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J. Biol. Chem., Vol. 278, Issue 49, 49079-49084, December 5, 2003

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The Coenzyme Q₁₀ Analog Decylubiquinone Inhibits the Redox-activated Mitochondrial Permeability Transition

ROLE OF MITOCHONDRIAL RESPIRATORY COMPLEX III^*

Jeffrey S. Armstrong, Matthew Whiteman, Peter Rose¶, and Dean P. Jones||

From the Department of Biochemistry, National University of Singapore, Singapore 117597, Singapore, the ^¶Department of Community, Occupational and Family Medicine, National University of Singapore, Singapore 117597, Singapore, and the ^||Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322

Received for publication, July 21, 2003 , and in revised form, August 25, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The mitochondrial permeability transition (MPT) is a key event in apoptotic and necrotic cell death and is controlled by the cellular redox state. To further investigate the mechanism(s) involved in regulation of the MPT, we used diethylmaleate to deplete GSH in HL60 cells and increase mitochondrial reactive oxygen species (ROS) production. The site of mitochondrial ROS production was determined to be mitochondrial respiratory complex III (cytochrome bc₁), because 1) stigmatellin, a Q_o site inhibitor, blocked ROS production and prevented the MPT and cell death and 2) cytochrome bc₁ activity was abolished in cells protected from the redox-dependent MPT by stigmatellin. We next investigated the effect of pretreating cells with coenzyme Q₁₀ analogs decylubiquinone (dUb) and ubiquinone 0 (Ub0) on the redox-dependent MPT. Pretreatment of HL60 cells with dUb blocked ROS production induced by GSH depletion and prevented activation of the MPT and cell death, whereas Ub0 did not. Since we also found that dUb did not inhibit cytochrome bc₁ activity, the mechanism of protection against redox-dependent MPT by dUb may depend on its ability to scavenge ROS generated by cytochrome bc₁. These results indicate that dUb, like the clinically used ubiquinone analog idebenone, may serve as a candidate antioxidant compound for the development of pharmacological agents to treat diseases where there is an oxidative stress component.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Mitochondria play a key role in apoptotic and necrotic cell death and therefore an understanding of mitochondrial function is important to determine the mechanisms regulating cell death in diseases such as cancer, the neurodegenerative diseases, and the mitochondrial genetic diseases (1–4).

One of the key mitochondrial events during cell death is the mitochondrial permeability transition (MPT)¹ which is due to the permeabilization of the inner mitochondrial membrane resulting in loss of mitochondrial transmembrane potential (_m), organelle swelling, and membrane rupture (5–7). The MPT results in either the release of cytochrome c from the mitochondrial inner membrane and the activation of caspases in a process known as apoptosis (8–10), or it can lead to a loss of cellular energy (ATP) production due to the disruption of the inner mitochondrial membrane integrity, which results in necrosis (9, 10).

The factors involved in MPT regulation are of considerable importance in cell biology because the MPT is central to the cell death process. In this respect, the cellular redox environment is one such critical regulatory factor (11–13), which is, in part, controlled by the tripeptide glutathione (GSH). Cellular depletion of GSH creates an increasingly oxidized environment in both the cytosol and the mitochondria with increased formation of mitochondrial reactive oxygen species (ROS) (14–16). Although a number of different sites within mitochondria have been proposed to be important in ROS production, including complexes I, II, and III (cytochrome bc₁) (17–23), cytochrome bc₁ has been found to play a central role in ROS formation in both isolated mitochondria and in mitochondria in situ (23, 24). Furthermore, in the absence of mitochondrial GSH, ROS production by cytochrome bc₁ reaches toxic levels resulting in activation of the MPT and cell death (15, 24).

Given this scenario, and the key role of cytochrome bc₁ in ROS production in redox modulated cells, pharmacological strategies aimed at inhibiting mitochondrial ROS production by cytochrome bc₁ may be useful in preventing oxidative stress dependent activation of the MPT and cell death in a number of biological settings. In this respect, much interest has been shown in the natural lipid soluble antioxidant compound coenzyme Q (CoQ₁₀) (18, 25–27). CoQ₁₀ is a lipid-soluble compound that contains a redox active quinoid nucleus and a hydrophobic trans-isoprenoid side chain (28). Its hydrophobicity causes it to be inserted into the membrane phospholipid of the mitochondrial inner membrane (29) where it is converted to the reduced form by cellular reductases (30, 31). Although CoQ₁₀ is a natural lipid antioxidant, it is extremely insoluble in water and is therefore not taken up by cells to any significant degree which hinders its use as a therapeutic agent (32, 33). One approach to circumvent this problem has been to develop mitochondrially targeted ubiquinone analogs including the compound Mito Q, which has been shown to possess both antioxidant and anti-apoptotic properties (33). Yet another approach has been to synthesize ubiquinone analogs with a reduced number of carbons in the side chain compared with CoQ₁₀. Included in this group is the clinically used synthetic compound idebenone (6-(10-hydroxydecyl)-2,3-dimethoxy-5-methyl-1,4-benzoquinone) (32). However, it has been suggested that many short-chain CoQ₁₀ analogs, including idebenone, enhance superoxide formation by respiratory complex I (30). Because idebenone is used in a number of clinical cytopathies and in neurodegenerative diseases, its potential pro-oxidant effects raise doubts regarding its safety as a drug (35). From this foreword it is apparent that the identification of alternative compounds that may be employed therapeutically in diseases with an oxidative stress component is warranted.

In this study we show that mitochondrial ROS production by cytochrome bc₁ activates the MPT in redox sensitive HL60 (B) cells after GSH depletion. The redox-dependent MPT is blocked by inhibition of ROS production in cells pretreated with the CoQ₁₀ analog decylubiquinone (dUb).

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—All chemicals were reagent grade and were obtained from Sigma. Tetramethylrhodamine methyl ester (TMRM) and dichlorofluorescein diacetate (DCFDA) was obtained from Molecular Probes (Eugene, OR). Bongkrekic acid (BA) was obtained from Calbiochem.

Cell Culture—HL60 cells transfected with bcl-2 (bcl-2) (HL60 B cells) were cultured as described previously (24). These cells were used in the experiments outlined below because we have previously shown that 1) Bcl-2 overexpression in the HL60 cell line confers a high sensitivity to GSH depletion and 2) that these cells are highly reproducible in their response to L-buthionine-S,R-sulfoximine and diethylmaleate (DEM) (24). Cells were passaged daily to maintain them in log-phase and kept at a concentration between 2.5–5 x 10⁵/ml.

Measurement of Intracellular GSH Redox Change—Cells were treated with DEM (1 mM) for 0, 15, and 30 min. They were then pelleted by centrifugation and extracted with 5% perchloric acid/saturated boric acid. Intracellular GSH was measured with high performance liquid chromatography as described previously (24). The amount of acid-insoluble protein was determined by the Bradford method with -globulin as a standard.

Measurement of ROS Production—After treatment with DEM (1 mM) for 0, 15, 30, 45, and 60 min, cells were stained with 50 µM DCFDA for 15 min, washed with phosphate-buffered saline containing 10 mM glucose, and analyzed immediately by flow cytometry using CellQuest software (BD Biosciences). DCFDA is a non-fluorescent ester of the dye fluorescein. DCFDA is cleaved by intracellular esterases and entrapped within the cell as the oxidant-sensitive DCF. ROS oxidize DCF to the fluorescent product fluorescein (36). The green fluorescence of fluorescein was measured using the FL-1 setting (log mode) after cell debris were electronically gated out. In each analysis, 10,000 events were recorded.

Measurement of _m—_m was measured with the fluorescent lipophilic cationic dye TMRM (250 nM), which accumulates within mitochondria according to the _m. After treatment with DEM for 0, 60, and 120 min, cells were stained with TMRM for 15 min and red fluorescence was measured by flow cytometry using the FL-2 setting. The protonophore CCCP (1 µM) was used to dissipate the chemiosmotic proton gradient (µH⁺) and served as a control for loss of _m. In each analysis, 10,000 events were recorded.

Electron Microscopic Examination of HL60 Cells—HL60 cells in the logarithmic proliferation phase were cultured with RPMI and DEM (1 mM) ± stigmatellin for 120 min and were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, at room temperature for 1 h. The cells were washed with 0.1 M cacodylate buffer and post-fixed with 1% osmium tetraoxide in 0.1 M cacodylate buffer. Finally, the cells were dehydrated with graded series of ethanol and embedded in LX112. Thin sections were prepared and stained with uranyl acetate. Specimens were examined on a JEOL 1000X electron microscope operating at 80 kV.

Respiratory Complex III (Cytochrome bc₁) Activity Measurements— Cytochrome bc₁ activity measurements were made as described in Ref. 37 with modifications. Briefly, cytochrome bc₁ complex activity from digitonin permeabilized and sonicated HL60 cell fractions were assayed in 50 mM potassium phosphate, pH 7.0, 250 mM sucrose, 0.2 mM EDTA, 1 mM NaN3, 0.1% (w/v) and 0.01% Tween 20 at 23 °C, using 50 µM 2,3-dimethoxy-5-methyl-6n-decyl-1,4-benzoquinol(decylubiquinol) (dUb) as substrate and 50 µM cytochrome c. Decylubiquinonol was synthesized in the laboratory from decylubiquinone by reduction with sodium borohydride (NaBH₄) (37). Reduction of cytochrome c was monitored in a spectrophotometer at 550 versus 539 nm in dual wavelength mode. Data are expressed as percentage of control activity and were determined from five individual isolations that were assayed in triplicate.

Statistical Analysis—Statistical analyses were performed using Student's t test for unpaired data, and p values <0.05 were considered significant. Data are presented as mean ± S.E.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
After DEM Treatment, GSH Is Depleted and Levels of Intracellular ROS Increase—HL60 (B) cells were treated with DEM (1 mM), and GSH levels were determined on cell aliquots at 0, 15, and 30 min after DEM treatment (Fig. 1A). Fig. 1 shows that DEM treatment caused the depletion of GSH in the HL60 (B) cell line. GSH was 95% depleted 30 min after DEM treatment. Fig. 1B shows that an increase in cellular ROS production was detected 15 min after cells were treated with DEM.

View larger version (38K): [in this window] [in a new window]   FIG. 1. A, aliquots of HL60 (B) cells (4 x 106/ml) were treated with DEM (1 mm) or RPMI (control) for 0, 15, and 120 min. GSH levels were determined on these samples by HPLC assay described under "Experimental Procedures." The GSH concentration was plotted as nmol/mg of protein. Data are expressed as mean ± S.E. (n = 3). B, representative flow cytometric analysis of HL60 (B) cells stained with DCFDA and analyzed using FL1-H channel as described under "Experimental Procedures." HL60 cells (2 x 106/ml) were treated with DEM (1 mM), DEM + rotenone (1 µM), DEM + stigmatellin (1 µM), or DEM + antimycin A (1 µM) for 0, 15, 45, and 60 min, washed in PBS, and suspended in PBS containing 10 mM glucose. Cells were loaded with DCFDA (50 µM) for 15 min, and green fluorescence was measured by flow cytometry using the FL1-H setting. The figure shows representative example of at least five experiments. In each analysis, 10,000 events were recorded. C, upper panel, representative scan (550 versus 539 nm) of cytochrome c reduction by cytochrome bc1. Cytochrome bc1 activity measurements were made as described under "Experimental Procedures." Trace A represents cytochrome bc1 activity in control mitochondrial fractions, trace B represents cytochrome bc1 activity in mitochondrial fractions treated for 120 min with DEM (1 mM), and trace C represents cytochrome bc1 activity in control mitochondrial fractions pretreated for 20 min with stigmatellin (1 µM) prior to the analysis. Lower panel, bar graph showing cytochrome bc1 activity in mitochondrial fractions from HL60 (B) cells that were treated with either 1 mM DEM for 60 and 120 min, DEM + rotenone (1 µM) for 120 min, or DEM + stigmatellin (1 µM) for 120 min. Results are expressed as percentage of control cytochrome bc1 activity ± S.E. and were determined using three individual mitochondrial isolations that were each assayed in triplicate.

Inhibition of Cytochrome bc₁ with Stigmatellin Blocks Loss of _m and Cell Death in DEM-treated Cells—_m was determined in cells by monitoring the fluorescence of the cationic potentiometric dye TMRM. Fig. 2A shows the _m at0h(panel a), 1 h (panel b), 2 h (panel c), 2 h + stigmatellin (1 µM) (panel d), 2 h + BA (25 µM) (panel e), 2 h + BA (50 µM) (panel f), 2 h + rotenone (1 µM) (panel g), control cells + CCCP (1 µM) (panel h). _m was decreased after DEM treatment which was prevented by BA and stigmatellin. Rotenone failed to prevent loss of _m induced by DEM. CCCP was used as control for loss of _m. Inhibition of loss of _m, after GSH depletion by BA, suggested that cytochrome bc₁ activity was a critical factor leading to the activation of the MPT in GSH depleted cells. To confirm this electron microscopy was performed to determine the mitochondrial ultrastructure in cells after GSH depletion. Fig. 2B shows representative electron micrographs of mitochondrial ultrastructure in control cells (panel a), cells treated with DEM for 150 min h (panel b), and cells treated with DEM for 2 h + stigmatellin (panel c). Co-incubation of cells with DEM + stigmatellin preserved mitochondrial structure and prevented changes (mitochondrial swelling) associated with activation of the MPT). We next hypothesized that since stigmatellin blocked ROS production and mitochondrial changes associated with activation of the MPT in GSH depleted cells, that stigmatellin should protect cells from loss of viability after GSH depletion. Fig. 2C shows that stigmatellin, but not the complex I inhibitor rotenone, significantly protects cells from loss of viability induced by GSH depletion.

View larger version (53K): [in this window] [in a new window]   FIG. 2. A, representative flow cytometric analysis of HL60 (B) cells stained with TMRM and analyzed using FL2-H channel as described under "Experimental Procedures." Cells (2 x 106/ml) were treated with DEM (1 mM) for 0 h (panel a), 1 h (panel b), 2 h (panel c), 2 h + stigmatellin (1 µM) (panel d), 2 h + BA (25 µM) (panel e), 2 h + BA (50 µM) (panel f), 2 h + rotenone (1 µM) (panel g), control cells + CCCP (1 µM)(panel h). Experiment repeated at least 5 times on different days. B, representative electron micrographs of HL60 cell mitochondria at a magnification of 45,600 showing the effects of DEM and DEM + stigmatellin (1 µM) on mitochondrial ultrastructure. Electron microscopy was performed as described under "Experimental Procedures." The figure shows control mitochondria in panel a, mitochondria after treatment with DEM in panel b, and mitochondria after treatment with DEM + stigmatellin in panel c. Similar results were obtained in two other experiments. C, HL60 (B) cells were treated with either DEM (1 mM), DEM (1 mM) + stigmatellin (1 µM), or DEM (1 mM) + rotenone (1 µM) for up to 150 min. Cell viability was determined at 15-min intervals during the time course of the experiment. Cell viability was determined by trypan blue analysis. Data are expressed as mean ± S.E. (n = 3).

View larger version (36K): [in this window] [in a new window]   FIG. 3. A, flow cytometric analysis of HL60 (B) cells stained with DCFDA and analyzed using FL1-H channel as described under "Experimental Procedures." Cells were treated with H2O2 (5 mM) for 0, 5, and 10 min and immediately analyzed by flow cytometry by measuring the green fluorescence using the FL-1 setting. In each analysis, 10,000 events were recorded. Data are expressed as mean ± S.E. (n = 3). B, flow cytometric analysis of HL60 (B) cells stained with DCFDA and analyzed using FL1-H channel as described under "Experimental Procedures." HL60 cells (2 x 106/ml) were incubated with RPMI (control), dUb (10 µM), or Ub0 (10 µM) for 6 h, washed in medium, and treated with DEM (1 mM) for 150 min. Cells were loaded with DCFDA (50 µM) for 15 min, and green fluorescence was measured by flow cytometry using the FL1-H setting. In each analysis, 10,000 events were recorded. Data are expressed as mean ± S.E. (n = 3). C, top panel, representative flow cytometric analysis of HL60 (B) cells stained with TMRM and analyzed using FL2-H channel as described under "Experimental Procedures." HL60 cells (2 x 106/ml) were incubated with RPMI (control), dUb (10 µM), or Ub0 (10 µM) for 6 h, washed in medium, and treated with DEM (1 mM) for 150 min. Cells were loaded with TMRM (250 nM) for 15 min, and red fluorescence was measured by flow cytometry using the FL2-H setting. The figure shows representative example of at least five experiments. In each analysis, 10,000 events were recorded. Bottom panel, flow cytometric analysis of HL60 (B) cells stained with TMRM and analyzed using FL2-H channel as described under "Experimental Procedures." HL60 cells (2 x 106/ml) were incubated with either RPMI (control), dUb (10 µM), or Ub0 (10 µM) for 6 h, washed in medium, and treated with DEM (1 mM) for 150 min. Aliquots of DEM treated cells were also co-incubated with BA (50 µM) for 150 min. Cells were loaded with TMRM (250 nM) for 15 min, and red fluorescence was measured by flow cytometry using the FL2-H setting. As a positive control for loss of m, control cells were co-incubated with CCCP (1 µM) for 5 min prior to flow cytometric analysis. D, HL60 (B) cells were incubated with either RPMI (control), dUb (10 µM), or Ub0 (10 µM) for 6 h, washed in medium, and treated with DEM (1 mM) for 150 min. Cell viability was determined 150 min after DEM treatment. Cell viability was determined by trypan blue analysis. Data are expressed as mean ± S.E. (n = 3). E, HL60 (B) cells were incubated with either RPMI (control) or dUb at either 100 nM, 10 µM, or 50 µM) for 6 h, washed in medium, and treated with DEM (1 mM) for 120 min. Cytochrome bc1 activity was assayed in mitochondrial fractions from HL60 (B) cells according to the "Experimental Procedures." Results are expressed as percentage of control cytochrome bc1 activity ± S.E. and were determined using three individual mitochondrial isolations that were each assayed in triplicate.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The MPT, although a poorly understood phenomenon, is activated in response to either increased levels of matrix calcium or oxidative stress (5–7). Depletion of mitochondrial GSH creates oxidative stress via increased mitochondrial ROS production; therefore, it is clearly important to define both the mitochondrial site and mechanism responsible for ROS production. Using a simple pharmacological approach we found that inhibition of cytochrome bc₁ activity blocked ROS production in GSH depleted HL60 cells, whereas inhibition of respiratory complex I, in the same experimental setting, did not. These results implicated cytochrome bc₁ as being a critical site in ROS production in redox modulated HL60 cells. The importance of cytochrome bc₁ in ROS production was further supported by the observation that its inhibition blocked the MPT and cell death in GSH depleted cells and was confirmed with the finding that in cells pharmacologically protected from the MPT cytochrome bc₁ activity was abolished.

Cytochrome bc₁, an essential component of the mitochondrial electron transport chain, catalyzes the transfer of electrons from ubiquinol to cytochrome c while concomitantly translocating protons across the mitochondrial inner membrane during the process of redox energy conservation (41). The mechanism of action of cytochrome bc₁, although incompletely understood, is suggested to involve separate catalytic sites for quinol oxidation (Q_o site) and quinone reduction (Q_i site) (42–44). Quinol oxidation requires the bifurcated transfer of two electrons in cytochrome bc₁. This process depends on alternating movement of the iron sulfur protein, which is blocked by the Q_o site-selective inhibitor stigmatellin (38). We found that antimycin A, a Q_i site inhibitor of cytochrome bc₁ (40), failed to block ROS production, whereas stigmatellin, a Q_o site inhibitor, abolished ROS production, which suggested that the intermediate responsible for ROS formation by cytochrome bc₁ was the ubisemiquinone radical as previously suggested (17, 18, 23).

We next determined the effects of ROS production by cytochrome bc₁ on _m and mitochondrial ultrastructure (Fig. 2, A and B). DEM treatment caused loss of _m, which was effectively blocked with the adenine nucleotide translocator ligand BA. This indicated that the loss of _m observed after DEM treatment was due to activation of the MPT (17, 45–47). This conclusion was confirmed using electron microscopy, since we observed that DEM treatment caused mitochondrial swelling reminiscent of the MPT (48) (Fig. 2B, panel b), which was prevented by inhibition of cytochrome bc₁ activity (Fig. 2B, panel c).

We next determined whether pretreatment with the ubiquinone analogs, dUb and Ub0, would prevent the activation of the MPT by cytochrome bc₁ in redox modulated HL60 cells, because these ubiquinone analogs have previously been shown to block the MPT in isolated mitochondria (49). We found that dUb inhibited both the DCF fluorescence increase resulting from H₂O₂ treatment (Fig. 3A) and the DCF fluorescence increase resulting after GSH depletion using DEM (mitochondrially generated ROS) (Fig. 3B), whereas Ub0 did not. Thus, cellular pretreatment with dUb was shown to be effective in decreasing ROS levels after treatment with both exogenous oxidants as well as endogenous ROS generated by the mitochondrial electron transport chain. This suggested that dUb, but not Ub0, by blocking ROS production, would also prevent the redox-dependent MPT. We found that dUb was effective in preventing the loss of _m induced by DEM treatment, whereas Ub0 was not (Fig. 3C). These results are supported by a study reported by Walter et al. (49) who also found that dUb was effective at preventing the MPT in isolated mitochondria; however, these investigators also found that Ub0 was effective at blocking the MPT (49). In our studies, we found that Ub0 was cytotoxic to HL60 (B) cells after either extended treatment ( 24 h) or when used at high concentrations (data not shown), which may be due to predominant partitioning of Ub0 in aqueous cell compartments where it can redox cycle. In this respect, Ub0 has been previously shown to mediate similar toxicity to menadione, a known redox cycling agent, in filarial parasites by interrupting mitochondrial electron transfer, ATP synthesis, and oxygen consumption (34). Since the MPT is generally regarded as a phenomenon preceding and culminating in cell death, if Ub0 blocked the MPT in vivo, it would not be expected to be cytotoxic per se, which it is in our experimental setting.

Next we considered the mechanism of inhibition of MPT by dUb. Since dUb was effective at blocking mitochondrial ROS production which preceded MPT activation, we reasoned that the mechanism either involved inhibition of cytochrome bc₁, which would concomitantly decrease ROS production (Fig. 1B) or that dUb was inhibiting or scavenging ROS produced by cytochrome bc₁. If dUb blocked complex III activity, then it would be of little value as a candidate antioxidant compound in the biological setting, since inhibition of cytochrome bc₁ activity blocks redox-energy conservation and the production of ATP. Our results (Fig. 3F) show that dUb does not inhibit complex III activity, even at high concentrations, leading us to conclude that its inhibitory effect on the MPT is a consequence of its antioxidant action, either preventing ROS formation or scavenging ROS generated by cytochrome bc₁.

It is known that by manipulating ubiquinone hydrophobicity, it is possible to produce analogs that partition to various cell compartments (32), which, in part, determines their efficiency as antioxidants in vivo (32). Since the major structural difference between dUb and Ub0 is the 10 carbon side chain, this indicates that both the presence of the hydrocarbon side chain, and its length, are critically important factors in conferring antioxidant or pro-oxidant properties to the ubiquinone analog. Because dUb is structurally quite similar to idebenone, it is possible that it functions in a similar manner. In this respect, it may localize at the surface of the mitochondrial membrane (32) where it intercepts and scavenges superoxide radicals generated by ubisemiquinone autoxidation within cytochrome bc₁.

In conclusion, the compound dUb provides a structural platform for the design and development of ubiquinone analogs that may be used as potential therapeutic antioxidants in age-related diseases where there is oxidative stress component. It also highlights the importance of the hydrophobic side chain during the drug design process.

	FOOTNOTES

 
^* This work was supported in part by National Institutes of Health Grants ES09047 and EY07892 and Core Grant P30 EY06360. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence and reprint requests should be addressed: Dept. of Biochemistry, National University of Singapore, Kent Rd., Singapore 117597, Singapore. Tel.: 65-6874-5996; Fax: 65-6779-1453; E-mail: bchjsa{at}nus.edu.sg.

¹ The abbreviations used are: MPT, mitochondrial permeability transition; ROS, reactive oxygen species; CoQ₁₀, coenzyme Q₁₀; idebenone, 6-(10-hydroxydecyl)-2,3-dimethoxy-5-methyl-1,4-benzoquinone; dUb, decylubiquinone; Ub0, ubiquinone 0; TMRM, tetramethylrhodamine methyl ester; DCF, dichlorofluorescein; DCFDA, dichlorofluorescein diacetate; BA, bongkrekic acid; DEM, diethylmaleate; CCCP, carbonyl cyanide p-chlorophenylhydrazone.

	ACKNOWLEDGMENTS

 
We acknowledge Professor Barry Halliwell for his critical review of this manuscript.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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