The Coenzyme Q10 analog decylubiquinone inhibits the redox-activated mitochondrial permeability transition: role of mitcohondrial [correction mitochondrial] complex III.

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 Qo 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.

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 bc 1 ), because 1) stigmatellin, a Q o site inhibitor, blocked ROS production and prevented the MPT and cell death and 2) cytochrome bc 1 activity was abolished in cells protected from the redox-dependent MPT by stigmatellin. We next investigated the effect of pretreating cells with coenzyme Q 10 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 bc 1 activity, the mechanism of protection against redox-dependent MPT by dUb may depend on its ability to scavenge ROS generated by cytochrome bc 1 . 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.
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)(2)(3)(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)(6)(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)(12)(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 bc 1 ) (17-23), cytochrome bc 1 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 bc 1 reaches toxic levels resulting in activation of the MPT and cell death (15,24).
Given this scenario, and the key role of cytochrome bc 1 in ROS production in redox modulated cells, pharmacological strategies aimed at inhibiting mitochondrial ROS production by cytochrome bc 1 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 (CoQ 10 ) (18, [25][26][27]. CoQ 10 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 CoQ 10 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 antiapoptotic properties (33). Yet another approach has been to synthesize ubiquinone analogs with a reduced number of carbons in the side chain compared with CoQ 10 . 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 CoQ 10 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 bc 1 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 CoQ 10 analog decylubiquinone (dUb).

EXPERIMENTAL PROCEDURES
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 ϫ 10 5 /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 M 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 M) was used to dissipate the chemiosmotic proton gradient (⌬H ϩ ) 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) Ϯ 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 bc 1 ) Activity Measurements-Cytochrome bc 1 activity measurements were made as described in Ref. 37 with modifications. Briefly, cytochrome bc 1 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 M 2,3-dimethoxy-5-methyl-6n-decyl-1,4-benzoquinol(decylubiquinol) (dUb) as substrate and 50 M cytochrome c. Decylubiquinonol was synthesized in the laboratory from decylubiquinone by reduction with sodium borohydride (NaBH 4 ) (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 Ͻ0.05 were considered significant. Data are presented as mean Ϯ S.E.

RESULTS
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 ϳ95% 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.
The Respiratory Site of ROS Production Is Cytochrome bc 1 -To determine the cellular site regulating ROS-production after GSH depletion, cells were co-incubated with DEM, DEM ϩ rotenone, DEM ϩ stigmatellin, or DEM ϩ antimycin A. Rotenone selectively blocks respiratory complex I (38), stigmatellin blocks the Q o site of cytochrome bc 1 (39), and antimycin A blocks the Q i site of cytochrome bc 1 (40). Whereas rotenone and antimycin did not prevent ROS increase (Fig. 1B), stigmatellin blocked ROS production during the time course of the experiment indicating the involvement of the Q o site of cytochrome bc 1 in ROS formation. To confirm the role of cytochrome bc 1 in mitochondrial ROS production, cells were digitonin permeabilized and sonicated as described in the methods. Extracts from cells treated with DEM or DEM ϩ stigmatellin (protected from ROS production and cell death) were assayed for cytochrome bc 1 activity. Fig. 1C shows a representative example of cytochrome bc 1 activity in control, DEM-treated, and control ϩ stigmatellin-treated cells. Fig. 1D shows 1) that GSH depletion did not significantly alter cytochrome bc 1 enzyme activity and 2) that cytochrome bc 1 enzyme activity in both control cells ϩ stigmatellin or DEM ϩ stigmatellintreated cells was decreased ϳ95%.
Inhibition of Cytochrome bc 1  Co-incubation of cells with DEM ϩ 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. Cellular Pretreatment with dUb Blocks ROS Production,

Loss of ⌬ m , and Loss of Cell Viability after DEM Treatment-
Previously it has been found that the coenzyme Q analogs dUb and ubiquinone 0 (Ub0) block the MPT in isolated mitochondria. To test whether these analogs blocked the MPT in mitochondria in situ, we pretreated cells with either dUb (10 M) or Ub0 (10 M) for 6 h, washed the cells in fresh medium, and incubated the cells with DEM. Fig. 3A shows that cells pretreated with dUb blocked the increase in DCF fluorescence induced by H 2 O 2 compared with untreated cells, indicating that dUb pretreatment increased the cellular antioxidant potential of these cells. By contrast, pretreatment with Ub0 increased the level of ROS compared with untreated cells after treatment with H 2 O 2 . We next determined whether pretreatment of cells with dUb or Ub0 blocked the increased mitochondrial ROS produced by GSH depletion using DEM. dUb atten- uated the increase in ROS mediated by DEM, whereas levels of ROS in cells pretreated with Ub0 did not significantly differ from unconditioned cells (Fig. 3B). Since dUb pretreatment attenuated the ROS increase in HL60 (B) cells, we next determined the effect of pretreatment of cells with dUb and Ub0 on ⌬ m after DEM treatment. Fig. 3C (top panel) shows a representative example of ⌬ m in cells either pretreated with dUb, Ub0, or RPMI (control) after DEM treatment. Pretreatment with dUb blocked the loss of ⌬ m induced by DEM, whereas pretreatment with Ub0 did not. Fig. 3C (bottom panel) shows that the percentage of cells with intact ⌬ m either pretreated with dUb followed by treatment with DEM, or co-incubated with DEM ϩ BA, is not significantly different from the percentage of control cells with intact ⌬ m . Since the pretreatment of HL60 (B) cells with dUb blocked both ROS increase and activation of the MPT, we hypothesized that pretreatment with dUb would protect cells against loss of viability induced by GSH depletion with DEM. Fig. 3D shows that dUb pretreatment protected cells against loss of viability induced by DEM treatment, whereas pretreatment with Ub0 had no protective effect. We next determined whether the mechanism of protection against ROS increase and activation of the MPT by dUb was due to inhibition of cytochrome bc 1 activity or was the result of either inhibition of ROS production by cytochrome bc 1 or was due to ROS scavenging. To differentiate between these possibilities we pretreated cells with a range of concentrations of dUb (100 nM to 50 M), incubated them with DEM for 150 min, and measured cytochrome bc 1 activity. Fig. 3E shows that cytochrome bc 1 activity was not inhibited by dUb (even at concentrations of dUb well in excess of those used to protect cells from ROS-dependent activation of the MPT and cell death). These results indicated that dUb exerted its effects on ROS by either inhibition of ROS production by cytochrome bc 1 or that it scavenged ROS produced. DISCUSSION Determining the cellular mechanisms controlling life and death is of central importance in understanding the pathogenesis, progression, and possible modes of intervention in many age-related diseases and disorders. Mitochondria, because of their role in energy transduction, are central to the health of the cell; however, they also play a crucial role in mediating cell death via activation of the MPT.
The MPT, although a poorly understood phenomenon, is activated in response to either increased levels of matrix calcium or oxidative stress (5)(6)(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 bc 1 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 bc 1 as being a critical site in ROS production in redox modulated HL60 cells. The importance of cytochrome bc 1 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 bc 1 activity was abolished.
Cytochrome bc 1 , 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 bc 1 , although incompletely understood, is suggested to involve separate catalytic sites for quinol oxi-dation (Q o site) and quinone reduction (Q i site) (42)(43)(44). Quinol oxidation requires the bifurcated transfer of two electrons in cytochrome bc 1 . This process depends on alternating movement of the iron sulfur protein, which is blocked by the Q o siteselective inhibitor stigmatellin (38). We found that antimycin A, a Q i site inhibitor of cytochrome bc 1 (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 bc 1 was the ubisemiquinone radical as previously suggested (17,18,23).
We next determined the effects of ROS production by cytochrome bc 1 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)(46)(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 bc 1 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 bc 1 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 H 2 O 2 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 (ϳ 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 bc 1 , which would concomitantly decrease ROS production (Fig. 1B) or that dUb was inhibiting or scavenging ROS produced by cytochrome bc 1 . 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 bc 1 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 bc 1 .
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 bc 1 .
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 agerelated diseases where there is oxidative stress component. It also highlights the importance of the hydrophobic side chain during the drug design process.