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Michael AC, Borland LM, editors. Electrochemical Methods for Neuroscience. Boca Raton (FL): CRC Press/Taylor & Francis; 2007.

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Electrochemical Methods for Neuroscience.

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Chapter 13Oxidative Stress at the Single Cell Level

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Introduction

Oxidative Stress Processes

Aerobic cells derive most of their energy from the controlled oxidation of CH bonds via oxygen-atom transfer catalyzed by metalloenzymes. In particular, these metalloenzymes are central actors of the respiratory chain in mitochondria. However, the same enzymes are generally good reducing agents, prone to open a side route (6–8% yield), leading to oxygen reduction into superoxide ion (O2•−, see Figure 13.1), the precursor of a whole series of hazardous species for living systems coined the “reactive oxygen species” (ROS) [1–4].

FIGURE 13.1. Scheme describing the formation of superoxide anion as a side reaction of oxygen reduction in the respiratory chain of mitochondria.

FIGURE 13.1

Scheme describing the formation of superoxide anion as a side reaction of oxygen reduction in the respiratory chain of mitochondria. (Adapted from Raha, S. and Robinson, B. H., Trends in Biochemical Sciences, 25, 10, 2000.)

At physiological pH, the superoxide ion is readily scavenged through its disproportionation (k = 2 × 105 M −1 s −1 , T = 25°C, pH = 7.4) into hydrogen peroxide and oxygen:

O2-+H+HOO         (KpH7<<1)
13.1
HOO+O2-HOO-+O2         (fast)
13.2
HOO-+H+H2O2         (fast)
13.3

which may be even more rapid in living cells when catalyzed ( k = 2 × 109 M −1 s −1 , T = 25°C, pH = 7.4) by superoxide dismutases (SOD):

2O2-(+2H+)H2O2+O2
13.4

Two main types of SOD are present in mammalian aerobic cells: a Mn-based enzymatic center form (Mn SOD) located in mitochondria and a Cu/Zn-based form (Cu/Zn SOD) located in cytoplasm in specific granules, and possibly in membranes [5–11]. These enzymes are prone to protect cells from superoxide oxidation of R–H bonds, though this reaction is generally considered as slow, and also from the reaction of superoxide with other radicals such as nitric oxide that may generate more hazardous compounds (Figure 13.2 and explanations below).

FIGURE 13.2. Main reactive oxygen species (ROS) and reactive nitrogen species (RNS) derived from the biological conversion of oxygen into superoxide ion (O2•− ) and nitric oxide (NO•).

FIGURE 13.2

Main reactive oxygen species (ROS) and reactive nitrogen species (RNS) derived from the biological conversion of oxygen into superoxide ion (O2•− ) and nitric oxide (NO).

H2O2 formed by O2•− disproportionation is an even more potent cytotoxic chemical than superoxide, since its lifetime is sufficient to allow its diffusion to almost any cellular compartment, where it may act as a potential source of hydroxyl radicals OH (Figure 13.2) through the Fenton reaction in the presence of iron-II free ions or metalloenzymes [1,2]. Because hydroxyl radicals are among the best hydrogen atom acceptors, they are prone to induce a large variety of biological modifications, among which are the peroxidation of cell membrane bilipids through the Haber–Weiss reaction, DNA bases, and backbone oxidation and hydroxylation [1,2,12].

In living cells, H2O2 is scavenged primarily by catalase and secondly by peroxidases (glutathione peroxidase), which catalyze respectively its fast disproportionation into oxygen and water (or into oxidized organic substrates and water), so that its concentration is usually maintained at nanomolar steady-state levels and the lethal routes mentioned above prevented [3,13–16]. In spite of its considerable efficiency at the nanomolar and sub-nanomolar physiological concentrations of hydrogen peroxide, catalase is inhibited by its substrate at higher concentrations. More active scavengers designed to perform in such higher concentration ranges may be produced by a cell through protein expression, but this requires at least half an hour after signaling of the ROS increase [1,17,18]. Thus, aerobic cells might remain unprotected against H2O2 and other ensuing ROS whenever its concentration rises rapidly at levels significantly beyond normal physiological ones.

Besides, the production of superoxide and other ROS may not only result from side-routes of the normal oxidative or energetic metabolism of cells. Specific processes are used purposefully by many cell types for communication, for the control of redox sensitive mechanisms (gene expression) leading eventually to cell death by apoptosis, or for defense against xenobiotics (bacteria, virus, etc.) in the case of phagocytes (macrophages and neutrophils). Indeed, a cell may change its normal oxygen metabolism so as to produce deliberately important quantities of superoxide through the involvement of specific enzymes, such as NADPH oxidases, which catalyze the following reaction [19–22]:

2O2+NADPH2O2-+NADP++H+
13.5

NADPH oxidase (from the NOX family) activity is of particular importance in the phagocytosis process of foreign bacteria and viruses by macrophages or neutrophils, since the bactericidal activity is provided essentially by ROS (and RNS). However, excess of ROS production by active or passive mechanisms will amplify cell damages with time. These damages might be prevented by enzymes (SOD, catalase, and peroxidases mentioned above), soluble antioxidant molecules (vitamins, glutathione, uric acid, etc.), or repaired by specific enzymes (DNA repair systems, lysosomes fusion for membrane repair, etc.). This critical situation termed oxidative stress basically describes an acute or chronic imbalance between the production of ROS and antioxidant capacities of living cells and organisms. Oxidative stress processes are suspected to be involved directly or indirectly (apoptosis) in many human pathologies (aging, cancers, Parkinson and Alzheimer diseases, auto-immune pathologies, arthritis, etc.) and have stressed in the last two decades, the interest of many research groups in several disciplines of Biology and Medicine.

From recent studies, the imprecation of ROS metabolism with that of nitric oxide (NO ) in oxidative stress processes appears ubiquitous in many physiological or pathological situations. Nitric oxide (or nitrogen monoxide) displays very different effects (positive or negative) in organisms as a function of its concentration and of the local conditions in vivo. NO mostly originates from the activation of enzymes, the NO synthases (three different mammalian types), which catalyze the reaction [23–26]:

L-arginine+2NADPH+2O2+2H+NO+citrulline+2NADP++2H2O
13.6

Nitric oxide is a weak oxidant by itself and should not cause oxidative stress. In fact, it is at most, a weak reductant associated with the strong oxidizing species NO+ [27]. Nevertheless, if O2•− and NO are produced in the same cell environment, or in closely related ones, they may couple at a rate close to the diffusion limit (approximately 3–19× 109 M −1 s −1 ) [27–29] to form the potent peroxynitrite anion:

O2-+NOONOO-
13.7

Peroxynitrite was generally ignored in the past. Contrastingly, it is now suspected to be the source of many oxidative, nitrating, or nitrosating processes involving several cell components and has focused a broad research in the last decade. At physiological pH, peroxynitrite may decompose through the intermediate formation of its conjugated acid ONOOH (pKa≈6.8) [30–34]:

ONOO-+H3O+ONOOH+H2O
13.8

ONOOH undergoes two facile decomposition routes. One goes through the formation of the very reactive radicals NO2 and OH and is considered as biologically feasible, especially in non-polar environments (e.g., membranes). Besides, peroxynitrite anion ONOO may react in biological conditions with carbon dioxide (overall constant k = 3–6× 104 M −1 s −1 , Equation 13.9 through Equation 13.12) [32,35–37]:

ONOO-+CO2[ONOOCO2]
13.9
[ONOOCO2]CO2+NO3-         (70%)
13.10
[ONOOCO2-]NO2+CO3-         (30%)
13.11
NO2+CO3-CO2+NO3-
13.12

This mechanism competes with the direct decomposition of ONOO , since CO2 concentration in vivo is quite high (12–30 m M in blood). In this process, it appears that a significant fraction of the short time living species ONOOCO2 ends into the potent nitrating and caboxylating radicals NO2 and CO3•−.

Similarly, when diffusing over significant distances from its source, NO is expected to react significantly ( k = 2 × 106 M −1 s −1 ) [38,39] with oxygen (Equation 13.13) since this species is present at rather important concentrations (mean of 0.24 m M ) in extracellular fluid or in blood:

4NO+O2+2H2O4NO2-+4H+
13.13

The side reactions of nitric oxide oxidation may form species such as NO2• and N2O3, which are also very efficient nitrating and nitrosating compounds. Finally, though the in vitro decomposition of nitric oxide and peroxynitrite leads ultimately to very stable nitrite and nitrate ions (which, in vivo, may accumulate in cells and their environment), these processes generate very reactive intermediates, such as NO2, N2O3 or NO+ (suspected to be at the origin of nitrated, nitrosated derivatives of amino acids, thiols, and other important biological components).

The existence of such a series of complex and intricately interconnected pathways, which necessarily follow any simultaneous production of superoxide and nitrogen monoxide by living cells, explains why the very nature of the ROS and/or RNS compounds has not yet been fully characterized in the majority of oxidative stress processes. The origin and mechanism of these processes has been mostly hypothesized based upon the observation of cell metabolites (requiring anywhere from hours to years to appear) or upon effects on cellular activation mechanisms such as gene expression (hours to days). Despite the apparent ubiquity and importance in aerobic cells of the ROS and RNS, few analytical methods have been developed that allows for direct monitoring of their production by living cells, i.e., for investigating the processes at the very origin of oxidative stress and its severe consequences. Electrochemistry on microelectrodes became a method of choice in this regard during the last 15 years owing to the electroactivity of several important ROS and RNS. While, initial studies were conducted on groups of cells or cell monolayers, they have finally evolved to allow direct measurements at the single cell level.

Electroactive Species Implicated in Oxidative Stress

The ability of the electrochemical methods to detect in biological conditions, some of the ROS and RNS mentioned above (or presented in Figure 13.1) obviously depends on two main factors: the half-wave potential of the redox couple (implicating a given ROS or RNS), and the time-life of this compound when produced by a living cell, since this latter commands its permanence and diffusion into cells or tissues. Moreover, with the ultimate goal here being the analysis on a single aerobic living cell, measurements have to be conducted in aqueous medium (buffer) in the presence of oxygen (solutions in equilibrium with normal atmosphere). This condition mostly precludes the use of the reduction domain for electrochemical analysis, given that the reduction of oxygen takes place at low reductive potentials on most electrode surface, e.g. − 0.1 V vs. (saturated sodium calomel electrode SCE) on platinum microelectrodes, and oxygen concentration in the close environment of the cell may vary during oxidative stress processes due to its consumption. Thus, direct reductive analysis of any other electroactive species would be generally convoluted with oxygen detection.

Hydroxyl radical is among the most reactive radicals found in biochemistry because it can abstract a hydrogen atom to any CH bond. This species is, thus, definitively not able to diffuse from its generation point in the cell toward the electrode surface without reacting with other molecules, such as the cell membrane constituents. The eventuality of its detection would require positioning the electrode surface at a nanometric distance from its source, which is outside of our present experimental limits in electrochemistry at living cells. Nitrogen dioxide, NO2, time-life is reported to be significantly longer under biological conditions than that of hydroxyl radical (a microsecond domain versus a nanosecond domain, respectively). This would be compatible with a direct electrochemical detection, though it still represents an analytical challenge. However, the standard potential of the redox couple for the oxidation of NO2 into NO2+ has been measured as E′(NO2+/NO O2) = + 1.56 V vs. NHE [27,28,40,41], a high potential close to the water oxidation wall. These conditions strongly limit the probability for a direct detection of NO O2 through its oxidation, and to the best of our knowledge, such results have never been reported into biological conditions.

Conversely, the primary radical species which lead to the complex family of ROS and RNS, i.e., superoxide and nitric oxide, are certainly an easier target for their electrochemical detection by oxidation. The thermodynamic data providing the following standard potential of the redox couple for the oxidation of NO ( E′(NO+ /NO ) = + 1.21 V vs. NHE) and a standard potential of the redox couple for the oxidation of O2•− ( E′(O2/O2•−) = − 0.33 V vs. NHE) do not preclude a priori detection [27,28,40]. From a kinetic point of view, the life span of each of these primary species strongly depends on the local conditions in cells for their production. As mentioned above, superoxide disappearance by disproportionation depends on the species concentration (order two kinetic) and is much accelerated in presence of SOD. Similarly, the nitric oxide auto-oxidation process is dependent on the species concentration and controls the availability of NO in conjunction with many possible reactions with biological targets in cells. Yet, nitric oxide at nano- to micromolar local concentration may diffuse over micrometric distances, thus its detection in the extracellular space is feasible. Moreover, the high lipophilic character of NO and the slight lipophilic character of O2•− allow their diffusion through cell membranes and their detection by a microelectrode placed outside the cell. Besides, we may consider the possibility that these species can be produced by enzymatic systems located in cell membranes, such as the NADPH oxidases (NOX family) for superoxide, and constitutive NO synthases for NO . Under such conditions, the local geometry disfavors possible reactions between NO or O2•− and the cell components, thus providing a better probability for their observation.

Several other neutral or anionic ROS and RNS are also good candidates for their detection and the characterization of oxidative stress at living cells. Concerning ROS, hydrogen peroxide is probably the most stable compound among this family of species; its time-life in vivo depends essentially on its possible reaction with free metallic ions such as iron(II) or catalase present in the medium (see also detailed explanations about diffusion of H2O2 in vivo in Chapter 11). The redox potential of its couple [E′(H2O2/O2) = − 0.146 V vs. NHE] makes it a good candidate for direct detection on cells. Nitrite is a very stable compound in vivo, and the redox potential of its couple [E′(NO O2/NO2) = + 0.99 V vs. NHE] is a priori compatible for direct oxidation. Nitrite is essentially the end product of nitric oxide metabolism, and is classically used as an indirect marker of oxidative or nitrosative stress with the problem that its basal levels may be quite high. Nevertheless, a large area of studies defining our present understanding of NO physiology has been based on the detection of nitrite and nitrate by Griess reaction. Nitrate ions are difficult to oxidize [ E′ (NO3/NO3) = + 2.50 V vs. NHE] in absence of an enzymatic transducing system and are also stable end products of RNS [27,28,40]. These two compounds did not focus much interest for their direct analysis on single cells. Conversely, the intermediate and reactive species, peroxynitrite anion, is an ideal target for electrochemical measurements. Other methods reported in the literature for peroxynitrite detection are routed on fluorimetric and chemiluminescence analysis, though they are indirect and not effectively selective [42–45]. Quantization and kinetic profiles of species release by cells is also impossible by such methods. In contrast, electrochemistry at microelectrodes may offer the considerable advantage of a direct detection of peroxynitrite by its oxidation, since its formal redox potential is quite low ( E′ (ONOO /ONOO) = + 0.20 V vs. NHE), and its decomposition kinetic is in the sub-second range [27,40,46]. Consequently, considering an electrochemical sensor positioned close to the cell source of peroxynitrite, i.e., under some conditions equivalent to the ones used for NO or O2•− detection (the actual species that lead to ONOO), it may be possible to detect and monitor that species before it reacts with its biological targets or before being decomposed into its daughter species.

Direct Electrochemical Detection of Oxidative Release on Single Cells

The average amount of oxidants that may be released by a single cell during an induced oxidative stress could be estimated from studies on cell populations (that number in the millions), by submiting the whole colony to a chemical or immunological stimulation. Then following with spectrophotometric analysis or fluorimetric assays [45,47,48]. For instance, neutrophils in culture may produce about 0.1 μmol of O2•− /h/106 cells, and macrophages in culture may produce about 1 μM of RNS/h/106 cells. These values lead to the estimation of a rate of release in the range of 10−16–10−13 mol/s/cell. Such infinitely small quantities represent a priori, a considerable challenge compared with the actual analytical standards. The difficulty is even greater whenever the whole event is completed within a very short time, and the timing is “chosen” by the cell itself. Both factors emphasize the huge difficulty of the analytical detection in terms of signal-to-noise ratio, and easily explain why, despite their extreme biological importance and huge medical consequences they have. the study of oxidative stress primary events has seldom been investigated by biologists at the single cell level.

The quality of any analytical information requires a sufficient signal-to-noise ratio. Taking for granted that the analytical device has been selected to adequately perform, both thermodynamically and kinetically, a sufficient signal-to-noise ratio imposes a sufficient concentration of the chemical to be detected given that a minimal noise level cannot be avoided. It is important to stress here that a large concentration does not mean a large quantity, since a concentration is a quantity of chemicals divided by the sampling volume. Thus, an infinitely small number of molecules may be easily converted into a large concentration by restricting the volume in which these molecules are released. This is the simple arithmetic solution retained by nature in its biological synapses, so as to detect kinetically (viz., concentration), with a high signal-to-noise ratio, the release of zepto-to attomoles of chemical messengers on a millisecond time-scale [49,50].

This principle was first adapted to electroanalytical purposes by Wightman’s group and our group [50–53]. Indeed, whenever the released molecules are electroactive and identifiable electrochemically, placing an electrode at micrometric or sub-micrometric distances from a living cell ensures an adequate signal-to-noise ratio by restricting the extracellular volume in which the molecules produced by the cell are released. For example, one femtomole delivered in a volume of a thousand μm [3] creates a millimolar concentration rise, i.e., produces a concentration variation which can be monitored kinetically via electrochemical techniques with an excellent signal-to-noise ratio, provided that the electrode does not pick up too much noise through its surface.

Ultimately, electrodes pick up electrical noise through their capacitance, viz., through their overall conducting surface area, while the analytical information (viz., the Faradaic current) may arise only from the surface area exposed to the cell release. Thus, using an electrode with an active surface matching that of the examined cell decreases the noise, while not affecting the quality and intensity of the analytical information. This increases the signal-to-noise ratio and ensures, simultaneously, a quantitative collection efficiency, since the artificial synaptic cleft covers the whole cell emitting surface. It is understood that positioning an ultramicroelectrode at micrometric distances from an isolated living cell (Figure 13.3), cells being of micrometric dimensions, ensures the most adequate signal-to-noise ratio and guarantees that the release of the collection of electroactive chemicals at the surface of a single living cell is total [54–56].

FIGURE 13.3. Principle of analysis by a microelectrode of a single cell’s secretion of electroactive species.

FIGURE 13.3

Principle of analysis by a microelectrode of a single cell’s secretion of electroactive species.

Based on these analytical principles, studies were initiated by several groups at the end of eighties and beginning of the nineties in order to detect either superoxide and its first derivatives hydrogen peroxide, or nitric oxide released by a single cell or a small group of cells. More recent studies have led to the fine analysis of cross-reaction products, such as peroxynitrite, between the two primary compounds (NO and O2•−). The following examples have been chosen in order to illustrate different types of applications and different technologies for the microelectrode preparation and their sensing properties.

Microelectrode Detection of Superoxide and Hydrogen Peroxide Released by Living Cells

Detection of Superoxide by Carbon and Gold Microelectrodes on Phagocytes and Vascular Cells

Pioneer work in the field of detection of species implicated in oxidative stress processes was reported by the group of Hill (Oxford University, U.K.) in the mid eighties. This group worked previously on SOD activity and investigated the possibility to detect superoxide anion release by human blood neutrophils [57–59]. Two series of results were reported. In the first one, they proposed to detect superoxide anion by its oxidation wave at low potential ( + 50 mV vs. Ag/AgCl) on the surface of a millimetric pyrolytic graphite electrode. Then, the electrode was “opsonized”, i.e., its surface was modified by a deposit of an immunological factor, the immunoglobulin G (IgG). This was obtained through a simple procedure, dipping the electrode (2 min) into a solution of IgG (30 mg/mL−1 in Hanks buffer, 37°C). Measurements were made into a buffer solution containing neutrophils previously purified and used in suspension. The neutrophils that met the electrode surface were activated by the deposit of IgG and rapidly initiated a respiratory burst response. Such typical response of neutrophils and macrophages during immunological processes has been known for decades to be a key factor of phagocytosis. The increase of oxygen consumption (the respiratory burst) by activated phagocytes correlates with the activation of their NADPH oxidases (NOX-2 type) that catalyze the formation of superoxide and hydrogen peroxide. Hill et al. have shown that O2•− released by neutrophils in their environment was detected on the graphite electrode surface. The selectivity for O2•− of their analysis was supported by the observation of a strong decrease of response when SOD or when N -ethyl-maleimide (NEM, a non selective inhibitor of NADPH oxidases) was injected into the medium.

In a second series of experiments, a gold microelectrode (10 μm in diameter) was used instead of the classic pyrolytic graphite millimetric electrode [58]. The microelectrode surface was opsonized by IgG and placed into a suspension of fresh neutrophils, as described previously. The contact between cells and the microelectrode surface induced very low variations of current, in the range of 1–1.5 pA, with a spike-shaped kinetic profile (see Figure 13.4). Control experiments in the presence of SOD or NEM (see above) showed that superoxide was responsible for this oxidation current, or at least was related to it. Measurements of the oxygen consumption rate by the neutrophils and its evaluation for a single cell led the authors to conclude that the oxidative bursts detected on their opsonized microelectrode originated in most of cases from the activation of a single neutrophil being in contact with the electrode surface.

FIGURE 13.4. Detection on an opsonized (IgG modified) gold microelectrode of superoxide release by a single neutrophil.

FIGURE 13.4

Detection on an opsonized (IgG modified) gold microelectrode of superoxide release by a single neutrophil. At time t(Neuts.), neutrophils were added to the buffer solution in which the microelectrode was placed with the intention that one of them would (more...)

These seminal results seem to be the first demonstration of the perfect suitability of electrochemical methods for the detection of ROS species implicated in oxidative stress at the level of one living cell.

In 1996, Tanaka et al. reported studies [60,61] that confirmed and refined the work of Hill et al. Superoxide production by phagocytes in culture (HL-60 cell line) was detected on a 10 μm-diameter carbon fiber microelectrode. The microelectrode surface was opsonized by dipping it into a solution of human IgG (80 mg mL−1 in PBS, 3 dips of 15 min at 37°C), a procedure akin to that of Hill’s group, or modified by adsorption of an activator, the phorbol ester PMA (4-phorbol-12-β myristate-13-acetate at 80 n M in PBS, 3 dips of 15 min at 37°C). Cells were immobilized on a collagen modified coverslip and consequently, easy to localize for analysis without relying on a hypothetical interaction between cells in suspension and the electrode surface. When the microelectrode surface was put in contact with the surface of a phagocyte, the cell shape was changed and a phagocytosis process was initiated. As reported in Figure 13.5, amperometric currents detected at + 0.1 V vs. Ag/AgCl, a potential sufficient to oxidize superoxide into oxygen on the carbon surface, provided slow developing signals in both condition of cell activation. Their maximum amplitude was in the range of a few picoampers. The authors assumed that the collection efficiency of superoxide secretion from the cell attached to the electrode surface was total, so that they could estimate the maximum rate and total amount of release: 1.2 fmol min−1 and 15 fmol total for IgG activation; and 0.4 fmol min −1 and 30 fmol total for PMA. The very low noise level of their background current, i.e., about 0.1 pA, led them to draw the conclusion that the oscillations on the cell signal analyzed for one to three hours originated from variations of NADPH during cell response. Indeed, this may be used by cells to modulate their NADPH oxidase activity or other enzymes activity also dependent on NADPH metabolism.

FIGURE 13.5. Detection on (a) an opsonized (IgG modified) or (b) PMA-modified carbon microelectrode of superoxide release by single phagocytes (of the HL-60 cell line).

FIGURE 13.5

Detection on (a) an opsonized (IgG modified) or (b) PMA-modified carbon microelectrode of superoxide release by single phagocytes (of the HL-60 cell line). The microelectrode surface was positioned in contact with one isolated cell at t = 0 min. (Adapted (more...)

Based on the work of Tanaka et al., several groups have used carbon fiber microelectrodes to monitor superoxide release by living cells into biological situations different from the one exposed above [62–65]. In particular, Privat et al. [66] have reported measurements of O2•− produced by vascular cells (endothelial and smooth muscle cells). Indeed, oxidative stress following overproduction of superoxide (and possibly nitric oxide) at the vascular level may lead to local dysfunctions. This has been recognized as a key factor in atherosclerosis, ischemia/reperfusion injury, etc. The authors have carefully calibrated their carbon fiber microelectrodes (7 μm in diameter) against superoxide solutions prepared from dissolved KO2 over a wide range of pHs (7.4–14). The electrode surfaces were electrochemically pretreated into PBS by cycling the potential, and measurements were conducted by differential pulse amperometry between 0 and + 0.1 V vs. SCE (40 ms step length and sampling for 6 ms). This procedure offered an excellent sensitivity for O2•− (0.8 n M /pA−1 ) while the low potential applied provided selectivity. In these experiments, the sensing property of the electrodes was separated from the cell stimulating step without including adsorption of chemical or immunological agents on the electrode surface. Vascular cells in culture were stimulated by a biological agent, the cytokine IL-1β, after positioning (via a micromanipulator) the microelectrode surface at a close distance to the cells, supposedly a few tens of microns. The configuration ensured an efficient collection of superoxide release, without a priori any significant loss of response due to the formation of hydrogen peroxide by disproportionation or due to reaction with NO , which was not supposed to be produced in these conditions. These hypotheses were supported by the low local concentrations, in the range of tens of nanomolar of superoxide detected (see Figure 13.6). This means that the second order kinetics of reactions Equation 13.4 and Equation13.8 are slow enough and henceforth do not affect O2•− concentration before these molecules reach the electrode surface. Yet, it may be noted that this may be a circular argument, given that if superoxide were to be produced at larger amounts, most of it would be consumed during the time-of-flight required to reach the electrode surface. Then, only a minimal fraction would survive and be detected, i.e., that for which spontaneous disproportionation becomes negligible.

FIGURE 13.6. Detection with a carbon fiber microelectrode of superoxide release from unstimulated and IL-1 β-stimulated endothelial vascular cells.

FIGURE 13.6

Detection with a carbon fiber microelectrode of superoxide release from unstimulated and IL-1 β-stimulated endothelial vascular cells. SOD was injected in another experiment to verify the signal origin. (Adapted from Privat, C., Stepien, O., David-Dufilho, (more...)

Detection of Superoxide by Cytochrome C Modified Gold Electrodes on Neuronal Cells

Let us present, now, another seminal study from the group of C. J. McNeil (University of Newcastle upon Tyne, U.K.). Starting in 1993 these authors have proposed a new concept for electrode preparation in order to improve their selectivity toward superoxide. These studies were based on millimetric electrodes and did not apply to single cells but instead to confluent cell layers.

We believe, nevertheless, that the present principle of electrode modification may also be used for the preparation of microelectrodes and that single cell analysis would be possible with such sensors. Hence, the authors have developed millimetric gold electrodes modified by a deposit of ferricytochrome c [67–70], a protein classically used for indirect spectrophotometric measurements of superoxide produced by living cells in culture since the reaction between the two compounds is very fast (k = 2 × 106 M −1 s −1 ): [47]

ferricytochrome c(Fe3+)+O2-ferrocytochrome c(Fe2+)+O2
13.14

Ferricytochrome c was covalently immobilized by a thiol derivative (DTSSP) linker interacting with the protein and the gold surface (SAM formation through Au–S bonds). This was decided to provide a better selectivity for O2•−, particularly against NO , than that offered by simple carbon or gold electrodes. A complete in vitro study with solutions of superoxide generated enzymatically by a system of xanthine/xanthine oxidase, demonstrated the interest of the concept based on the electro-oxidation of reduced cytochrom c , thus providing a catalytic effect on the response. Consequently, these modified microelectrodes offered a high sensitivity for O2•−, e.g., 2 pM /pA−1 [68, 69]. Each electrode was used in conjunction with a second electrode, a commercial NO sensor, for simultaneous measurements of O2•− and NO produced by neuronal cells in culture (different cell lines were analyzed).

The data presented in Figure 13.7 were obtained from simultaneous measurements with the two types of sensor over a layer of adherent glioblastoma cells from the A172 cell line [68]. Their oxidative response was stimulated by addition of PMA (a phorbol ester activating NADPH oxidases, as explained before) or (lipopolysaccharide- LPS α prepared from fragments of bacteria walls, known to activate NO synthases) to the buffer solution. Based on their in vitro tests, the authors assumed no possible interference between the two sensors, i.e., superoxide was not detected by the NO sensor, and the O2•− sensor was insensitive to NO . However, simultaneous measurements gave them the ability to observe the cross reaction between O2•− and NO in their experimental conditions. Following addition of the stimulus (LPS in Figure 13.7), a rapid increase of NO was detected, and even possibly amplified, under conditions of NO synthases pre-activation (inducible NOS induction by the cytokine interferon-γ), while superoxide was not detected.

FIGURE 13.7. Detection of O • −2 on a ferricytochrome c modified gold electrode and NO• on a commercial selective electrode at a sub-millimetric distance from a layer of 106 glioblastoma cells (of the A172 line).

FIGURE 13.7

Detection of O • −2 on a ferricytochrome c modified gold electrode and NO on a commercial selective electrode at a sub-millimetric distance from a layer of 106 glioblastoma cells (of the A172 line). The LPS stimulation was applied (more...)

Then, when the production of NO decreased, superoxide started to be observed showing that in the initial phase of the cell response O2•− was titrated by excess of NO to form ONOO. Consequently, these studies reported one of the first examples of direct and selective measurement of primary species of oxidative stress at living cells and the observation of reaction between these species at origin of RNS formation. Nevertheless, the applicability of the present analytical method and sensor to quantitative biological studies was restricted by the low collection efficiency of superoxide at sub-millimetric distance separating the electrode surface and the layer of cells. The spontaneous formation (without catalase involvement) of H2O2 in these conditions is not negligible, though it was not evaluated. Similarly, peroxynitrite formed by coupling between NO and O2•− could not be measured directly. Yet, ONOOmay re-oxidize ferricytochrome c and interfere with measurement, though this reaction is quite slow [71]. Consequently, these studies on cell colonies led to the conclusion that the detection of side-products of superoxide and nitric oxide would also be of great importance for characterizing, in real-time, the nature and origin of oxidative stress processes occurring in a cell.

Detection of Hydrogen Peroxide by Platinized Carbon Fiber Microelectrodes on Skin Fibroblasts

Among the different derivatives of superoxide anion, hydrogen peroxide is the most stable one and is, consequently, an essential marker of oxidative stress. Our group decided to focus on its detection through the development of microelectrodes, allowing its monitoring on single cells [72]. It was previously established that H2O2 oxidation is difficult on any type of carbon surfaces, while its oxidation on bare polished platinum surfaces induces the formation of platinum oxide and peroxide and, consequently, unstable electrochemical responses. Meanwhile, several groups working in the development of glucose sensors have shown that hydrogen peroxide produced by glucose oxidase could be easily and quantitatively detected by oxidation on black platinum or platinized electrode surfaces [73,74]. Black platinum was thus electro-deposited from a Pt(IV) solution following a 4e reduction into Pt(0) on the electrode surface:

PtCI62-+4ePt0+4CI-
13.15

A stable, irreversible, voltammetric wave (2e oxidation process, E 1/2 = + 0.25 V vs. SSCE) for H2O2 could be observed on our platinized carbon fiber microelectrodes (measuring 1–30 μm in diameter). Moreover, as the platinized surface was a rough deposit, the microelectrodes possessed a huge active area, which ensured a high sensitivity for H2O2 detection. The limit of sensitivity determined by amperometry at + 0.55 V vs. SSCE was a few nanomolars, the selectivity being provided by the electrode potential, as attested to by control experiments in the presence of added catalase or o -dianisidine.

Owing to their excellent analytical properties, these modified microelectrodes were applicable for monitoring oxidative stress on single human skin fibroblasts. These cells are the main constituents of skin dermis and, as a consequence, are the site of some of the oxidative damages induced by UV-A and -B from sunlight and other chemicals in contact with the skin. Under oxidative stress conditions, DNA alterations of fibroblasts might be amplified and cause the cell to shift toward a cancerous state [1,75,76].

We have shown for the first time, that fibroblasts are able to produce oxidative bursts under depolarisation of the cell membrane induced by a mechanical stress [72,77], the puncturing of the membrane with the tip of a microcapillary without inducing cell disruption or death. The shafts of the microelectrodes were insulated in order to detect electroactive species only at their very tip surface. Owing to their small size (10–15 μm in diameter), the tips could be positioned via a micromanipulator at a few tens of microns above the cell surface, i.e., in experimental conditions for which any loss of H2O2 through its reaction with other compounds should not occur. The variation of amperometric current detected immediately after the microcapillary puncture on the cell membrane corresponded to an active response of the cell and not to the passive release of electroactive species diffusing from the cytosol or from storage vesicles. Experiments in the presence of catalase, the natural scavenger of H2O2 , or in the presence of a substrate for peroxidases, o -dianisidine, showed that hydrogen peroxide was the major component of the cell response (see Figure 13.8c and Figure 13.8d). In later studies, inhibitors of NADPH oxidases confirmed that H2O2 detected by the microelectrodes had an enzymatic origin [77] and that the kinetic profile of the oxidative bursts measured on fibroblasts or lymphocytes was dependent on metabolic factors governing NADPH oxidases activity [77–79].

FIGURE 13.8. Detection on a platinized carbon fiber microelectrode (measuring 10–15 μm in diameter) of hydrogen peroxide release by a single human fibroblast in culture.

FIGURE 13.8

Detection on a platinized carbon fiber microelectrode (measuring 10–15 μm in diameter) of hydrogen peroxide release by a single human fibroblast in culture. (a) scheme and (b) view under microscope of the experimental setup. Typical responses (more...)

Nevertheless, several results led us to consider that oxidative bursts detected on fibroblasts or other cell types were not exclusively related to hydrogen peroxide production by cells. First, a residual signal was always observed in experiments with catalase, and then, the burst amplitude was increasing at amperometric potentials higher than the one of H2O2 plateau potential. The hypothesis of a simultaneous production of O2•− /H2O2 and NO has been examined. An exhaustive study of the cell response has allowed a full delineation of the origin and nature of ROS and RNS produced under these conditions and established the intimate relationships and coupling between NADPH oxidases and NO synthases activities during cellular oxidative stress processes. These results will be presented in the last part of this chapter.

Microelectrode Detection of Nitric Oxide and Peroxynitrite Releases by Living Cells

Detection of Nitric Oxide by Porphyrin Modified Microelectrodes at Single Cells from Cardiac Endothelium

Malinski’s group (Oakland University, U.S.A.) was one of the first to describe an electrochemical NO sensor and its application to cells measurements of nitric oxide release. In 1992 they reported the development of a carbon fiber microelectrode, which had a surface modified by the electrodeposit of a film of nickel porphyrin [80]. We wish not to detail the preparation and characterization of this sensor as this will be documented later, in chapter 21 [81]. Malinski et al. modified their electrodes to match the specific conditions of a single cell analysis on cells in culture or located on the surface of the endocardium (of a rabbit heart). The active tip of the electrochemical sensor was reduced to 2–5 μm in diameter instead of 50–60 μm, the diameter of the sensor previously used [82–84]. Secondly, the electrode was inserted into a catheter, so that the tip of the sensor was recessed 5– 6 μm inside of the catheter tip, and thus, was protected in the case of experiments performed directly in the tissue. Each electrode was tested and calibrated with pure NO solutions of concentration ranging from 5 × 10to 3 × 10−6 mol L −1 .

Experiments were performed on cells mechanically removed from the surface of the left heart ventricle of an anesthetized rabbit. Cells were plated on usual coverslips and maintained in buffer. The tip of the porphyrinic microelectrode was then positioned via a micromanipulator over the surface of a single endothelial cell; the zero distance was obtained when touching the cell membrane and henceforth a signal artifact was detected. Then, the electrode tip was placed at a 10 μm height. The cell was subsequently stimulated following the injection of a calcium ionophore solution (compound A23187, which induces transient opening of calcium channels, with a 8 μM final concentration) into its environment, leading to the instant activation of NO synthases (constitutive enzymes of endothelial type). The release of NO detected by amperometry started in the first second following the cell stimulation (see Figure 13.9a). The signal reached its maximum in about 1 s and returned to baseline after about 10 s. Its amplitude (in concentration of NO ) was highly dependent on the distance between the sensor tip and the cell surface. Logically, the maximum value, close to 1 μM , was obtained when placing the sensor at the membrane surface (see Figure 13.9b). This observation is consistent with the principles described above in the Direct Electrochemical Detection of Oxidative Release on Single Cells section of this chapter. The quantification of release from a single living cell is highly dependent on the collection efficiency of the electrode surface and of the volume of analysis defined by the distance between that sensor surface and the cell source of active species. Consequently, minimizing this distance ensures better conditions for analysis of the true nature of the biological information, though this may lead to an indirect blasting of the cell surface by the products resulting from the electrochemical detection.

FIGURE 13.9. (a) Release of nitric oxide detected by a porphyrin modified microelectrode placed 10 μm from the surface of an isolated endothelial cell (an endocardium cell of a rabbit) stimulated by a calcium ionophore (A23187) at t = 0 s.

FIGURE 13.9

(a) Release of nitric oxide detected by a porphyrin modified microelectrode placed 10 μm from the surface of an isolated endothelial cell (an endocardium cell of a rabbit) stimulated by a calcium ionophore (A23187) at t = 0 s. (b) Exponential (more...)

This first series of studies allowed comparing the response of endothelial cells obtained from the surface of the endocardium of normal or hypertensive rats. Nitric oxide release was lower in hypertensive rats (NO is the major vaso-relaxing compound) in agreement with former studies reporting a weaker relaxation of smooth muscle cells from these animals compared to the wild type ones. However, the authors have shown that this lower NO response was very sensitive to SOD, indicating that a large part of NO produced by cells was not detected by the electrode because it was consumed through its reaction with O2•− before diffusing to smooth muscle cells and inducing their relaxation. Consequently, these studies demonstrated that the formation of RNS occurred preferentially in the hypertensive rats, this event leading potentially to the pathophysiological consequences observed in this animal model, as well as in humans.

Detection of Peroxynitrite by Modified Microelectrodes during Ischemia of Endothelial Cells from Heart

The direct electrochemical detection on living cells of peroxynitrite anion release was first reported by two groups in 2000, each one using a different type of electrode and focusing on a different biological application. We will first emphasize studies reported by the L. T. Jin’s group (ECNU, Shanghai, China), while our work will be presented in the last part of this chapter. L. T. Jin et al. had previously reported the development of microelectrodes modified by metallophtalocyanines or Schiff bases as sensors for nitric oxide and superoxide [65,85–87]. In these studies they prepared cylindrical carbon fiber or platinum microelectrodes, which had a surface modified by the deposit of tetraaminophtalocyanine manganese(III) (Mn(TAPc)), as a catalyst for peroxynitrite reduction, and subsequently modified by a membrane of the catanionic polymer poly-vinylpyridine (PVP). Briefly, cylindrical carbon fiber microelectrodes (7 μm in diameter) were chemically etched to provide electrodes of 1 μm in diameter at the tip and measuring tens of microns long. Then, their surfaces were electrochemically treated (potential cycling into H2SO4), dried and modified by the electrodeposition of poly-Mn(TAPc), and conducted into a solution of 5 m M Mn(TAPc) by voltammetric scanning (100 mV s −1 ) of the potential between + 0.5 and + 1.0 V vs. Ag/AgCl. Prior to in vitro tests or cell experiments, the electrodes were modified by a layer of PVP, working as a cation repulsive barrier, as well as, as a diffusional barrier for large biological molecules, two characteristics that should significantly improve the selectivity of these microelectrodes for ONOO . PVP was simply deposited from a solution allowed to dry onto the electrode surface.

In vitro tests with pure peroxynitrite solutions have shown that this species was detectable by its reduction into nitrite and nitrogen dioxide electrocatalyzed by Mn(TAPc) at low potential ( + 0 V vs. Ag/AgCl). This potential value in conjunction with PVP layer properties afforded a large immunity to many interferents, including several ROS (O2•−, H2O2), RNS (NO , NO2), dioxygen or other molecules that may be present in cell cytosol (l-Arginine, NADPH, ascorbic acid, etc.). These modified microelectrodes displayed a limit of sensitivity of 18 n M ONOO when used in conjunction with differential pulse amperometry (DPA). The useful range was comprised between this lower limit up to a few hundreds of micromolars.

These electrodes were then used for intracellular measurements of ONOO produced into myocardial cells isolated from the heart ventricle of neonatal rats. Peroxynitrite is suspected to be a major component of cardiac injury in the situation of heart ischemia followed by reperfusion, though this hypothesis was previously inferred only the basis of indirect markers of a nitrosative stress (staining of nitro-tyrosine residues). The authors chose to insert the micrometric size tip of their microelectrodes into the beating heart cell. Despite the stress it may induce to the cell (see below), they did not observe any significant change of baseline level of peroxynitrite concentration. After continuous ischemia of the cell, the signal due a priori to peroxynitrite, progressively increased to reach a plateau after 15 min that stayed at the same level of concentration (70 n M ) for about 15 more minutes (see Figure 13.10). Afterwards, the signal decreased until reperfusion of the cell that induced an increase of peroxynitrite concentration higher than the initial ischemic step. Control experiments made with an inhibitor of NO synthase or SOD or melatonin, the most potent pharmaceutical inhibitor of ONOO in 2000, fully supported the conclusion that the variations of signal detected intracellularly by the modified microelectrodes were due to peroxynitrite. These results provided the first direct evidence that peroxynitrite was the actual mediator of cardiac injuries occurring during ischemia/reperfusion process.

FIGURE 13.10. Time-course of peroxynitrite detected by a Mn(TAPc)/PVP modified microelectrode inside a single beating heart cell in culture after its ischemia/reperfusion.

FIGURE 13.10

Time-course of peroxynitrite detected by a Mn(TAPc)/PVP modified microelectrode inside a single beating heart cell in culture after its ischemia/reperfusion.

Detection of Peroxynitrite by Platinized Microelectrodes on Single Human Fibroblasts

Beginning with the results reported in the Detection of Hydrogen Peroxide by Platinized Carbon Fiber Microelectrodes on Skin Fibroblasts section showing that platinized carbon fiber microelectrodes (10 μm in diameter) were adequate tools to measure the release of hydrogen peroxide bursts by single human skin fibroblasts, our group further demonstrated through the development of an original experimental protocol, that the oxidative bursts refiected the production and release of several important ROS and RNS, which could be identified and quantified [46,88,89]. In our second series of studies, the distance between the microelectrode tip and cell surface where it was depolarized, was varied and optimized to ensure the detection of species with short time-life that would not be able to diffuse to the electrode surface before decomposing under the conditions of Figure 13.8 ( d ≈ 50 μm). Furthermore, we have confirmed that under these new conditions, the collection efficiency of the cell response increased and was maximal (greater than 90%) when the distance was lower than 5 μm, indicating that species released by the cell in its environment were quantitatively detected [89]. Then, a large series of different experiments conducted at different amperometric oxidative potentials on about 1000 cells from the same wild type control strain, demonstrated that their response increased in amplitude with the potential and consequently, that this response included the measurement of several electroactive species (Figure 13.11a). These data enabled us to construct an equivalent steady-state voltammogram of the average cell oxidative burst contents by measuring the current versus the potential (one point at each 25 mV over the potential range between + 150 and + 900 mV vs. SSCE) (Figure 13.11b).

FIGURE 13.11. Determination by platinized carbon fibre microelectrodes of the species released by single human fibroblasts during oxidative burst responses induced by mechanical depolarization of the cell membrane.

FIGURE 13.11

Determination by platinized carbon fibre microelectrodes of the species released by single human fibroblasts during oxidative burst responses induced by mechanical depolarization of the cell membrane. (a) Amperometric measurement of the cell response (more...)

The voltammogram clearly evidenced three different plateaus corresponding to at least three oxidation waves (labeled I, II and III in Figure 13.11b). However, when spikes were normalized versus their maximum current, no difference of time-profile could be observed, indicating that the different species were released following the same overall kinetic. The slowly developing kinetic (rise-time close to 1 s and total time-length of about 90 s) could not correspond to the time-course of a passive release by diffusion of ROS and RNS initially located in the cell cytosol, but rather to the kinetic of biological production of these compounds or of their mother species (compare also with other studies reported above) stimulated by the cell membrane depolarization. The voltammogram constructed from cell experiments was then compared to the oxidative response in vitro of stable solutions of several ROS and RNS potentially derived from superoxide and nitric oxide [46,88,89]. It was determined that waves II and III correspond, in terms of their potential position and transfer kinetic to the detection of NO and NO2, respectively. Conversely, wave I did not correspond to the detection of a single species such as H2O2, which had a slightly lower oxidation half-wave potential (see Figure 13.11b), or ONOO , which had a slightly higher oxidation half-wave potential. An exhaustive study of peroxynitrite anion electrochemistry [46] on platinum and the platinized surface of electrodes enabled us to show that the oxidation wave I corresponded to the mixing of current of H2O2 and ONOO oxidation waves, both located in a near range of potentials. Finally, the arithmetic addition of the voltammetric response of four different species (H2O2, ONOO, NO , and NO2) adjusted in concentration was in perfect agreement with the voltammogram determined from the cell oxidative response (see the comparison between data points and the solid line figuring the predicted voltammogram in Figure 13.11b). This analysis has demonstrated that the oxidative bursts of fibroblasts corresponded to the emission of a cocktail of several ROS and RNS, among which the very reactive peroxynitrite was one, and that each species (though the presence of NO3 ions could not be detected due to their electro-inactivity in our experimental conditions) could be determined and selectively quantified by amperometric measurements at several potentials.

Further studies on cells treated with different pharmacological agents such as enzymatic inhibitors have defined that enzymatic systems of NADPH oxidase and NO synthase types were simultaneously activated following the cell stimulation by the microcapillary stress and were responsible for the production of the two primary species, O2•− and NO (Figure 13.12a) [88]. Obviously, these primary species cannot be detected directly at the microelectrode surface, given that when present in the same solution they react together to form ONOO, which cannot survive long since it also rapidly evolves into NO2 (this later being observed into the extracellular medium by the microelectrode). However, the two types of enzymatic systems are located at different cell sites (presumably cytosol versus membrane) allowing superoxide anion to disproportionate and form hydrogen peroxide before reacting with nitric oxide. These hypotheses in conjunction with the previous characterization of a maximal collection efficiency of the species released by the fibroblasts have enabled us to convert the amperometric currents detected at the plateaus potentials of waves I, II and III into fluxes of emission (Figure 13.12b) according to Faraday’s laws:

FIGURE 13.12. (a) Determination from amperometric curves in Figure 13.

FIGURE 13.12

(a) Determination from amperometric curves in Figure 13.11a of the flux of each species (H2O2, ONOO, NO 2 , and NO°) released by single human fibroblasts during oxidative burst responses. (b) Evaluation of the flux of the primary (more...)

Ij=njF×Φj
13.16

The conversion takes into account for each species (j ) the known number of electrons exchanged during its electrochemical oxidation at the electrode surface. This provides a better measurement than simple oxidation currents of the true exchanges between a single cell and its environment during an oxidative stress process (Figure 13.12b).

The determination of the individual fluxes of the different species released by the cells allowed us to reconstruct the fluxes of production of the primary species giving rise to these ROS and RNS. Indeed, because all species originate from O2•− and NO , fluxes of the latter could be evaluated according to the conservation of matter Equation 13.17 and Equation 13.18:

(ΦO2-)prod=2(ΦH2O2)mes+(ΦONOO-)mes+(ΦNO2-)mes
13.17
(ΦNO°)prod=(ΦNO°)mes+(ΦONO2-)mes+(ΦNO2-)mes
13.18

Each curve was subsequently integrated and provided a measurement of the total amount of each compound produced during the oxidative burst (values in parenthesis in Figure 13.12c and Figure 13.12d). For both primary species, the quantities are in the range of tens of femtomoles and may be considered as infinitely small from a macroscopic point of view and considering actual analytical standards. Nevertheless, when considering the “living space” of a single cell, equivalent at maximum to a few times its own dimensions, these amounts are not negligible in terms of the local concentrations (by way of the kinetic effects) they produce locally. They correspond to local high, or at least efficient concentrations, of messenger, activator, or toxic molecules.

The results presented here have led ultimately to several applications for biomedical purposes, such as, studies of the oxidative stress implication in HIV infection of blood lymphocytes [79,90], studies of the oxidative stress implication in the carcinogenesis of skin fibroblasts [78,91], or even to studies of primary biological mechanisms, such as, ROS and RNS production by macrophages during phagocytosis.

Conclusions and Perspectives

The studies described here demonstrate that the electrochemical measurements at microelectrodes are methods of high interest for studying biological processes implicating superoxide and/or nitric oxide and their derivatives. The advantages of the electrochemical analysis over other biophysical methods include the possible direct detection, the fast response-time and selectivity of the sensors, and, above all, a single cell analysis. Clearly, when the microelectrode surface can be positioned at close distance to the cell (or the biological source), i.e., in an artificial synapse configuration, the infinitely low amplitude of the biological information can even so be detected and studied.

Future developments of the electrochemical methodologies for studies of oxidative stress processes should focus on (1) the improvement of microelectrodes selectivity for each ROS and RNS of interest; (2) multiple analysis on several cells or of several species done in parallel by several electrodes eventually integrated into microsystems; (3) intracellular measurements owing to nanometer size electrodes; (4) detection of a single molecule activity such as the one of an enzymatic system generating O2•− or NO , possibly located into the cell cytosol or the cell membrane.

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