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Effect of Glutathione Depletion on Sites and Topology of Superoxide and Hydrogen Peroxide Production in Mitochondria

USC-UCLA Research Center for Alcoholic and Pancreatic Disease, University of Southern California Research Center for Liver Diseases, Keck School of Medicine (D.H., N.K.), and Department of Molecular Pharmacology & Toxicology, School of Pharmacy (R.C. D.R.), University of Southern California, Los Angeles, California

Received June 12, 2003; accepted August 4, 2003

	Abstract

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In this work, the topology of mitochondrial and H₂O₂ generation and their interplay with matrix GSH in isolated heart mitochondria were examined. We observed that complex I releases into the matrix (where it is converted to H₂O₂ by Mn-SOD) but not into the intermembrane space. No free radical generation was observed from complex II, but succinate treatment caused H₂O₂ generation from the matrix through a reverse electron flow to complex I. Complex III was found to release into the matrix and into the intermembrane space. Antimycin, which increases steady-state levels of (ubisemiquinone at the Qo site) in complex III, enhanced both H₂O₂ generation from the matrix and production from the intermembrane space. On the other hand, myxothiazol, which inhibits formation, completely inhibited antimycin induced toward the intermembrane space and inhibited H₂O₂ generation from the matrix by 70%. However, myxothiazol alone enhanced H₂O₂ production from complex III, suggesting that other components of complex III besides the can cause generation toward the matrix. As expected, mitochondrial GSH was found to modulate H₂O₂ production from the matrix but not generation from the intermembrane space. Low levels of GSH depletion (from 0—40%, depending on the rate of H₂O₂ production) had no effect on H₂O₂ diffusion from mitochondria. Once this GSH depletion threshold was reached, GSH loss corresponded to a linear increase in H₂O₂ production by mitochondria. The impact of 50% mitochondrial GSH depletion, as seen in certain pathological conditions in vivo, on H₂O₂ production by mitochondria depends on the metabolic state of mitochondria, which governs its rate of H₂O₂ production. The greater the rate of H₂O₂ generation the greater the effect 50% GSH depletion had on enhancing H₂O₂ production.

Mitochondrial GSH depletion occurs in several pathological conditions, including long-term ethanol intake (Fernandez-Checa et al., 1987; Hirano et al., 1992), cerebral ischemia (Anderson and Sims, 2002), exposure to environmental toxins, such as cadmium (Nigam et al., 1999), and during liver regeneration (Vendemiale et al., 1995). In rats receiving longterm ethanol treatment, mitochondrial GSH levels were found to be selectively decreased by up to 65% after 8 weeks of treatment (Fernandez-Checa et al., 1987). These findings lead to an intriguing question: what is the physiological consequence of moderate mitochondrial GSH depletion seen in vivo in these pathological situations? Although in cultured cells, severe GSH depletion results in cell necrosis, moderate mitochondrial GSH depletion is generally not cytotoxic (Nagai et al., 2002) and physiological effects are not always evident (Uhlig and Wendel, 1992). In terms of mitochondrial H₂O₂ production, the present understanding of the GSH peroxidase system predicts that small changes in mitochondrial GSH levels would have little consequence to H₂O₂ release by mitochondria. GSH peroxidase (E-Cys-Se^-) is a selenium-based enzyme that reduces H₂O₂ to water (Flohe et al., 1976):

(1)

(2)

(3)

Mitochondria contain high amounts of GSH (5—10 mM), whereas steady-state levels of H₂O₂ in the matrix are believed to be more than 1000-fold lower (Boveris and Cadenas, 1997). Kinetic data suggests that because of this large stoichiometric difference between GSH and H₂O₂ in the matrix, even a 50% decrease in GSH levels may not have a significant impact on H₂O₂ detoxification (Flohe et al., 1976). Theoretically, within this GSH range, these enzymes are fully reduced with their active selenol site, ready to scavenge any H₂O₂ molecule. However, extrapolation of kinetic studies to the in vivo situation may be misleading. If concentrations of H₂O₂ are highly localized in certain areas of the matrix, the large stoichiometric difference between GSH and H₂O₂ would be partially offset. If we take into consideration the compartmentalization of the matrix and the fact that H₂O₂ can diffuse rapidly through membranes, even small decreases in GSH peroxidase kinetics—caused by lower GSH levels—may lead to an increase in H₂O₂ escape from mitochondria. Because a detailed study of the effects of GSH concentration on H₂O₂ production in mitochondria has not been performed, an understanding of the consequences of mitochondrial GSH depletion that occurs in vivo in certain pathological conditions remains incomplete.

The mechanism of H₂O₂ production by mitochondria, on the other hand, has been extensively studied both in physiological and pathological situations such as aging (Chance et al., 1979; Cadenas and Boveris, 1980; Turrens et al., 1985; Hansford et al., 1997). These studies have shown that complexes I and III, ascribed to flavoprotein (Turrens and Boveris, 1980) and ubisemiquinone autoxidation (Boveris and Cadenas, 1975; Cadenas et al., 1977), respectively, are responsible for generation by the respiratory chain. Complex I is believed to generate into the matrix, where Mn-SOD converts into H₂O₂, which in turn is either reduced by GSH peroxidase or diffuses from mitochondria (Chance et al., 1979). In complex III, ubisemiquinone is formed at both the Q_I site that lies near the matrix and at the Q_O site near the intermembrane space (Brandt, 1998). (ubisemiquinone at the Q_O site) rather than (ubisemiquinone at the Q_I site) has been the focus of generation by complex III (Demin et al., 1998). Recently, we demonstrated that in complex III generates into the intermembrane space (Han et al., 2001), a portion of which diffuses into the cytoplasm through voltage dependent anion channels (Han et al., 2003). Although the topology of and H₂O₂ by mitochondria is becoming better understood, several key questions still remain. For example, it is still not completely clear whether complex I or II is also a source of in the intermembrane space.

The purpose of this work was to gain further insight on the topology of mitochondrial and H₂O₂ production and to study the consequences of intramitochondrial GSH depletion on their production using isolated heart mitochondria.

	Materials and Methods

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Chemicals and Biochemicals. 5,5'—Dimethyl-1-pyrroline-N-oxide (DMPO), 1-chloro-2,4 dinitrobenzene (CDNB), NADP, superoxide dismutase, myxothiazol, glutamate, malate, succinate, Nagarse, antimycin A, catalase, TTFA (thenoyltrifluoroacetone), and rotenone were obtained from Sigma Chemical Co. (St. Louis, MO).

Isolation of Heart Mitochondria. Heart mitochondria were isolated from adult male Wistar rats by differential centrifugation using Nagarse (Cadenas and Boveris, 1980). Rat hearts were excised, washed, and chopped into fine pieces. Chopped hearts were suspended in an isolation buffer consisting of 230 mM mannitol, 70 mM sucrose, 1 mM EDTA, and 5 mM Trizma/HCl buffer, pH 7.4. The chopped heart was treated with Nagarse (1 mg/heart) for 5 min and then homogenized by 10 strong strokes of a loose-fitting Potter-Elvehjem Teflon pestle. The homogenate was centrifuged at 800g for 8 min, the pellet removed, and the centrifugation process was repeated. The resulting supernatant was centrifuged at 8000g for 10 min, washed with isolation buffer and the centrifugation repeated. Isolated mitochondria were resuspended in isolation buffer and placed in ice for the duration of experiment.

Determination of Mitochondria Integrity. To determine the integrity of isolated heart mitochondria, oxygen uptake was measured and the respiratory control ratio (RCR) was determined. RCR was defined as the state 3/state 4 ratio. Oxygen uptake was measured polarographically with a Clark-type electrode (Hansatech, King's Lynn, Norfolk, UK) in respiration buffer containing 230 mM mannitol, 70 mM sucrose, 30 mM Tris-HCl, 5 mM KH₂PO₄, 1 mM EDTA, and 0.1% bovine serum albumin, pH 7.4. For all experiments, mitochondria with an RCR greater than 4 using glutamate/malate as substrates were used.

Depeletion of Mitochondrial Glutathione. Mitochondrial GSH was depleted using CDNB, a favored substrate for glutathione transferases localized in the mitochondrial matrix (Jocelyn and Cronshaw, 1985). Glutathione transferase catalyzes the specific conjugation of GSH with CDNB. GSH was depleted using CDNB as follows: isolated mitochondria (5 mg/ml) suspended in isolation buffer were incubated with various concentrations of CDNB dissolved in ethanol for 2 min at room temperature. The pellets were spun at 8000g for 5 min and washed once more in isolation buffer. Control mitochondria were treated with an equal amount of ethanol. Treatment of isolated heart mitochondria (5 mg/ml) with 35 µM CDNB resulted in 90 ± 7% depletion of mitochondrial GSH. No changes in mitochondrial respiration or in the respiratory control ratio were observed with the maximum amount of CDNB used (35 µM), in agreement with previous results (Jocelyn and Cronshaw, 1985).

We find the ratio of nanomoles of CDNB to milligrams of heart mitochondria to be important in determining amount of GSH depletion; for lower amounts of mitochondria, lower concentrations of CDNB must be used. No more than 35 µM CDNB can be used for heart mitochondria (5 mg/ml). Higher levels of CDNB will have secondary effects on complex I and may stimulate H₂O₂ production (Zoccarato et al., 1988, 1990). It is important that samples are washed carefully to remove excess CDNB as to not affect complex I generation of H₂O₂ (Zoccarato et al., 1988).

Determination of Glutathione. Samples of mitochondria were stored in metaphosphoric acid 2% at —80°C until the analysis. GSH levels were quantified by HPLC, using coulometric electrochemical detector (Harvey et al., 1989). The HPLC conditions were performed using an Alltech C18 column 250 x 4.6 mm and an electrochemical detector ESA Coulochem II. The potential settings were: guard cell, + 0.95 V; electrode 1, +0.45 V. The mobile phase consisted of 50 mM NaH₂PO₄ adjusted to pH 2.7 with phosphoric acid.

Superoxide Determination. Superoxide was determined by electron paramagnetic resonance (EPR) with the spin trap, DMPO. EPR with DMPO measures only diffusing from mitochondria through the voltage-dependent anion channel, not in the matrix (Han et al., 2003). For EPR measurements, heart mitochondria were placed in buffer consisting of 230 mM mannitol, 70 mM sucrose, 20 mM Tris, and 200 units of catalase, and pH was adjusted to 7.4 with MOPS in the absence or presence of mitochondrial respiratory substrates or inhibitors. DMPO was present at a concentration of 160 mM. EPR spectra were recorded with a Bruker ECS106 spectrometer (operating at X-band) equipped with a cylindrical room temperature cavity operating in TM₁₁₀ mode. Aliquots of samples were promptly transferred to bottom-sealed Pasteur pipettes and measured at room temperature with the following settings: receiver gain, 5 x 10⁵; microwave power, 20 mW; microwave frequency, 9.77 GHz; modulation amplitude, 1 G; time constant, 164-ms scan time, 87 s; scan width, 80 G. The DMPO-OH signal generated from heart mitochondria was quantified by comparison with a TEMPOL standard after double integration of both signals as described previously (Han et al., 2003). All spectra shown are an accumulation of seven scans.

Hydrogen Peroxide Measurements. H₂O₂ production by heart mitochondria was measured by monitoring fluorescence of p-hydroxyphenylacetate oxidation in the presence of horseradish peroxidase (Hyslop and Sklar, 1984). Fluorescence measurements (_ex = 320 nm; _em = 400 nm) were performed with an LS-5 spectrofluorometer (PerkinElmer Life Sciences, Boston, MA) equipped with a thermal-controlled and magnetic stirring sample compartment. For all measurements, mitochondria were incubated in 230 mM mannitol, 70 mM sucrose, and 20 mM Tris (pH was adjusted to 7.4 with MOPS) at 25°C.

The rates of H₂O₂ production by heart mitochondria in this work are much greater than those we previously reported (Han et al., 2003). In our previous work, we used-higher amounts of mitochondria, which interferes with fluorescence and underestimates H₂O₂ production and effect of such inhibitors as myxothiazol.

Determination of Aconitase and Fumarase Activities. For aconitase and fumarase measurements, control and GSH depleted mitochondria were treated with glutamate/malate, and at indicated times, mitochondria were spun down (8,000g for 8 min). The pellets were stored at —80° until time of analysis. Mitochondria pellets were resuspended in 0.5% Triton X-100 to disrupt the membranes.

Aconitase activity was assayed by following the formation of NADPH at an absorbance of 340 nm at 25°C. The reaction mixture was composed of 30 mM Tris-HCl, pH 7.4, 30 mM sodium citrate, 0.6 mM MnCl₂, 0.2 mM NADP⁺, 1 U/ml of isocitrate dehydrogenase, and 0.1 mg of mitochondria (Gardner and White, 1995).

Fumarase activity was assayed spectrometrically by monitoring the increase in absorbance at 240 nm at 25°C. The reaction mixture was composed of 30 mM KH₂PO₄, pH 7.4, 0.01 mM ETDA, and 5 mM malate (Gardner and White, 1995).

Data Analysis. Statistical analysis was performed using paired t test, with the minimum level of significance defined at p < 0.05. Where appropriate, means ± S.D. are presented in the text.

	Results

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Dose-Dependent Depletion of Mitochondrial GSH by CDNB. GSH forms a conjugate with CDNB in the mitochondrial matrix, a process believed to be catalyzed by GSH S-transferase (Jocelyn and Cronshaw, 1985). The addition of CDNB to isolated heart mitochondria caused a dose-dependent loss of GSH from the matrix (Fig. 1). CDNB treatment caused no measurable changes in mitochondria; parameters checked included state III and IV respiration and generation from complex I (data not shown). Previous studies have reported that high levels of CDNB (60 µM) can induce generation from complex I, a phenomenon that can be prevented by washing CDNB out of mitochondria (Zoccarato et al., 1988), as done in this work.

View larger version (9K): [in this window] [in a new window]   Fig. 1. Dose-dependent depletion of GSH by CDNB in isolated heart mitochondria. Isolated mitochondria (5 mg/ml), suspended in isolation buffer, were incubated with various concentrations of CDNB dissolved in ethanol for 2 min at room temperature. The pellets were spun at 8000g for 5 min and washed once more in isolation buffer. Control mitochondria were treated with the same amount of ethanol. GSH was determined by HPLC using electrochemical detection.

H₂O₂ Production Using Complex I Substrates: Effect of GSH Depletion. To assess H₂O₂ production by complex I, mitochondria were treated with complex I substrates (glutamate/malate), and kinetics of H₂O₂ production was monitored fluorometrically in the presence of p-hydroxyphenylacetate and horseradish peroxidase. The complex I inhibitor rotenone was also added to mitochondria to observe the effect of inhibition of complex I on H₂O₂ production. The consequences of GSH depletion (90 ± 7%) on H₂O₂ production using glutamate/malate as substrates are summarized in Table 1. Rotenone was effective in causing a large increase in H₂O₂ production in both control and GSH-depleted mitochondria. It is believed that rotenone blockage of complex I allows for greater flavoprotein autoxidation (Turrens and Boveris, 1980).

A dose-dependent relationship between GSH depletion and H₂O₂ production in the presence of glutamate/malate and rotenone is shown in Fig. 2. A biphasic curve is observed—the first phase, in which GSH depletion has no effect on H₂O₂ production, is followed by a second phase, in which GSH depletion is linearly related to increases in H₂O₂ production. These data show that a certain threshold of GSH depletion must be reached before H₂O₂ diffusion from the mitochondrial matrix increases.

View larger version (8K): [in this window] [in a new window]   Fig. 2. Dose-dependent effect of GSH depletion on H2O2 production by complex I. GSH was depleted in isolated heart mitochondria using various concentrations of CDNB. Mitochondria were treated with glutamate/malate plus rotenone, and H2O2 production was monitored using HRP (10 U) and p-hydroxyphenylacetate (1 mM) in buffer [230 mM mannitol, 70 mM sucrose, and 20 mM Tris/HCl (pH adjusted to 7.4 with MOPS)] at 25°C.

H₂O₂ Production Using Complex II Substrate: Effect of GSH Depletion. The addition of the complex II substrate succinate to heart mitochondria also caused dramatic increases in H₂O₂, an effect that was curtailed by rotenone treatment (Table 1). GSH-depleted (90 ± 7%) mitochondria exhibited an increase of more than 2-fold in H₂O₂ production compared with control. The fact that rotenone, a complex I inhibitor, decreased H₂O₂ production by a complex II substrate suggests that a reversal of electron flow was occurring, with H₂O₂ production resulting from oxidation of the flavoprotein in complex I (Turrens and Boveris, 1980; Hensley et al., 1998). This reversed electron flow in the presence of succinate has been observed in other systems and represents another pathway of H₂O₂ production by complex I. The addition of the complex II inhibitor, TTFA, resulted in complete elimination of H₂O₂ production by mitochondria. The effect of TTFA is in agreement with previous observations (Cadenas and Boveris, 1980) and supports the notion that inhibition of complex II does not induce generation.

Decreasing mitochondrial GSH levels also resulted in increased H₂O₂ production in mitochondria treated with succinate (Fig. 3). The GSH-H₂O₂ dose curve with succinate treatment was biphasic, similar to the GSH-H₂O₂ dose curve observed with glutamate/malate plus rotenone treatment. The GSH-H₂O₂ dose curve with succinate had a slightly lower threshold of GSH depletion (27%) needed before H₂O₂ production increased, compared with the threshold observed with glutamate/malate plus rotenone (37%). The fact that succinate treatment was associated with higher H₂O₂ generation suggests that the threshold of GSH depletion needed before increased H₂O₂ production may vary according to the rate of H₂O₂ production.

View larger version (8K): [in this window] [in a new window]   Fig. 3. Dose-dependent effect of GSH depletion on H2O2 production in the presence of succinate. GSH was depleted in isolated heart mitochondria using various concentration of CDNB. Mitochondria were treated with succinate and H2O2 production was monitored using HRP (10 U) and p-hydroxyphenylacetate (1 mM) in buffer at 25°C.

Effect of 50% Mitochondrial GSH Depletion on H₂O₂ Production by Mitochondria. The effect of 50% mitochondrial GSH depletion, as seen in vivo in certain pathological conditions such as ethanol exposure (Fernandez-Checa et al., 1987), on mitochondrial H₂O₂ production was analyzed. Figure 4 shows that the greater the rate of H₂O₂ production, the more 50% GSH depletion enhanced H₂O₂ generation in heart mitochondria. The succinate plus rotenone plus antimycin-treated values represent measurements of short, high bursts of H₂O₂ that occur after antimycin treatment. In presence of glutamate/malate (a very low range of H₂O₂ production, 0.010 ± 0.008 nmol/min/mg), 50% GSH depletion had no affect on H₂O₂ production (data not shown). Mitochondria treated with glutamate/malate may not have had enhanced H₂O₂ production with 50% GSH depletion because it may not have reached the threshold of GSH depletion needed to affect H₂O₂ levels. However, it must be noted that small changes in H₂O₂ levels are difficult to measure when H₂O₂ production rates are so low as with mitochondria treated with glutamate/malate.

View larger version (16K): [in this window] [in a new window]   Fig. 4. Effect of 50% mitochondrial GSH depletion on H2O2 production by mitochondria. The rates of H2O2 production were compared in control and 50% GSH-depleted mitochondria. The values noted in the figure represent the percentage increase as a consequence of 50% GSH depletion. The succinate plus rotenone plus antimycin-treated values represent measurements of short, high bursts of H2O2 that occur in mitochondria after antimycin treatment. Heart mitochondria (0.08 mg) were incubated in a buffer containing 230 mM mannitol, 70 mM sucrose, 20 mM Tris/HCl (pH adjusted to 7.4 with MOPS) at 25°C with HRP (10 U) and p-hydroxyphenylacetate (1 mM). When present, succinate = 7.5 mM, and rotenone = 2 µM. *, P < 0.05 compared with control.

Superoxide Generation by Heart Mitochondria: Effect of Inhibitors and GSH Depletion. EPR experiments with the spin trap DMPO were performed to examine the topology of production in mitochondria and to determine the effect of GSH depletion on production. In the absence of respiratory substrates, no observable EPR signal was observed in mitochondria treated with DMPO (Fig. 5A). The addition of complex I (glutamate/malate 10 mM; Fig. 5B) or complex II (succinate 10 mM + rotenone 2 µM; Fig. 5C) substrates to heart mitochondria resulted in a low-intensity EPR signal characteristic of the DMPO-OH spin adduct (quartet signal with intensity ratios of 1:2:2:1; a^N = a^H = 14.9). The DMPO-OH signal is formed by spontaneous decay of the DMPO-superoxide adduct (DMPO-OOH) in mitochondria (Finkelstein et al., 1980). Both complex I and complex II substrates induced formation, indicating that a low level of is generated by complex III. The addition of rotenone in the presence of glutamate/malate caused no substantial increase in production (Fig. 5D), suggesting that complex I is not a source of into the intermembrane space. The DMPO-OH signal after succinate treatment (data not shown) was similar for mitochondria treated with succinate plus rotenone, suggesting that reverse electron flow did not induce generation toward the intermembrane space. The treatment of mitochondria with the complex II inhibitor TTFA and succinate also failed to generate a DMPO-OH signal (data not shown), indicating that complex II is not a source of into the intermembrane space.

View larger version (31K): [in this window] [in a new window]   Fig. 5. Superoxide generation by heart mitochondria: effect of inhibitors and GSH depletion. Assay conditions: the reaction mixture consisted of mitochondria (0.04 mg of protein/ml) in buffer (230 mM mannitol/70 mM sucrose/20 mM Tris, and 200 U catalase) pH 7.4, supplemented with 160 mM DMPO and respiratory substrate and/or inhibitor. A, no treatment; B, glutamate/malate; C, succinate plus rotenone; D, glutamate/malate plus rotenone; E, succinate plus rotenone plus antimycin; F, succinate plus rotenone plus antimycin—GSH-depleted mitochondria (by 90%); G, succinate plus rotenone plus antimycin plus myxothiazol; H, succinate plus rotenone plus antimycin plus SOD. When present, glutamate/malate = 7.5 mM; succinate = 7.5 mM; rotenone = 2 µM; SOD = 2 µM; myxothiazol = 2 µM; and antimycin = 1 µg/mg protein.

Antimycin, which inhibits complex III to cause increases in levels but inhibits levels (de Vries, 1986), caused an increase of the EPR signal intensity (Fig. 5E) in mitochondria treated with succinate plus rotenone. As expected, no difference in the DMPO-OH signal was observed in control and GSH depleted mitochondria (90 ± 7%) in either succinate plus rotenone-treated mitochondria (data not shown) or mitochondria treated with succinate plus rotenone and antimycin (Fig. 5, E and F). Myxothiazol, a complex III inhibitor that inhibits formation (de Vries, 1986), was found to abolish the EPR signal generated by antimycin (Fig. 5G). The DMPO-OH signal was confirmed to be by the addition of exogenous Cu,Zn-SOD, which abolished the EPR signal generated by mitochondria (Fig. 5H).

Effect of Antimycin and Myxothiazol on and H₂O₂ Production by Complex III: Effect of GSH Depletion. The generation of and H₂O₂ by complex III and their modulation by GSH was further analyzed. As seen with , the production of H₂O₂ from complex III can be enhanced by the addition of the antimycin, an effect largely inhibited by myxothiazol (Fig. 6A). However, myxothiazol did not completely inhibit antimycin-induced H₂O₂ generation, and myxothiazol alone induced H₂O₂ generation from mitochondria. This contrasts with mitochondrial generation, where myxothiazol treatment alone failed to induce generation and almost completely inhibited from the intermembrane space (Fig. 6B). Taken together, these results suggest that induced by antimycin, can release both toward the intermembrane space (where it diffuses through voltage dependent anion channels) and toward the matrix (where it is converted to H₂O₂ by Mn-SOD in the matrix). Myxothiazol, by inhibiting formation, caused a nearly complete inhibition of antimycin-induced toward the intermembrane space and inhibited H₂O₂ generation from the matrix by 70%. The fact that myxothiazol treatment alone enhanced H₂O₂ production from complex III suggests that another component of complex III besides the contributes to generation toward the matrix, in agreement with previous studies (Starkov and Fiskum, 2001; Young et al., 2002). Furthermore, even in the presence of antimycin, it is possible that other components besides contribute to release into the matrix, because H₂O₂ production was higher with antimycin plus myxothiazol (0.43 nmol/min/mg) than with myxothiazol alone (0.28 nmol/min/mg) (Fig. 6A).

View larger version (13K): [in this window] [in a new window]   Fig. 6. Effect of antimycin and myxothiazol on and H2O2 production by complex III: effect of GSH depletion. A, effect of GSH depletion (90 ± 7%), antimycin, and myxothiazol on H2O2 production by heart mitochondria in presence of succinate plus rotenone. Heart mitochondria (0.08 mg) were incubated in a buffer containing 230 mM mannitol, 70 mM sucrose, 20 mM Tris/HCl (pH adjusted to 7.4 with MOPS) at 25°C with HRP (10 U) and p-hydroxyphenylacetate (1 mM). B, effect of antimycin and myxothiazol on production by heart mitochondria in presence of succinate plus rotenone. Assays were carried in buffer (230 mM mannitol/70 mM sucrose/20 mM Tris, and 20 U catalase), pH 7.4, supplemented with 160 mM DMPO using 0.04 mg of protein/ml of mitochondria. levels were estimated using TEMPOL as standard. When present, succinate (succ) = 7.5 mM, rotenone (rot) = 2 µM, myxothiazol (myx) = 2 µM, and antimycin (anti) = 1 µg/mg protein. *, P < 0.05 compared with control. *, P < 0.05 compared with mitochondria treated with succinate plus rotenone plus antimycin.

The depletion of GSH (90 ± 7%) resulted in an 83% increase in H₂O₂ production by mitochondria in presence of antimycin (Fig. 6A). Myxothiazol in certain cases can inhibit complex I and modulate generation, so complex II substrate succinate plus complex I inhibitor rotenone were used in these experiments.

Effect of GSH Depletion on the Activity of Matrix Enzymes Aconitase and Fumarase. To determine the consequences of H₂O₂ production caused by GSH depletion in the mitochondrial matrix, the activities of two matrix enzymes, aconitase and fumarase, were determined. Aconitase contains an iron-selenium cluster in the catalytic site that makes it very susceptible to inactivation by reactive oxygen species such as H₂O₂ (Nulton-Persson and Szweda, 2001). Fumarase, on the other hand, lacks any distinct features, making it vulnerable to H₂O₂. Incubation of mitochondria with glutamate/malate resulted in a decrease of up to 35% in aconitase activity at 45 min in GSH-depleted mitochondria (90 ± 7%), whereas aconitase of control mitochondria displayed only an 11% decrease (Fig. 7A). As expected, much less decrease in fumarase activity was observed in both control and GSH-depleted mitochondria (Fig. 7B). Control mitochondria exhibited only a 7% decrease in fumarase activity, whereas GSH depleted mitochondria exhibited a 15% decrease.

View larger version (11K): [in this window] [in a new window]   Fig. 7. Effect of GSH depletion on the mitochondrial matrix enzyme activity. Control and GSH depleted mitochondria (90 ± 7%) were incubated with glutamate/malate at 25° in buffer containing 230 mM mannitol, 70 mM sucrose, 20 mM Tris-HCl (pH adjusted to 7.4 with MOPS). At the indicated times, mitochondria were treated with 0.5% Triton X-100, and enzyme activity was measured. A, aconitase activity was assayed in a reaction mixture containing 50 mM Tris-HCl, pH 7.4, 30 mM sodium citrate, 0.6 mM MnCl2, 0.2 mM NADP+, and 1 U/ml of isocitrate dehydrogenase. Isocitrate formation was followed at 340 nm at 25° for 10 min. B, fumarase activity was assayed spectrometrically by monitoring the increase in absorbance at 240 nm at 25°C. The reaction mixture was composed of 30 mM KH2PO4, pH 7.4, 0.01 mM ETDA, and 5 mM malate. *, P < 0.05 compared with control.

	Discussion

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Extensive studies using isolated mitochondria and various inhibitors have identified the flavoprotein component in complex I (Turrens and Boveris, 1980) and ubisemiquinone in complex III (Cadenas et al., 1977; Turrens et al., 1985) as the major sources of in the respiratory chain. The stimulatory effects of antimycin, which increases levels but inhibits formation (de Vries, 1986), suggests that not , is important in generation by complex III. X-ray crystallography studies have shown that the Qo site in complex III lies near the intermembrane space (Iwata et al., 1998), whereas flavoprotein of NADH dehydrogenase is suspected to be oriented toward the matrix side (Walker, 1992). Thus, mitochondrial topology predicts that complex I generates toward the matrix, whereas complex III generates toward the intermembrane space. Our results are in agreement with what is predicted by topology of mitochondria. In the presence of complex I substrates, H₂O₂ was observed diffusing from the matrix (caused by generation into the matrix which is converted to H₂O₂ by Mn-SOD), and the generation of by complex I toward the intermembrane space was not observed. On the other hand, in the presence of either complex I or II substrates, a small signal that is enhanced by antimycin is observed, indicating complex III generation of into the intermembrane space.

Our findings also shows that in complex III does releases not only toward the intermembrane space but also to the matrix. However, in the presence of antimycin, it is possible other components besides might contribute to release into the matrix. Looking at the relative nanomoles of (0.32 nmol/min/mg) and H₂O₂ (2.4 nmol/min/mg) induced by antimycin treatment in GSH-depleted mitochondria, it seems that most is generated toward the matrix rather than the intermembrane space. However, because can interact with intermembrane space constituents such as cytochrome c before detection by EPR (Han et al., 2003), we are probably underestimating the amount of being generated into the intermembrane space. In addition, these values may be misleading if some of the measured H₂O₂ generated after antimycin treatment is formed in the intermembrane space from spontaneous dismutation or catalyzed by an intermembrane space Cu,Zn-SOD (intermembrane Cu,Zn-SOD has reported in liver but has never been confirmed in heart mitochondria) (Okado-Matsumoto and Fridovich, 2001; Inarrea, 2002). The topology of and H₂O₂ in heart mitochondria based on this and other work is summarized in Fig. 8. The relative percentages of generated toward the intermembrane space and into the matrix by remain an important unresolved question.

View larger version (34K): [in this window] [in a new window]   Fig. 8. Scheme showing the topology of superoxide and hydrogen peroxide production by heart mitochondria. Complex I seems to generate only toward the matrix, whereas Complex II seems to play no role in generation by the electron transport chain. Complex III, on the other hand, generates toward both the intermembrane space and the matrix. In presence of antimycin, seems to generate toward both the intermembrane space and the matrix. and cytochrome b are alternative candidates for generation of into the matrix, as observed with myxothiazol treatment.

Although is generally considered the major source for in complex III, our work with myxothiazol supports other studies suggesting that components other than in complex III can generate into the matrix (Starkov and Fiskum, 2001; Young et al., 2002). Ubisemiquinone at the Q_I site () and cytochrome b are reasonable candidates for release into the matrix induced by myxothiazol and merit further investigation (Fig. 10). The iron-selenium clusters in complex III are believe to lie near the intermembrane space and so seem to be less likely candidates for production into the matrix, although they cannot be ruled out. Topologically, myxothiazol is a unique complex III inhibitor because it can inhibit generation toward the intermembrane space and yet stimulate generation into the matrix.

GSH peroxidase is considered the major detoxification system for H₂O₂ in the mitochondrial matrix (Chance et al., 1979; Antunes et al., 2002). Although mitochondrial catalase has been described in heart mitochondria (Nohl and Hegner, 1978; Radi et al., 1991), its activity is extremely low and does not seem to play a significant role in H₂O₂ detoxification (Antunes et al., 2002). Similarly, mitochondrial peroxiredoxins, because of their low rate constant with H₂O₂, cannot compensate for the loss of GSH peroxidase activity in mitochondria (Hofmann et al., 2002). This study provides strong experimental evidence for the importance of mitochondrial GSH levels in modulating H₂O₂ levels in the mitochondrial matrix, which had previously only been hypothesized (Flohe et al., 1976). An interesting aspect of this work was the observation of a threshold effect—a certain level of GSH needed to be depleted before H₂O₂ diffusion from the matrix was increased. This observation is in line with the large stoichiometric differences between GSH and H₂O₂ that normally favor detoxification by GSH in the matrix. Small changes in GSH levels are predicted not to affect the catalytic activity of GSH peroxidase because GSH levels are in excess in the matrix. We observed that the GSH-H₂O₂ dose curve with succinate required a slightly lower threshold of GSH depletion (27%) before H₂O₂ production increased than the threshold observed with glutamate/malate plus rotenone (37%). This suggests that the rate of H₂O₂ production may determine the GSH depletion threshold, a fact that may have important physiological relevance in vivo.

Disorders such as long-term ethanol intake and cerebral ischemia are associated with decrease in mitochondrial GSH levels of more than 50% (Fernandez-Checa et al., 1987). What are the expected consequences when GSH levels decrease by approximately 50%? Our results show the changes in H₂O₂ production by GSH-depleted mitochondria depend on the level of H₂O₂ generated by mitochondria in vivo. Under normal conditions, H₂O₂ generation by mitochondria is difficult to measure without the aid of inhibitors because basal levels of H₂O₂ production by mitochondria are low. In addition, oxygen tensions in vivo may be up to a magnitude lower than in in vitro system (240 µM in buffer) using isolated mitochondria in which most H₂O₂ measurements are made. Because H₂O₂ generation is linearly related to O₂ tension (Boveris and Chance, 1973), H₂O₂ production in vivo is expected to be lower than measured using isolated mitochondria. At low measured rates of H₂O₂ production (0.02—0.10 nmol/min/mg), we were unable to observe any change in H₂O₂ production in 50% GSH-depleted mitochondria. Thus, under basal conditions, 50% mitochondria GSH depletion is expected to have little or no impact on H₂O₂ diffusion from the mitochondrial matrix.

H₂O₂ production depends on the metabolic state of mitochondria and may become exaggerated under certain conditions. H₂O₂ production may be modulated by NADH levels, substrate levels, and ADP/ATP levels (Boveris and Chance, 1973; Han et al., 2001), and endogenous regulator molecules, such as ceramides, have antimycin like effects; binding to complex III causes increased H₂O₂ production from mitochondria (Garcia-Ruiz et al., 1997). In these conditions of elevated mitochondrial H₂O₂ production that can occur in many pathological conditions, 50% mitochondrial GSH depletion may have profound effects on H₂O₂ diffusion from the matrix in vivo.

It has recently been observed that generation by complex I can be enhanced by glutathionylation (Taylor et al., 2003). Thus, GSH/GSSG redox status may modulate H₂O₂ production from complex I. Conversely, the amount of GSSG in the matrix is determined by H₂O₂ levels, along with GSSG reductase activity, NADP⁺/NADPH status, and GSH concentrations. A complex inter-relationship between GSH levels, GSH redox status, NADP/NADPH status, and H₂O₂ production seems to exist in mitochondria and needs to be further explored. There is no evidence that glutathionylation of complex III can occur. In our work, increased H₂O₂ production from complexes I and III was observed after CDNB treatment, suggesting that changes in GSH levels, not glutathionylation, were responsible for increases in mitochondrial H₂O₂ production.

H₂O₂ and released by mitochondria have been implicated in signal transduction, pathophysiology, and aging (Cadenas and Davies, 2000). The present studies have addressed the effect of mitochondrial GSH depletion on mitochondrial H₂O₂ and generation. release into the intermembrane space by complex III is not influenced by GSH status but H₂O₂ release, which is a result of matrix-directed from complex I or III, is greatly influenced by GSH status. Moderate (50%) depletion of matrix GSH amplifies H₂O₂ release when directed to the matrix is increased, as may occur in pathological conditions. Moderate depletion of mitochondrial GSH therefore may be of considerable importance in sensitizing cells to disease conditions in which H₂O₂ and production are greatly stimulated, such as tumor necrosis factor signaling and effects of sphingolipids. This may contribute to the development of apoptosis or necrosis as well as redox effects on signal transduction.

	Footnotes

 
The study was supported primarily by Project 1 (N.K.) of the USC-UCLA Research Center for Alcoholic Liver and Pancreatic Diseases [National Institute on Alcohol Abuse and Alcoholism (NIAAA) grant P50-AA11199 (to N.K.)]. D.H. was also supported in part by NIAAA-sponsored postdoctoral training grant T32-AA07578. Resources of the Cell Biology Core, USC Research Center for Liver Diseases, supported by National Institute of Diabetes and Digestive and Kidney Diseases grant P30-DK48522, were used for mitochondrial preparations.

ABBREVIATIONS: DMPO, 5,5'-dimethyl-1-pyrroline-N-oxide; CDNB, 1-chloro-2,4 dinitrobenzene; RCR, respiratory control ratio; EPR, electron paramagnetic resonance; MOPS, 4-morpholinepropanesulfonic acid; UQ, ubisemiquinone; SOD, superoxide dismutase; HRP, horseradish peroxidase; HPLC, high-performance liquid chromatography.

Address correspondence to: Dr. Derick Han, School of Medicine, University of Southern California, Los Angeles, CA 90089-9121 USA. E-mail: derickh{at}usc.edu

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