Measurement of Cellular Oxidation, Reactive Oxygen Species, and Antioxidant Enzymes during Apoptosis

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[38] M e a s u r e m e n t of Cellular Oxidation, Reactive Oxygen Species, and Antioxidant Enzymes during Apoptosis

By LISA

M . ELLERBY a n d D A L E E . BREDESEN

Introduction Programmed cell death is a form of cellular suicide that involves a series of characteristic morphological changes, 1 and is deliberately invoked by the cell to ensure that unnecessary or harmful cells are eliminated during development or cellular assault. These morphological changes include chromatin condensation, oligonucleosomal D N A fragmentation, plasma membrane blebbing, and cell shrinkage. Along with morphological changes during apoptosis, a number of biochemical changes occur such as alterations in reactive oxygen species ( R O S ) , 2-4 a decrease in oxygen consumption, 5 loss of normal cytochrome c function, 5'6 and changes in the cellular oxidation-reduction s t a t u s . 7-9 The exact role ROS, antioxidant enzymes, and cellular redox changes play in signaling events in apoptosis is complex, depending on both cell type and apoptotic stimuli. Some experimental work indicates ROS are important regulators of apoptosis in upstream signaling pathways, while other studies clearly indicate reactive oxygen species are generated in downstream events after the activation of caspases and the release of cytochrome c. In Fig. 1, we present a model that can account for three phases of apoptosis in which ROS and cellular redox changes may occur and we note possible feedback loops through the mitochondrial permeability transition, which is known to be modulated by reactive oxygen species as well as numerous antioxidant enzymes. ROS and cellular redox changes can have profound effects on numerous 1 j. F. R. Kerr, A. H. Wyllie, and A. R. Currie, Br. J. Cancer 26, 239 (1972). 2 H. Albrecht, J. Tschopp, and C. V. Jongeneel, FEBS Lett. 351, 45 (1994). 3 D. J. Kane, T. A. Sarafian, R. Anton, H. Hahn, E. B. Gralla, J. S. Valentine, T. Ord, and D. E. Bredesen, Science 262, 1274 (1993). 4 S. Tan, Y. Sagara, Y. Liu, P. Maher, and D. Schubert, J. Cell Biol. 141, 1423 (1998). 5 A. Krippner, A. Matsuno-Yagi, R. A. Gottlieb, and B. M. Babior, J. Biol. Chem. 271, 21629 (1996). 6 j. Yang, X. Liu, K. Bhalla, C. N. Kim, A. M. Ibrado, J. Cai, T.-I. Peng, D. P. Jones, and X. Wang, Science 275, 1129 (1997). 7 G. B. Pierce, R. E. Parchment, and A. L. Lewellyn, Differentiation 46, 181 (1991). s L. M. Ellerby, H. M. Ellerby, S. M. Park, A. L. HoUeran, A. N. Murphy, G. Fiskum, D. J. Kane, M. P. Testa, C. Kayalar, and D. E. Bredesen, J. Neurochem. 67, 1259 (1996). 9 j. Cai and D. P. Jones, J. Biol. Chem. 273, 11401 (1998).

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FIG. 1. Reactive oxygen species (ROS) are generated at various points in the cell death process. Amplification of ROS can occur at various stages through cell-cell mediated ROS generation, mitochondrial generation, and destruction of critical organeUes in the cell.

signaling pathways, and evidence indicates that these alterations are involved in all three phases or steps of apoptosis--initiation, decision phase, and effector/destruction phase. For example, activation of redox sensitive kinases, transcription factors, depletion of glutathione (GSH), and redoxsensitive enzymes such as sphingomyelinase (Smase) all take place during the initiation phase or effector/decision phases of apoptosis. 1°-13 Evidence for this kind of regulation in apoptosis is as follows: (1) Apoptosis signalregulating kinase 1 (ASK1) is activated (dimerized) by hydrogen peroxide and inhibited by antioxidants; (2) redox-regulated transcription factor p53 induces apoptosis with the expression of a variety of proteins involved in the generation of ROS11; (3) generation of reactive oxygen species and changes in cellular redox state have been proposed to be critical events in 10y. Gotoh and J. A. Cooper, J. BioL Chem. 273, 17477 (1998). 11 K. Polyak, Y. Xla, J. L. Zweler, K. W. Kinzler, and B. Vogelstein, Nature ( L o n d o n ) 389, 300 (1997). 12L. Ghibelli, C. Fanelli, G. Rotilio, E. Lafavia, S. Coppola, C. Colussi, P. Civitareale, and M. R. Ciriolo, F A S E B J. 12, 479 (1998). 13 B. Liu, N. Andrieu-Abadie, T. Levade, P. Zhang, L. M. Obeid, and Y. A. Hannun, J. Biol. " Chem. 273, 11313 (1998).

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tumor necrosis factor a (TNF-a)-induced death14; and (4) depletion of GSH has been shown to precede the onset of apoptotic cell death in a number of apoptotic paradigms. 12 Interestingly, GSH has been shown to inhibit Smase activity directly. Smase catalyzes the hydrolysis of sphingomyelin to ceramide) 5 Other stimuli such as growth factor withdrawal, 16 human immunodeficiency virus infection,~7and ceramide TM result in the production of reactive oxygen species from the mitochondria, and it is likely that these events will correlate with the release of mitochondrial apoptotic factors such as cytochrome c and apoptosis-inducing factor (AIF). 6'~9 Release of cytochrome c during apoptosis results in a change in normal mitochondrial transport, which features a low level (1-5%) of superoxide production (approximately 1-5% of dioxygen consumption), to a state in which electrons are increasingly transferred to dioxygen, with a resulting increase in production of superoxide. Apoptosis during the destruction phase can also result in global changes in both glutathione and pyridine redox status. Oxidation of the cellular glutathione pool and the depletion of NADH and release of NAD ÷ may be common redox alterations necessary for the completion of the apoptotic pathway. Under normal conditions, pyridine nucleotides inside the mitochondria are more reduced than in the cytosol. Only a few percent of the total cellular NADH concentration is located in the cytosol of a cell. The NADH/NAD ÷ ratio in the heart cytosol has a standard redox potential of -226 mV. Substantial effects on cellular activity would result from the release of NAD ÷ into the cytosol because of the particularly high levels of pyridine nucleotides in the mitochondria when compared with the cytosol. Superoxide production resulting from the release of cytochrome c from the mitochondria, conditions that provoke the mitochondrial permeability transition (MPT), or damage to mitochondria may contribute to release of NAD ÷. Because of its uncoupling effect on the respiratory chain, MPT causes an immediate depletion of NADH and NADPH. This change, along with oxidation of the cellular GSH/GSSG pool, affects many different enzymatic reactions, as well as gene expression.

14 K. Schulze-Osthoff, A. C. Bakker, B. Vanhaesebroeck, R. Beyaert, W. A. Jacob, and W. Fiers, J. Biol. Chem. 267, 5317 (1992). 15 I. Singh, K. Pahan, M. Khan, and A. K. Singh, J. Biol. Chem. 273, 20354 (1998). 16 L. J. S. Greenlund, T. L. Deckwerth, and E. M. Johnson, Neuron 14, 303 (1995). 17 G. H. W. Wong, T. McHugh, R. Weber, and D. V. Goeddel, Proc. Natl. Acad. Sci. U.S.A. 88, 4372 (1991). 18 C. Garcia-Ruiz, A. Colell, M. Mari, A. Morales, and J. C. Fernandez-Checa, J. Biol. Chem. 272, 11369 (1997). 19 S. m. Susin, N. Zamzami, M. Castedo, T. Hirsch, P. Marchetti, A. Macho, E. Daugas, M. Geuskens, and G. Kroemer, J. Exp. Med. 184, 1331 (1996).

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Either of these events could contribute to the destruction phase in which the dying cells are broken down. In this chapter, we describe methods to measure reactive oxygen species and cellular redox changes during apoptosis. These parameters are clearly affected during apoptosis, and in many cases appear to play central roles in the apoptotic process. Assessment of Levels of Free Radicals Free radicals generated in cultured cells can be quantified by several approaches. We have measured the level of free radicals in apoptotic cells by monitoring the oxidatively sensitive compound 2,7-dichlorofluorescin diacetate (DCFH-DA; Molecular Probes, Eugene, OR). 3 D C F H - D A enters cells, where it is cleaved by esterases to produce DCFH, which is convened to the fluorescent product 2,7-dichlorofluorescein (DCF) derivatives by peroxides generated in the cultured cells. Cells are plated at a density of 105/well in 96-well tissue culture plates that have been coated with polyL-lysine. Cells are washed three times with Hanks' balanced saline solution (HBSS) and then with HBSS buffer containing D C F H - D A (1/zg/ml). Each well contains a 100-/zl total volume. Plates are read on a fluorescence plate reader (i.e., plate format fluorimeter), with an excitation wavelength of 485 nm and an emission wavelength of 530 nm, at 15-min intervals for 90 min at room temperature. General Considerations for Processing Cultured Cells Undergoing Apoptosis There are a number of important factors that should be considered when collecting mammalian cells undergoing apoptotic cell death for biochemical analysis. For example, it is critical when determining various biochemical parameters that the conditions for apoptotic induction result in a substantial population of apoptotic cells and that the process of collecting these cells result in minimal losses of the apoptotic cells. Another important consideration is that the initial conditions for growth of the mammalian cells to be utilized in biochemical assays during cell death should start out under conditions in which >98% of the cells are not apoptotic. This can be determined by staining the cells with trypan blue, acridine orange, annexin V, or other stains. A third consideration is the confluence of the cells. For typical experiments with adherent cells, cells should be plated on 100-mm culture plates at 15-20% confluence (with equal numbers of cells), and then allowed to grow to 50-70% confluence. Experiments should be carried out under similar conditions, and growth of cells to high confluency (>95%)

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for biochemical measurements is not recommended because of alterations in serum levels and other important biochemical parameters. To induce cell death, conditions should be utilized that maximize the percentage of apoptotic cells relative to nonapoptotic cells. Collection of ceils is described below.

Protein Determination Protein concentrations are determined by the Coomassie assay (Pierce, Rockville, MD) or another similar assay. Bovine serum albumin (BSA) may be used as a protein standard.

Enzymatic Assays For determination of enzymatic activities, monolayers of cells on 100-mm cell culture plates are carefully scraped into medium with a sterile disposable cell lifter, and spun at 1200g in Eppendorf tubes for 10 min at 4°. Cells washed once with phosphate-buffered saline (PBS) are resuspended in 400/zl of phosphate buffer [50 mM sodium phosphate, 0.5% (v/v) Triton X-100, pH 7.5] and sonicated for two 15-s bursts. Sonicates are spun for 10 min at 15,000g and protein concentration determination of the resulting supernatants should be carried out immediately. Enzymatic assays can be performed immediately or after storage at -70 °. Catalase (HEO2:H202 oxidoreductase; EC 1.11.1.6) activity of the extracts (20/zl, 5-10 mg/ml) can be measured by monitoring the disappearance of hydrogen peroxide at 240 nm. 2° A stock hydrogen peroxide solution should be prepared: 25 ml of 0.05 M potassium phosphate buffer, pH 7.0, with 85 /zl of 30% (v/v) H202. Assay solution should be prepared in a cuvette by mixing 0.6 ml of 0.05 M phosphate, pH 7.0, with 0.3 ml of stock hydrogen peroxide solution. The absorbance of the hydrogen peroxide assay solution should be ~0.29 at 240 nm. Crude extract is added to assay solution and the change in absorbance as a function of time is measured for I min. The specific activity can be calculated from the following equation: k = 2.3/60 sec (log Ainitial/Afmal); units = k/6.9 × 10-3/mg of protein in crude extract. Superoxide dismutase (superoxide:superoxide oxidoreductase; EC 1.15.1.1) activity (10/.d, crude extract diluted to 0.1 to 0.2 mg/ml) can be measured by monitoring the autooxidation of 6-hydroxydopamine ac20H. Luck, "Methodsof EnzymaticAnalysis"(H. V. Bergmeyer,ed.), pp. 885-892. Verlag Chemie Press, Weinheim,1965.

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cording to Heikkila 21 and Heikkila and Felicitas. 22 Superoxide dismutase (SOD) will inhibit the autooxidation of 6-hydroxydopamine by consuming superoxide generated during this process. This enzymatic assay is particularly useful in measuring SOD activity in crude extracts (SOD assays based on cytochrome c are not specific for SOD and, therefore, although useful for assaying SOD activity in relatively pure preparations of SOD, are less useful for measuring SOD activity in crude lysates). 6-Hydroxydopamine (hydrobromide, MW 250 g/mol; Sigma, St. Louis, MO) solution should be prepared and used immediately. To prepare the solution, argon purge 10 ml of double-distilled H20 (ddH20) with 50/zl of concentrated perchloric acid (HCIO4) for 15 min. Add 25 mg of 6-hydroxydopamine (0.01 M) to the argon-purged acid solution and store the stock on ice in a foil-wrapped tube. Assays of cellular extracts should be carried out at 37 ° in 0.05 M sodium phosphate, 0.1 mM diethylenetriaminepentaacetic acid (DETAPAC; Sigma), pH 7.4. The kinetics of the autooxidation of 6-hydroxydopamine should be monitored at 490 nm (0-0.5 min) under conditions that result in linear kinetics. Typically, 15 /zl of stock 6-hydroxydopamine is added to 1 ml of 0.05 M sodium phosphate, 0.01 mM D E T A P A C (pH 7.4) with the absorbance range set at 0-0.2 absorbance units. Record the autooxidation of 6-hydroxydopamine at 490 nm in triplicate, and calculate the slopes. Assay crude extract (-10/zl, crude extract diluted to 0.1 to 0.2 mg/ml) under conditions that result in 50% inhibition of the autooxidation of 6-hydroxydopamine (repeat three to five times). Data can be presented as micrograms of protein resulting in 50% inhibition or a standard curve can be generated with purified superoxide dismutase. The glutathione reductase (NAD[P]H:oxidized-glutathione oxidoreductase; EC 1.6.4.2) activity of crude extracts (20/zl, 5-10 mg/ml) may be assayed spectrophotometrically by following N A D P H oxidation at 340 nm at 25o.23 The assay buffer contains the following: 1 ml of 0.05 M Tris, 1 mM E D T A (pH 8.0), 77/~l of GSSG (50-mg/ml stock GSSG in ddH20), 19/xl of N A D P H (NADPH at 10 mg/ml in 0.5% NaHCO3). Report data as glutathione reductase units per milligram of protein. Glutathione peroxidase (glutathione : hydrogen-peroxide oxidoreductase; EC 1.11.1.9) activity of crude extracts (20/zl, 5-10 mg/ml) may be determined with a coupled assay in which the rate of tert-butyl hydroperoxide-dependent N A D P H oxidation at 340 nm is monitored according to 21R. E. Heikkila, Autoxidation of 6-hydroxydopamine.In "CRC Handbookof Methods for Oxygen Radical Research" (R. A. Greenwald, ed.), pp. 233-235. CRC Press, Boca Raton, Florida, 1985. 22R. E. Heikkila and C. Felicitas, Anal. Biochem. 75, 356 (1976). 23j. W. Anderson, C. H. Foyer, and D. A. Walker, Planta 158, 442 (1983).

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Gunzler and Flohe. 24 Assays are carried out as follows: 0.96 ml of 0.05 M potassium phosphate, 1 mM E D T A (pH 7.0), 10/zl of GSH (100 mM), 10 tzl of yeast glutathione reductase (0.01 U/ml), and 10 tzl of N A D P H (15 mM in 0.1% NaHCO3) are mixed together in a cuvette. The hydroperoxide-independent N A D P H consumption rate at 340 nm is recorded for 3 min at 37 °. Then, 10/xl of tert-butyl hydroperoxide is added to the reaction, mixed, and the overall rate at 340 nm is recorded. The same procedure is repeated with crude extracts added to the reaction mixture. This process assesses the nonenzymatic rate of oxidation of GSH, i.e., by factors that consume either hydroperoxide or N A D P H other than glutathione peroxidase, and thus can be subtracted from the total rate. Reduced Glutathione and Protein Thiols For determination of glutathione, monolayers of mammalian cells on 100-mm plates are collected as described above. After removal of residual liquid, 300/zl of 5.5% (w/v) sulfosalicyclic acid is added directly onto the cells. The precipitated protein and acid extracts are transferred to an Eppendorf tube, briefly vortexed, and centrifuged at 15,000g for 2-5 min. The acid-soluble fraction is used to measure glutathione, while the protein pellet is used for protein thiol analysis and determination of protein concentration. Total glutathione is measured according to the method of Tietze. 25 First, a standard curve is generated by measuring various concentrations of GSSG in solution (1 mM in 0.5% NaHCO3, standard curve 0.05 to 10 nmol), utilizing the following recycling assay. In a disposable cuvette the following are mixed: 1.0 ml of 0.1 M potassium phosphate (pH 7.0), 50 tzl of N A D P H (6 mg of N A D P H in 1.5 ml of 0.5% NaHCO3), 20/zl of 5,5'-dithiobis-(2nitrobenzoic and dithiobis-(2-nitrobenzoic acid) (DTNB) (1.5 mg/ml of 0.5% NaHCO3), and sample or standard. Then, 20/zl of glutathione reductase (7 units) is mixed into the reaction mixture and the absorbance change at 412 nm is monitored for 2 min at 30°. After generation of a standard curve (slope versus GSH concentration), the concentration of total GSH can be determined. Oxidized glutathione (GSSG) is measured by the method of Griffith, z6 in which 2-vinylpyridine is used to derivatize the reduced form of glutathione. To derivatize the GSH, 100/zl of the sulfosalicyclic-extracted sample is incubated with triethanolamine (6/zl; mix well) and then with vinylpyridine (2/zl). Vortex for 1 min. The pH of the solution should be 24 W. A. Gunzler and L. Flohe, in "Handbook of Methods for Oxygen Radical Research" (R. A. Greenwald, ed.). CRC Press, Boca Raton, Florida, 1985. 25 F. Tietze, A n a l Biochem 27, 502 (1969). 26 O. W. Griffith, A n a l Biochem. 106, 207 (1980).

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verified (pH --6.0 to 7.0) and the reaction carried out at room temperature for 1 hr. Assay the sample as described with 30-50 /.d of sample, and prepare a standard curve with vinylpyridine-treated standards. Protein thiol samples are prepared by resuspending acid-precipitated protein pellets into 0.1 M Tris, 0.5% (w/v) sodium dodecyl sulfate (SDS), pH 7.0. Protein thiols are measured with DTNB as described. 27 DTNB solution is prepared as follows: DTNB (1.7 mg/ml) in 0.2 M Tris, 0.02 M EDTA, pH 8.0. A thiol standard curve is generated against known amounts of GSH solution (1 mg/ml) by measuring the DTNB reaction at 412 nm. The protein concentration of samples is measured with Coomassie reagent from Pierce. Measurement of Oxidized and Reduced Pyl-idine Nucleotide Levels and ATP Levels Pyridine nucleotides and ATP levels can be measured simultaneously by reversed-phase high-performance liquid chromatography (HPLC) as described by Stocchi e t al. 28 on an HPLC Supelcosil LC-18 column. Samples for HPLC analysis are prepared by rapid alkaline extraction. Prior to lysis, monolayers of cells on 100-mm plates are washed with ice-cold Hanks' buffer. The cells are then rapidly scraped and suspended in 300/zl of Hanks' buffer and i00/A of 1 M KOH. The sample is immediately deproteinized by vortexing. After 3 min on ice, 400/~l of ice-cold water is added to the sample, and the solution is spun in a CF 50A Amicon (Danvers, MA) membrane and centrifuged at 1400g for 10 min. The pH of the solution is immediately adjusted to pH 6.5 with 1 M KH2PO4. The CF 50A cones should be prepared before collecting samples as follows: Soak the cones in 0.1 N N a O H for i hr, then rinse thoroughly with distilled water, and set up in centrifuge tubes. The samples may be analyzed immediately by HPLC or stored at - 7 0 ° for later analysis. To verify proper sample handling, known amounts of ATP or other nucleotides should be included in the processing procedure to verify good recovery and stability of nucleotides. HPLC is carried out under two different conditions. To measure ATP, ADP, NAD ÷, NADH, NADPH, and NADP ÷, the mobile phase should be prepared with two eluants: 0.1 M KH2PO4, pH 6.0 (buffer A) and 0.1 M KH2PO4, pH 6.0 with 15% (v/v) methanol (buffer B). The conditions for resolution of the nucleotides and nucleosides are the following: 9 min at 100% buffer A, 6-min ramp to 25% buffer B, 2.5-min ramp to 90% buffer 27j. Sedlak and R. H. Lindsay,Anal. Biochem. 25, 192 (1968). 28V. Stocchi, L. Cucchiarini, M. Magnani, L. Chiarantini, P. Palma, and G. Crescentini,Anal. Biochem. 146, 118 (1985).

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B, 2.0-min ramp to 100% buffer B, then hold for 6 min. The flow rate is 1.3 ml/min and detection is carried out at 254 nm. Quantification should be carried out with injection of standard solutions. To measure N A D P H and NADH, 50/.d of sample is injected and separated with mobile phase [0.1 M KH2PO4 (pH 6.0), 10% (v/v) methanol] and monitored at 340 nm. Quantification can be carded out by injection of known amounts of standard solutions. Conclusion We have utilized a number of the methods described here to demonstrate that the cellular oxidation-reduction potential is shifted toward more reducing in association with the expression of bcl-2 in the neural cell lines GT1-7 and PC12. 8 This reductive shift includes an increase in the GSH/ GSSG ratio, which is the major determinant of the cellular thiol redox status; an enhancement of protein free thiol groups, which is a reflection of the increased GSH/GSSG ratio; and an increase in the ratio of reduced to oxidized pyridine nucleotides. These results now are likely to be related to the ability of Bcl-2 to block the release of cytochrome c from the mitochondria and thus the subsequent redox-related events in apoptosis. 6 Further studies utilizing the methods described here should be useful in ordering and elucidating the role cellular redox and reactive oxygen species play in apoptosis. Acknowledgment This research was supported by NIH AG12282 to D.E.B. and NIH NS40251 to L.M.E.

[39] V o l u m e R e g u l a t i o n a n d I o n T r a n s p o r t during Apoptosis

By CARL D. BORTNER and JOHN A. CIDLOWSKI The loss of cell volume is a defining characteristic of programmed cell death, which separates apoptosis from necrosis. Assessment of cell volume during apoptosis can easily be accomplished by flow cytometry, by examining changes in the light-scattering properties of living cells that are directly proportional to cell size and density. Flow cytometry quantitates only relative changes in cell size; however, when used in conjunction with Coulter

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