition of DPBS-GP containing the probe with or without PMA in the plate reader prewarmed to 37°C.
Fluorescence Plate Reader-Total fluorescence intensities were acquired using a plate reader (Beckman Coulter DTX-880) equipped with the appropriate excitation and emission filters. The instrument was kept at 37°C during the measurements, and fluorescence intensity read from the bottom of each well was integrated over 0.5 s every 90 s. The UV-visible absorption and fluorescence parameters of the fluorogenic probes used in this study and their products of interaction with ROS/ RNS are listed in Table 1.
HPLC Analyses-HPLC experiments were performed using an Agilent 1100 system equipped with UV-visible absorption and fluorescence detectors. To obtain UHPLC-like conditions, a fused core C18 column (Phenomenex, Kinetex C18, 100 ϫ 4.6 mm, 2.6 m) was used. Typically, a gradient elution using an aqueous mobile phase with increasing fraction of acetonitrile (from 10 -20% to 100% over 5 min) in the presence of 0.1% TFA was used. For the analysis of DAF-2 nitrosation, isocratic elution using an aqueous mobile phase containing 10 mM phosphate buffer, pH 7.4, and 5% acetonitrile was also used. The analytes were eluted using a flow rate of 1.5 ml/min. Mass spectra were obtained using a 7.0-tesla Fourier transform ion cyclotron resonance mass spectrometer, interfaced with an Agilent 1100 HPLC system.  . 1a), we monitored the products formed from oxidation of Simultaneous Analyses of Reactive Oxygen/Nitrogen Species HE (Fig. 2). The reaction between O 2 . and HE generates a fluorescent product, 2-OH-E ϩ . The fluorescence excitation-emission matrix spectrum of DNA-bound 2-OH-E ϩ is shown in supplemental Fig. S7. Nonspecific oxidation of HE generates another fluorescent product, ethidium (E ϩ ), and non-fluorescent dimeric products (e.g. E ϩ -E ϩ ) (Fig. 2a). We then monitored the formation of these products under different fluxes of O 2 . (derived from hypoxanthine/xanthine oxidase as described in Fig. 1a) and ⅐ NO (released slowly from NONOate-based ⅐ NO donor) (Fig. 1a). These experiments were performed in a 96-well fluorescence plate containing HE and varying levels of co-generated ⅐ NO and O 2 . (Fig. 2b), and the rates of increase in the fluorescence intensity were plotted against the fluxes of ⅐ NO and O 2 . (Fig. 2c). These products were also separated and quantitated using an HPLC technique coupled with absorption and fluorescence detection (Fig. 2, e-h, and supplemental Fig.  S1). Upon examining the total profiles of HE oxidation prod-ucts (Fig. 2, e- . flux alone, the fluorescence intensity due to 2-OH-E ϩ (independently confirmed by HPLC) increased (Fig. 2, c and f). With increasing ⅐ NO flux, the fluorescence intensity was not diminished (Fig. 2c), although the HPLC results indicated a decrease in 2-OH-E ϩ (Fig. 2f). This is, however, consistent with the HPLC data that showed an increase in E ϩ that has a similar fluorescence spectrum as 2-OH-E ϩ under these conditions (cf. Fig. 2g, Table 1, and supplemental Figs. S1 and S7). These results clearly show that the use of the plate reader for high-throughput screening is a feasible approach for detecting O 2 . -and ⅐ NO-derived oxidants. Importantly, the results should be verified by HPLC analysis to determine the mechanism whereby HE is converted to a fluorescent product as E ϩ and 2-OH-E ϩ cannot be easily distinguished by using the plate reader alone. , the increase in fluorescence intensity was attributed to 2-OH-E ϩ formation. Consistent with this analysis, the addition of superoxide dismutase, but not catalase, inhibited the rate of increase in fluorescence from macrophages activated by PMA (Fig. 3b). L-NAME did not inhibit the increase in red fluorescence (Fig. 3b). Similar to the cell-free system, co-stimulation of ⅐ NO and O 2 . production by combined treatment with IFN, LPS, and PMA decreased the yield of 2-OH-E ϩ with a concomitant increase in the intracellular levels of HE-derived dimeric products. Collectively, these results suggest that although in resting cells the fluorescence intensity reflects the sum of both E ϩ and 2-OH-E ϩ , the PMA-induced increase resulting from HE-derived fluorescence intensity in incubations containing RAW 264.7 macrophages was mediated predominantly by O 2 . . Kinetic measurements have previously shown that ONOO Ϫ reacts rapidly with most boronates (k ϳ10 6 M Ϫ1 s Ϫ1 ), yielding the corresponding phenolic products nearly stoichiometrically (9,14). The fluorogenic probes used in this study were: CBA (9) . from HX/XO, and ⅐ NO from DPTA-NONOate. b, stimulation of ONOO Ϫ from RAW 264.7 macrophages using a mixture of PMA (200 ng/ml, IFN-␥ (50 units/ml), and LPS (0.5 g/ml). ⅐ NO is produced from induction and activation of inducible nitric oxide synthase (iNOS), and superoxide is generated from activation of NADPH oxidase by PMA. and newly synthesized FlAmBE (Fig. 4, a and b). The reaction between these probes and ONOO Ϫ yields the corresponding phenolic products exhibiting blue and green fluorescence, respectively. The formation of ONOO Ϫ was monitored in real time during co-generation of ⅐ NO and O 2 . in cell-free systems using the same hypoxanthine/xanthine oxidase/ DPTA-NONOate system described above (Fig. 1a). As shown in Fig. 4, c and d, the two boronate probes exhibit a similar reaction profile under the same experimental conditions, although the fluorescence intensity formed from FlAmBE was several orders of magnitude higher than that formed from CBA. In the presence of O 2 . or ⅐ NO alone, there was no detectable fluorescence from the probes. However, with increasing ⅐ NO and O 2 . fluxes, both probes were oxidized to fluorescent products with rates of oxidation nearly plateauing at a 1:1 flux ratio of ⅐ NO and O 2 . (Fig. 4, c and d).  . in phosphate buffer (50 mM, pH 7.4) containing DTPA (0.1 mM). HE, E ϩ , 2-OH-E ϩ , and E ϩ -E ϩ were quantified using the appropriate standard. e-h denote global profiling of HE, 2-OH-E ϩ , E ϩ , and E ϩ -E ϩ levels, respectively, under the experimental conditions described in b. JANUARY 27, 2012 • VOLUME 287 • NUMBER 5 JOURNAL OF BIOLOGICAL CHEMISTRY 2987 macrophages and CBA or FlAmBE caused little or no increase in fluorescence (Fig. 4, e and f) as compared with the control (non-treated) cells. Concomitant HPLC analysis (Fig. 5, a-c) indicates that the fluorescence intensity was due to the formation of the corresponding phenolic products, COH or fluorescein N,N-dimethylamide (FlAmide). These results are attrib-  uted to ONOO Ϫ formation and its direct reaction with boronate probes forming the corresponding fluorescent product. As ⅐ NO production in response to IFN/LPS is dependent on activation of inducible nitric oxide synthase, we next determined the effect of L-NAME (nonspecific inhibitor of nitric oxide synthase enzymes) on ONOO Ϫ formed by activated macrophages. As shown in Fig. 4, g and h, L-NAME inhibited PMA-, LPS-, and IFN-␥-induced fluorescence. Similarly, inhibition of O 2 . production using ROS-specific antioxidant enzymes should inhibit ONOO Ϫ formation as well. As . . e, increase in the blue fluorescence intensity was monitored using a plate reader, derived from RAW 264.7 macrophages in DPBS-GP buffer containing CBA (20 M) and different stimulators. f, increase in green fluorescence intensity was monitored using a plate reader from incubations containing RAW 264.7 macrophages and FlAmBE (20M) in the presence of different stimulators. g, same as e but in the presence of L-NAME and ROS (O 2 . and H 2 O 2 )-detoxifying enzymes. h, same as f but in the presence of L-NAME and ROS (O 2 . and H 2 O 2 )-detoxifying enzymes. *, p Ͻ 0.05. expected, superoxide dismutase, but not catalase, inhibited CBA and FlAmBE fluorescence derived from ONOO Ϫ . Collectively, these results indicate that the boronate-based fluorogenic probes can be used to selectively detect ONOO Ϫ formed from macrophages activated to co-generate ⅐ NO and O 2 . .

Oxidation of Amplex Red in Cell-free and Cellular Systems Generating ⅐ NO and O 2 . as Measured Using HPLC and Fluores-
cence Analyses-The horseradish peroxidase (HRP)-dependent oxidation of Amplex Red to a fluorescent product (Fig. 6a) has been used to quantitate H 2 O 2 in biological systems (17). The overall profile of the fluorescent product (i.e. resorufin) formed from Amplex Red in incubations generating ⅐ NO and O 2 . in the presence and absence of HRP and catalase are shown (Fig. 6, b  and c). HPLC analysis of the incubation mixtures revealed the formation of a single major red fluorescent product, resorufin (supplemental Fig. S3). HRP enhanced the rate of the formation of resorufin by over 30-fold in incubations containing Amplex Red and HX/XO/NONOate, as observed by real-time monitoring of fluorescence intensity changes. Note that in the absence of HRP, oxidation of Amplex Red was enhanced during cogeneration of ⅐ NO and O 2 . . This indicates that 1-electron oxidants such as radicals derived from ONOO Ϫ are able to cause oxidation of Amplex Red to resorufin, although at a considerably lower yield as compared with HRP-mediated oxidation. Also, catalase increased the rate of Amplex Red oxidation in the absence of HRP and the ⅐ NO donor (Fig. 6c). We attribute this to the peroxidase activity of catalase as H 2 O 2 is produced directly from XO-catalyzed oxidation of HX and xanthine (X) and from the dismutation of O 2 . (Fig. 1a).

Real-time Monitoring of H 2 O 2 from Activated
Macrophages-Next, we used RAW 264.7 macrophages stimulated with IFN-␥/LPS and PMA. The cell culture conditions were identical to those used to detect O 2 . and ONOO Ϫ . Oxidation of Amplex Red was monitored in incubations containing macrophages and the proinflammatory mediators in the presence or absence of HRP (Fig. 6, d-g). The addition of PMA, or PMA plus LPS and IFN-␥ to incubations containing macrophages, Amplex Red, and HRP caused a time-dependent increase in fluorescence intensity (Fig. 6d). In the absence of PMA, no significant increase in fluorescence was detected. Results obtained in the presence of antioxidant enzymes (Fig. 6e) suggest that catalase significantly inhibited PMA-mediated oxidation of Amplex Red. L-NAME had no effect, indicating that H 2 O 2 was responsible for Amplex Red oxidation under these conditions. Interestingly, preincubation of macrophages with LPS and IFN-␥ caused an increase in the PMA-induced rate of oxidation of Amplex Red. As this oxidation was inhibited by catalase, but not by L-NAME, we tentatively attribute this effect to the depletion of the intracellular pool of antioxidants during prolonged generation of ⅐ NO by LPS/IFN-␥-stimulated macrophages. Similar experiments with activated macrophages were performed in the absence of exogenously added HRP (Fig. 6, f and a, after a 60-min incubation, CBA-derived product was analyzed by HPLC under the same conditions as in the plate reader (Fig. 3b) using the wavelengths as shown in the figure. exc., excitation; emi., emission. b, same as a except that the CBA-derived product was analyzed in the cell culture media. c, conditions same as a except that it gives a quantitative analysis of CBA and COH in cell lysates and media. g). As shown, the fluorescence intensity was considerably diminished; L-NAME and superoxide dismutase but not catalase inhibited Amplex Red-derived fluorescence (Fig. 6, f  and g). These results suggest that it is possible to monitor both H 2 O 2 -derived and ONOO Ϫ -derived oxidants formed from activated macrophages using the Amplex Red probe. However, distinguishing between ONOO Ϫ and H 2 O 2 -dependent mechanisms would require additional studies using several inhibitors (e.g. L-NAME, superoxide dismutase, and catalase).
Oxidative Nitrosation of DAF-2 in Cell-free and Cellular Systems Generating ⅐ NO and O 2 . as Measured Using HPLC and Fluorescence Analyses-The formation of a green fluorescent product, DAF-2T from DAF-2, has been used to monitor intracellular ⅐ NO formation (Fig. 7a) (18). The oxidative nitrosation profile of DAF-2T formed from the probe DAF-2 in incubations generating ⅐ NO and O 2 . shows that the rate of appearance of green fluorescence reached a maximum at ratios close to 1:1 (Fig. 7b). Independent HPLC analyses revealed that DAF-2T is responsible for the green fluorescence observed under these conditions (supplemental Fig. S4). Superoxide alone did not oxidize DAF-2 to DAF-2T, and ⅐ NO alone (in the presence of oxygen) caused only a modest increase in DAF-2T fluorescence (Fig. 7b). Clearly, these results suggest that in the presence of Real-time Monitoring of ⅐ NO using DAF-2 from Activated Macrophages-⅐ NO and ONOO Ϫ generation from activated macrophages was monitored using the probe DAF-2DA (the diacetate, cell-permeable analog of DAF-2 carboxylate) and DAF-2. The addition of IFN-␥/LPS to macrophages increased the green fluorescence of DAF-2T, which was further enhanced in the presence of PMA (Fig. 7c). HPLC analyses confirmed that the formation of DAF-2T was responsible for the increase in the fluorescence intensity (not shown) (19). L-NAME significantly inhibited the fluorescence intensity due to DAF-2T from macrophages activated by LPS/IFN-␥ or LPS/IFN-␥/PMA (Fig.  7d). However, superoxide dismutase did not affect IFN-␥/LPSinduced DAF-2T fluorescence, but significantly inhibited IFN-␥/LPS/PMA-induced DAF-2T fluorescence. Similar results were obtained with DAF-2 probe (not shown). These results strongly suggest that DAF-2 or DAF-2DA is not a suitable probe for measuring ⅐ NO selectively under conditions generating both ⅐ NO and O 2 . , although information with regard to RNS generation may be gained with the help of NOS inhibitors and ROS-detoxifying enzymes.

DISCUSSION
As discussed in recent reviews, ROS and RNS do not represent a single reactive species, but encompass a wide range of reactive species that exhibit oxidizing, nitrating, nitrosating, and halogenating properties (20,21). To better understand the physiological and pathological consequences of ROS and RNS and their inhibitory mechanisms, it is crucial to unequivocally identify and quantify the species that are specifically responsible for a given biologic effect. In this study, we show that the combined use of a multiwell fluorescence plate reader and HPLC will detect all the relevant oxidation products within 5-10 min (UHPLC-like conditions using a fused core C18 column), thus making it feasible to monitor ROS and RNS formation under a variety of experimental conditions. The global profiling of ROS and RNS-derived fluorescent products demonstrated herein gives additional insight into the chemical mechanisms of their formation. For example, the profiles generated from oxidation of boronate-based fluorogenic probes (CBA, FlAmBE) in the presence of ⅐ NO and O 2 . indicate a direct oxidation of boronates by ONOO Ϫ to a fluorescent product that reaches a plateau at nearly a 1:1 ratio of ⅐ NO and O 2 . (Fig. 4).
In contrast, oxidation/hydroxylation of HE under the same conditions shows ⅐ NO-mediated inhibition of 2-OH-E ϩ generated from the reaction of HE with superoxide. This could be explained by competitive scavenging of O 2 . by ⅐ NO. The concomitant formation of HE-derived dimeric products confirms the generation of a more potent, ONOO Ϫ -derived 1-electron oxidant under those conditions. We have shown recently that ONOO Ϫ directly and stoichiometrically reacts with boronates nearly a million times faster than H 2 O 2 and two hundred times faster than HOCl (9,14). Thus, under conditions that simultaneously induce ONOO Ϫ or HOCl formation in a biological setting, boronates cannot be used to measure H 2 O 2 . The lack of an effect by catalase on FlAmBE-and CBA-derived fluorescence from activated macrophages (Fig. 4) is in support of this kinetic analysis. However, boronates conjugated to a triphenylphosphonium moiety were used to measure H 2 O 2 production inside mitochondria using tandem LC/MS analysis (21). Under these conditions, it is likely that there is negligible formation of ONOO Ϫ or HOCl.
The global profiling of HE and HE-derived oxidation products is essential for proper interpretation of results obtained under different experimental conditions (22). Measurement of intracellular superoxide via monitoring of 2-OH-E ϩ , the diagnostic marker product of O 2 . /HE reaction, is dependent on HE uptake and its rate of oxidation to ethidium and dimeric products. Although E ϩ and HE-derived dimeric products are not formed in the presence of O 2 . alone, other 1-electron oxidants (perferryl iron or ONOO Ϫ -derived oxidants) induce their formation, and consequently, decrease intracellular HE levels (13). This can lead to a decrease in 2-OH-E ϩ formation. A previous study indicated that the ratio between 2-OH-E ϩ and HE is a better parameter for assessing O 2 . formation in cells (23). However, this approach is based on several kinetic assumptions that have not yet been totally validated (22). The fluorescent probes, dichlorodihydrofluorescein (DCFH) and dihydrorhodamine 123 (DHR), have been most widely used to measure intracellular oxidants (H 2 O 2 , ONOO Ϫ ). The pitfalls and artifacts of their use in biological systems have been previously discussed (24,25). These two probes do not directly react with H 2 O 2 or ONOO Ϫ . Iron or heme proteins with peroxidase activity are required for H 2 O 2dependent oxidation of dichlorodihydrofluorescein and dihydrorhodamine. ONOO Ϫ oxidizes these probes via a radical mechanism involving ⅐ OH, ⅐ NO 2 , or CO 3 . in the presence of bicarbonate. Most importantly, the intermediate radicals formed from these probes react with oxygen, forming superoxide and H 2 O 2 and thus artifactually enhancing product formation and fluorescence intensity (25,26). This is one of the major shortcomings of these probes. In contrast, HE-and Amplex Red-derived radicals do not react with molecular oxygen, thus preventing artifactual generation of O 2 . and H 2 O 2 . Boronate reaction with ONOO Ϫ is mostly direct (ϳ85-90%), involving a 2-electron oxidation/reduction. Only a minor fraction (ϳ10 -15%) occurs via a free radical pathway (27).
We have previously shown that the fluorescence spectral overlap between 2-OH-E ϩ and E ϩ makes it impossible to assign HE-derived red fluorescence to either 2-OH-E ϩ or E ϩ without guidance from HPLC measurements (22). As shown in Fig. 2b (15,28) or E ϩ (nonspecific oxidation product of HE) under these conditions (Fig. 2d). Extraneous factors (light, sonication, etc.) were shown to induce the formation of 2-OH-E ϩ and/or E ϩ (16). Many investigators still have the wrong notion that E ϩ is the primary product of the HE and O 2 . reaction. To properly establish the connection between species and signaling, it is essential to understand the mechanism of oxidation of fluorogenic probes by specific ROS/RNS (29). The schematic shown in Fig. 8 describes the approach an investigator may take to determine which reactive oxygen and nitrogen species are produced in their system of interest. In a 96-well plate, cells treated as desired can be assayed using HE, CBA, DAF-2DA, and Amplex Red. All of these probes are cell-permeable. The fluorescence signal results from both intracellular and extracellular compartments. The use of a fluorescence plate reader for the initial screening, when coupled with inhibitors of specific oxidative products as indicated, FIGURE 8. Suggested experimental design for identifying the reactive oxygen and nitrogen species generated simultaneously in response to a treatment of interest. The scheme describes the approach an investigator may take to determine which reactive oxygen and nitrogen species are produced in their system of interest. In a 96-well plate, cells treated as desired can be assayed using HE, CBA, DAF-2DA, and Amplexா Red. The use of a fluorescence plate reader for the initial screening, when coupled with inhibitors of specific oxidative products as indicated, allows for simultaneous detection of multiple reactive species. Information obtained from the plate reader can then be used to design more selective studies by UHPLC to verify and quantify the identity, location, and level of the products detected in the plate reader. DAF-2DA, diaminofluorescein-2 diacetate; SOD, superoxide dismutase. allows for real-time simultaneous detection of multiple reactive species. In addition to using exogenous superoxide dismutase and catalase antioxidant enzymes (that are cell-impermeable), the investigator should also use molecular biological approaches that will alter the intracellular levels of these enzymes. Information obtained from the plate reader can then be used to design more selective studies by UHPLC to verify the identity and quantify the amount of intra-and extracellular products. As additional ROS/RNS-specific probes evolve with their oxidation/nitration/nitrosation chemistry properly characterized, the investigator may substitute these probes in this experimental approach.
Caveats-There are several important caveats to consider when examining ROS/RNS production in any given system. Chief among these is the tissue-specific expression of antioxidant enzymes. These enzymes will compete for reaction with the oxidants in question, and thus could potentially lead to aberrant quantification of the ROS/RNS involved. For example, the reaction of O 2 . with superoxide dismutase (k ϳ1 ϫ 10 9 M Ϫ1 s Ϫ1 ) is significantly more rapid than the reaction with HE (k ϳ10 5 -10 6 M Ϫ1 s Ϫ1 ) (30,31). Similarly, performing ROS/RNS quantification studies in complete culture media can also affect the measurements. For example, ascorbate in culture media can react with multiple radical species (e.g. alkoxyl and alkyl peroxyl radicals) (32). Other components (phenol red, pyruvate, etc.) that are present in the culture media may react with ROS/RNS. Thus, it is advisable to maintain consistent culture conditions if comparisons between treatment groups are desired. ROS/RNS also participate in side reactions that can alter their apparent concentration or rate of production. A prime example of this is the reaction of peroxynitrite and carbon dioxide to form nitrosoperoxycarbonate (ONOOCO 2 Ϫ ). Another important factor in these studies is the interaction between probe-derived intermediates and products with intra-and extracellular components (e.g. thiols). These side reactions will affect ROS/RNS-dependent formation and quantitation of products formed from the fluorescent probes.
The reactions discussed here are but an example of the considerations that must be made by researchers who wish to examine the profile of ROS/RNS produced in their model system of interest. Alternative methods such as monitoring nitrite/nitrate production or non-mitochondrial oxygen consumption may assist in providing a global view of the levels of ROS/RNS production; however, these methods lack species identification and should be used for gathering general insight into the oxidative biology occurring in a given system.
Future Perspectives-The high-throughput global profiling methodology developed herein for detecting ROS/RNS will be most useful in enzymatic systems regulated by several co-factors and redox modification of proteins in cell signaling. In the future, it is likely that one can monitor real-time changes in intracellular glutathione (GSH:GSSG) redox potential (33), lipid hydroperoxide, lipid aldehyde, isoprostanes, nitrated lipid, and immunospin-trapped protein radical adduct, calcium imbalance, membrane potential, release of pro-inflammatory mediators, and oxidant-induced signaling molecules in response to real-time changes in oxidant generation. Results from such studies will provide a solid foundation for establish-ing the identity of ROS/RNS species involved in redox signaling, cell proliferation, or cell death.