Matthew Whiteman Yuktee Dogra Paul G Winyard and Jeffrey S Armstrong


Reactive oxygen intermediates (ROIs) play a key role in a number of human diseases either by inducing cell death, cellular proliferation, or by acting as mediators in cellular signaling. Therefore, their measurement in vivo and in cell culture is desirable but technically difficult and often troublesome. To address some of the key methodological issues in examining the formation of ROI in cells and mitochondria, this chapter discusses the following: (a) the cellular sources of ROI and their enzymatic removal, (b) common methods used to determine cellular and mitochondrial ROI such as chemiluminescence, electron paramagnetic resonance spectroscopy, fluorescence, and enzymatic techniques, and (c) some common problems associated with these assays and the interpretation of data. We also provide some simple protocols for the estimation of ROI production in cells and mitochondria, and when measuring ROI in cells and mitochondria, we emphasize the need for thorough understanding of results obtained and their interpretation.

Keywords: Assay, chemiluminescence, cytochrome c , electron paramagnetic resonance spectros-

copy, fluorescence, mitochondria, reactive oxygen intermediates.

1. Introduction

Reactive oxygen intermediates (ROIs), including superoxide anion (O2'-), hydroxyl radicals (* OH), and hydrogen peroxide (H2O2), are generated during aerobic metabolism. The mitochondrion is the most common source of ROI production, although there are other important cellular sources, including enzymes such as cytochrome P450 in the endoplasmic reticulum, lipoxygenases, cyclooxygenases, xanthine oxidase, and the

John T. Hancock (ed.), Methods in Molecular Biology, Redox-Mediated Signal Transduction, vol. 476 © 2008 Humana Press, a part of Springer Science + Business Media, Totowa, NJ DOI: 10.1007/978-1-59745-129-1_3

nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) oxidase of phagocytic cells. Approximately 1-3% of the electrons carried by the mitochondrial electron transport chain (ETC) under normal physiological conditions leak out of the pathway and pass directly to oxygen, generating O2'- (1). Mitochondrial NADH-ubiquinone oxidoreductase (respiratory complex I) is an important source of production especially in the reverse transport mode (2), whereas ubiquinol-cytochrome c oxidoreductase or cytochrome bc1 (respiratory complex III) and the coenzyme Q radical generated during the Q cycle is likely to be the most important site of mitochondrial O2'- production (1, 3, 4). High concentrations of the enzyme manganese superoxide dismutase (MnSOD) in the mitochondrial matrix ensure that basal levels of O2*- formed during normal electron transport are kept at a bare minimum to limit oxidative damage to mitochondrial matrix proteins involved in the regulation of metabolism, such as the iron-sulfur proteins aconitase and succinate dehydrogenase (1, 5). MnSOD catalyses the dismutation of O2'- to yield hydrogen peroxide (H2O2), which is an important signaling molecule generated throughout the animal and plant kingdoms. Under physiological conditions, H2O2 generated by the dismutation of mitochondrial O2*- is reduced by a glutathione (GSH)-dependent peroxidase enzyme (GSH/Px) to yield water whereas in the cytosol, H2O2, generated by peroxisomal P-oxidation of fatty acids, or by the enzyme xanthine oxidase (XO), is decomposed by catalase and GSH/Px, respectively.

However, when H2 O2 is produced in excess it is also a toxic molecule that can react with ferrous iron (Fe2+) to form *OH (either via the Fenton reaction, the metal catalyzed Haber-Weiss reaction or with the neutrophil oxidant hypochlorous acid) (1), which is a short-lived species that can react with molecules such as deoxyribonucleic acid (DNA; see Fig. 3.1. for a schematic representation of cellular and mitochondrial "ROI" production pathways). In the mitochondrion, the cooperative action of the enzymes such as MnSOD and the GSH/GSHPx system ensure that the ROIs (O2*- and H2O2) generated during normal aerobic metabolism are kept to nontoxic levels. Although cells possess extensive antioxidant defence systems (extensively reviewed in ref. 1) to combat ROI tox-icity, including catalase, SODs, and the GSH and GSH/GSHPx systems, when ROIs overwhelm cellular antioxidant defense systems and redox homeostasis is lost, the result is "oxidative stress," which can lead to cell damage and death.

An overwhelming body of evidence has accumulated that demonstrates that oxidative stress is a contributing factor in the pathogenesis or clinical progression of a wide range of human diseases, such as neurodegenerative disorders (i.e., Huntington's, Parkinson's disease, and Alzheimer 's disease), retinal degenerative disorders, AIDS, cancer, chronic inflammatory conditions

Fig. 3.1. Cellular and mitochondrial sources of ROS production and breakdown. The mitochondrial electron transport chain (ETC), NADPH oxidases, and the xanthine/xanthine oxidase (X/XO) systems are shown as important sources of O2*-. Superoxide dis-mutases (SODs) enzymatically convert O2*- to H2O2, which is broken down to H2O by catalases and the GSH/GSHPx systems.

Fig. 3.1. Cellular and mitochondrial sources of ROS production and breakdown. The mitochondrial electron transport chain (ETC), NADPH oxidases, and the xanthine/xanthine oxidase (X/XO) systems are shown as important sources of O2*-. Superoxide dis-mutases (SODs) enzymatically convert O2*- to H2O2, which is broken down to H2O by catalases and the GSH/GSHPx systems.

such as arthritis, colitis as well as the ageing process in general. Therefore, the ability to make qualitative and quantitative measurements of ROI formation by cells is of particular relevance in addressing disease aetiology, progression, and pathology.

2. Methods

2.1. Spectrophotometry Methods for the Estimation of ROI

The spectrophotometric methods outlined herein are often the simplest, most widely used, and well-accepted techniques for measurement of extracellular O2'- production by isolated enzymes, cell homogenates, and isolated phagocytic cells. In general, O2'- is measured as the SOD-sensitive reduction of a substrate as a result of the nonspecificity of many methods, although this technique does not always guarantee specificity for or.

2.1.1. Nitro Blue Tetrazolium

Nitro blue tetrazolium (NBT) is a nitro-substituted aromatic compound that can be reduced by O2*- to the monoformazan, whose formation can be readily monitored by standard bench top spectrophotometers at 550-560 nm. The reaction is a two-step process that proceeds via the formation of an intermediate NBT radical, which then undergoes either further reduction or dismutation to the monoformazan (6, 7). NBT detects intracellular O2*-; however, it is less sensitive and specific for O2*- than fluorimetric assays, such as those that use dihydroethidium. The NBT radical intermediate can also react with molecular oxygen under aerobic conditions and, in doing so, can generate artifac-tual O2*- that further reduces NBT (8). Importantly, because the formation of monoformazan is SOD-inhibitable, it illustrates that this technique does not unequivocally confirm that SOD-inhibitable NBT reduction is caused by O2*- because NBT is also susceptible to reduction by several tissue reductases and has been used to detect cellular enzymes, most notably nitric oxide synthase (9). For these reasons, the detection of O2*- in biological samples should not exclusively rely on NBT reduction but also should be supplemented with an additional and independent measure of O

2.1.2. Cytochrome c Reduction

Cytochrome c reduction is a widely used and well-accepted technique for measurement of O2*- production by isolated enzymes, cell homogenates, and activated phagocytes. Generally, O2*- is measured as the SOD-inhibitable reduction of cytochrome c, determined in a simple bench top spectrophotometer by the increase in absorbance at 550 nm. Because the cytochrome c is cell impermeable, it can be used only to measure extracellular O2*-. There are several precautions when one uses this reaction to detect O2*-. First, cytochrome c reduction is also nonspecific for O2*- and compounds such as ascorbate and glutathione, as well as cellular reductases catalyse cytochrome c reduction. In addition, cytochrome c can be "reoxidized" by cytochrome oxidase (COX), peroxidases, and a variety of oxidants (including H2O2 and ONOO-) and, as such, underestimates the true rate of O2*- production (10). Cyanide (CN-) can be added to the reaction mixture to inhibit COX activity, and ROI scavengers such as catalase will block oxidation by H2O2 and the addition of urate will prevent oxidation by ONOO-. Alternatively, specificity for O2*- may be improved by either measuring SOD-inhibita-ble cytochrome c reduction or using acetylated or succinoylated forms of cytochrome c which minimise artifactual reduction and oxidation without affecting the ability of O2*- to reduce cytochrome c (11, 12).

2.1.3. Aconitase

Aconitase is a citric acid cycle enzyme belonging to the family of dehydrogenases containing iron sulfur (4Fe-4S) centers that catalyse the conversion of citrate to isocitrate. The mitochondrial and the cytosolic forms of aconitase are inactivated by O2*_, and its activity has been proposed to reflect intracellular levels of O2*_ production and with low levels of enzyme activity reflecting high levels of O2*_ (13). Inactivation of the enzyme occurs because of the oxidation of the enzyme and the subsequent loss of Fe from the (4Fe-S) cluster. Unfortunately, other ROIs, 'NO, and ONOO- and hypochlorous acid (HOCl) have been shown to inactivate aconitase. As such, the assay is not generally considered specific for O2*_ (14). A serious disadvantage to this method is that because inactivation of the enzyme occurs over several hours or days, it is impossible to intervene with specific scavengers to restore its activity.

2.2. Fluorescence Dye Techniques for the Determination of Cellular ROI

2.2.1. Protocol 1: The Determination of General 'ROI' Formation Using DCFDA and Fluorescence Activated Cell Soiling Analysis Materials

Dichlorofluorescin diacetate (DCFH-DA) is taken up by cells and is hydrolyzed to 2' ,7'-dichlorofluorescin (DCFH), which is then trapped inside cells. Intracellular DCFH, a nonfluores-cent fluorescein analogue, is oxidized by ' ROI' to highly fluorescent 2',7'-dichlorofluorescein (DCF). DCFH-DA has been quantitatively used to detect ROI produced by PMA-activated phagocytes as well as in cultured cells. Because DCFH is also believed to be oxidized by H2 O2, or H2 O2-generating systems (i.e., glucose oxidase and glucose) and is inhibited by catalase but not by SOD, it was thought that intracellular DCFH oxidation was mediated by H' O2 (15) and, initially, that the oxidation of DCFH to the fluorescent compound DCF was relatively specific for H2 O2 formation. However, recent studies are emerging to show that this is not the case and that DCF yields fluorescence in response to a variety of ROIs (including H2O2 and other peroxides, ONOO-, see ref. 16, and hypochlorous acid [HOCl], see ref. 17, after intracellular oxidation mediated by GSH depletion (16, 18-20) and, most importantly, independently of a functional ETC (21), indicating that the ROIs generated under these conditions are not mitochondrial in origin. As a result, specific scavengers of 'ROI' must be used with due caution to understand the source of the DCF fluorescence signal.

1. DCFH-DA (Molecular Probes, Eugene, OR).

2. Phosphate-buffered saline (PBS), pH 7.4 in 10 mMglucose.

3. Fluorescence-activated cell sorter (FACS) machine or fluorescence plate reader (excitation wavelength 498 nm and emission wavelength 522 nm.

4. Cell culture media (serum free).

1. Culture human HL60 cells in RPMI media with 10% fetal calf serum (FCS) and supplements. Cells are maintained in log-phase and kept at a concentration between 2.5 and 5 x

2.2.2. Protocol 2: The Determination ofGeneral 'ROI' Formation With DCFDA and Fluorescence Microscopy: Induction by the Neutrophil Oxidant, Hypochlorous Acid Materials

105/mL. DCFH-DA is cell permeable because of the diac-etate ester. Upon entry into cells, cellular esterases cleave the diacetate to yield the cell-impermeable compound DCFH. Because serum supplements may contain esterases, it is imperative to load cells in serum-free media and centrifuge them to remove culture media before then washing them twice by centrifugation in PBS at 100^.

2. Suspend cells in PBS by light vortexing and load with 5-10 p.M DCFDA, incubating them for 15-20 min at 37°C.

3. Wash cells 2x in PBS (centrifuge for 1 min @ 100^ and suspend them in PBS containing 10 mM glucose).

4. Because we are performing a viable cell assay, immediate FACS analysis is required, with the use of appropriate software for analysis.

5. The FACS setting should be FL-2 (FITC) with log mode after cell debris have been electronically gated out.

1. DCFH-DA (Molecular Probes).

3. Fluorescence microscope with fluorescein filters (i.e., capable of excitation wavelength 498 nm and emission wavelength 522 nm).

4. Cell culture media (serum free).

5. Sodium hypochlorite solution (Sigma).

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