The reduction of glutathione disulfide produced by t-butyl hydroperoxide in respiring mitochondria

Free Radical Biology & Medicine, Vol. 20, No. 3, pp. 433-442, 1996 Copyright © 1996 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/96 $15.00 + .00 ELSEVIER

SSDI 0891-5849(95)02093-4

Original Contribution THE

REDUCTION

t-BUTYL

OF

GLUTATHIONE

HYDROPEROXIDE

DISULFIDE

IN RESPIRING

PRODUCED

BY

MITOCHONDRIA

HANLIN L I U and JAMES P. KEHRER Division of Pharmacology and Toxicology, College of Pharmacy, The University of Texas at Austin, Austin, TX, USA

(Received 27 June 1995; Revised 6 September 1995; Accepted 11 September 1995)

Abstract--Factors affecting the reduction of GSSG by rat liver mitochondria after a t-butyl hydroperoxideinduced (t-BOOH) oxidative stress were studied. The amounts of ADP and mitochondrial protein were adjusted to consume less than 50% of the available oxygen during the 8-min experimental period. A 4-min treatment of mitochondria with 24 nmol t-BOOH/mg protein (60 ~M) oxidized 91% of total glutathione. In the presence of glutamate/malate, succinate or ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) (state 4 respiration), 84, 84, and 28% of the GSSG formed during t-BOOH treatment was reduced after 4 min, respectively. A similar extent of reduction was seen during state 3 respiration (1.5 mM ADP) with glutamate/malate, but no reduction occurred during state 3 respiration with either succinate or ascorbate/TMPD. The succinate-supported reduction of GSSG was completely blocked by rotenone, antimycin A, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), or 1,3-bis (2-chloroethyl)- 1-nitrosourea (BCNU). In contrast, oligomycin potentiated GSSG reduction using either glutamate/malate or succinate as substrates. Rotenone partially blocked glutamate/ malate-supported GSSG reduction. NADPH levels were altered in direct proportion to the effects on GSSG reduction. The current data indicate that the reduction of GSSG in oxidatively- stressed isolated rat liver mitochondria occurs most efficiently during state 4 respiration and is independent of ATP synthesis. Both transhydrogenation and the transmembrane proton gradient appear to be important in NADPH regeneration and consequent GSSG reduction. Keywords--Mitochondria, Oxidative stress, tert-Butyl hydroperoxide, Glutathione, Glutathione Disulfide, Mitochondrial respiration, Reducing equivalents, Free radicals

glutathione reductase [ NADPH:GSSG oxidoreductase, E.C. 1.6.4.2], vitamin E, and glutathione.

INTRODUCTION

Leakage of partially reduced oxygen molecules from various pathways is a normal occurrence of oxidative metabolism. Evidence that mitochondria are a significant source of these reactive oxygen species (ROS) is extensive, 1-3 although the actual quantity generated under physiological conditions is not clear. 4 Because of the constant and unavoidable exposure to ROS, a battery of defense systems have evolved. Mitochondrial defenses against ROS are similar to, but independent of, those in the cytoplasm. These defenses include manganese superoxide dismutase, glutathione peroxidases (both selenoenzyme and nonselenoenzyme),

GSH is synthesized only in the cytoplasm but is present in higher concentrations in the mitochondrial matrix (10 to 12 mM) than in the cytoplasm (1 to 8 mM). 5'6 GSH is, therefore, transported across the mitochondrial inner membranes against a concentration gradient. There is no transport system for GSSG e f f l u x . 7 Thus, mitochondria need a large capacity to reduce GSSG in order to prevent the disruption of thiol and/or pyridine nucleotide redox balance that appears to be closely coupled to function. Glutathione peroxidase catalyzes the reduction of hydroperoxides produced by various reactions within cells, tert-Butyl hydroperoxide (t-BOOH) is an organic hydroperoxide analogous to naturally formed short chain lipid hydroperoxides. This water-soluble oxidant is largely reduced via the glutathione peroxi-

Address correspondence to: James P. Kehrer, Division of Pharmacology and Toxicology, College of Pharmacy, The University of Texas at Austin, Austin, TX 78712-1074, USA. 433

434

H. LJtJ and J. P. KEHRER

dase pathway to the relatively inert product tert-butyl alcohol. The GSSG formed in this reaction is reduced by glutathione reductase at the expense of NADPH. The kinetics of hydroperoxide degradation by the mitochondrial glutathione peroxidase system have been studied. 8"9 However, there are many competing pathways for reducing equivalents in mitochondria, and the efficiency of mitochondrial GSSG reduction under different respiratory conditions has not been clearly defined. For example, GSSG reduction in mitochondria isolated from AS-30D tumor cells, after treatment with t-BOOH, reportedly occurs in actively respiring organelles. 1° It has been also reported that exogenous oxidants are decomposed much slower during state 3 respiration (in the presence of ADP). ~'~ Other studies have demonstcated that most Krebs-cycle acids can prevent the oxidation of GSH and potentiate the reduction of GSSG formed during treatment with hydroperoxides. 5'8'9 Further, the rate of reduction of t-BOOH or diamide is diminished by inhibitors of mitochondrial function. 9,~ The availability of N A D P H should be the ultimate determinant of cellular and mitochondrial thiol redox status following an oxidative stress. In an attempt to clarify the relationship between the rnitochondrial respiratory state and disulfide reduction, the current study examined in greater detail and with lower oxidant loads than earlier work the redox status of rat liver mitochondrial glutathione and pyridine nucleotides during different respiration states following a tB O O H - i n d u c e d oxidative stress. The results showed that the reduction of GSSG required N A D P H and occurred predominantly during state 4 respiration. This reduction was not ATP dependent, but was largely proton-motive force dependent. Factors known to either affect the transmembrane proton gradient or interfere with the transhydrogenase activity were able to change the profile of mitochondrial GSSG reduction. EXPERIMENTAL

Animals Male S p r a g u e - D a w l e y rats (175 to 200 g) were supplied by Harlan S p r a g u e - D a w l e y (Houston, TX) and housed at the Animal Resources Center at the University of Texas at Austin for at least 1 week prior to use. The animals were maintained on a 12 h light/ dark cycle and were provided with food and water ad lib.

Reagents ADP, albumin (fraction V), antimycin A, t-BOOH, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), D,L-di-thiothreitol, EGTA, N-ethylmaleimide

( N E M ) , L-glutamic acid, GSH, GSSG, L ( - - ) malic acid (disodium salt), 3-(N-morpholino)propane sulfonic acid ( M O P S ) , NAD+, N A D H (disodium salt, from yeast, grade III), NADP + (sodium salt), NADPH (tetrasodium salt), oligomycin, rotenone, succinic acid, N,N,N',N'-tetramethyl-p-phenylenediamine ( T M P D ) , and Tris (hydroxymethyl)-aminomethane (Trizma base) were obtained from Sigma Chemical Company (St. Louis, M O ) . 1,3-Bis(2-chloroethyl )- l-nitrosourea ( B C N U ) was purchased from Bristol-Myers (Evansville, IN). o-Phthalaldehyde ( O P A ) was purchased from Fisher Scientific Company (Fair Lawn, NJ). All other chemicals were reagent or spectrophotometric grade. PeroXOquant T M quantitative peroxide assays kits were obtained from Pierce Chemical Company (Rockford, IL).

Isolation of mitochondria All experimental procedures were started by 0900 h to avoid circadian variation of tissue GSH levels. Rats were anesthetized with 50 m g / k g sodium pentobarbital and then given 200 IU of heparin via the inferior vena cava. One minute later, the liver was perfused through the portal vein with 50 ml of ice-cold mitochondrial isolation buffer (225 mM mannitol, 10 mM MOPS, 75 mM sucrose, and 1 mM EGTA, pH 7.4; MSE buffer). The liver was removed, rinsed in MSE buffer, and weighed. The liver tissue was then finely minced and homogenized in 30 ml cold MSEA buffer (MSE with 1 mg/ml of albumin). The homogenate was then diluted to 40 ml with ice-cold MSEA and centrifuged at 500 x g for 10 min (4°C). The resulting supernatant was centrifuged at 12,500 × g for 7 min to get a mitochondria-rich pellet that was carefully rinsed 2 to 3 times with 5 to 10 ml of cold MSEA to remove the fluffy layer. The remaining pellet was resuspended in 5 ml of MSEA, diluted with 35 ml MSEA, and centrifuged at 12,500 × g for 7 min. The resultant pellet was gently resuspended and centrifuged as described above. The final pellet was resuspended in ice-cold MSEA buffer (final volume of 2 - 2 . 5 ml; 3 0 - 4 0 mg mitochondrial protein/ml). Protein was determined by the microbiuret assay. ~2 Mitochondrial function was examined by measuring oxygen utilization with a Clarke-type electrode at 30°C in a 1.5-ml chamber. Mitochondrial protein (600 to 800 #g) was added to 1.5 ml incubation buffer (250 mM sucrose, 10 mM KH2PO4 and 10 mM Tris base, pH 7.5 ). State 4 respiration was measured in the presence of 1.3 mM malate and 13 mM glutamate, 5 mM succinate, or 5 mM ascorbic acid with 0.05 mM TMPD. State 3 respiration was initiated by adding ADP at an initial concentration of 500 nmol/ml. The respiratory control ratio ( R C R ) was calculated using

Mitochondrial reduction of glutathione oxygen consumption rates during state 3 and subsequent state 4 respiration.

435

and plotting the peak area against amounts. Data were calculated as nmol/mg protein.

Experimental system Pyridine nucleotide extraction and assay Mitochondria with initial RCRs --> 4 for succinate and -> 5 for glutamate/malate were incubated at 2.5 mg protein/ml in an open U-shape water jacketed chamber (50 ml) with magnetic stirring at 30°C to avoid anoxia, t-BOOH was added and after a 4 min incubation, state 3 or state 4 respiration was initiated. The amounts of mitochondria and substrate were proportional to those used in function measurements. Sufficient ADP was added to maintain state 3 respiration for the duration of the experiment (8 min). When used, rotenone (0.1 # M ) , antimycin A (7 # M ) , oligomycin (5 # M ) , or FCCP (0.4 # M ) (final concentrations) was added before substrate. In some studies, mitochondria were incubated with 0.1 mM BCNU for 10 min at room temperature before any other treatment.

Glutathione assay Mitochondrial glutathione levels were determined on 0.5 ml samples frozen in liquid nitrogen and stored at - 7 0 ° C overnight by modifying a previously reported method.~3 For total glutathione measurement, samples were homogenized in 25 mM sodium phosphate buffer (pH 7.0). GSSG was measured in a duplicate sample treated with 20 mM NEM to remove GSH. Homogenate (200 #1) was mixed with 200/.tl 25 mM DTT (in 25 mM sodium phosphate buffer, pH 7.0) and 100 #1 Tris buffer (100 mM, pH 8.5) and incubated for 30 min on ice. Proteins were precipitated by adding 0.5 ml 5-sulfosalicylic acid (3.75% [ w / v ] ) and the sample was centrifuged for 10 min at 3500 x g, 4°C. An aliquot (200 #1) of the resulting supernatant was mixed with 200 #1 OPA solution [50 mg OPA in 0.5 ml methanol, then diluted to 10 ml with 0.4 M potassium borate solution (pH 9.9)] and incubated for 2 min at room temperature. The mixture was neutralized with 200 #1 sodium phosphate buffer (250 mM, pH 7.0). The final solutions were filtered at 0.2 # and kept in darkness on ice for a maximum of 24 h before analysis by HPLC. The HPLC mobile phase was composed of 0.15 M sodium acetate buffer, pH 7.0 (92.5%), and methanol (7.5%). Samples (100 #1) were injected onto a C18 column with a flow rate of 1.5 ml/min. The retention time of the glutathione-OPA derivative was 6.5 min. Detection was achieved fluorometrically with excitation and emission wavelengths of 340 nm and 420 nm, respectively. Standard curves were generated by derivatizing known amounts of GSH and GSSG that were subjected to the same procedures as a mitochondrial sample

The mitochondrial incubation mixture (1 ml) was removed and mixed with 30 #1 10 M KOH. The samples were neutralized by adding 350/zl 1 M KH2PO4, centrifuged at 3500 × g for 10 min at 4°C, and the supernatant was filtered at 0.2 #m. The alkaline extract was either injected immediately into HPLC (500 #1/ injection) or stored at - 7 0 ° C for later analysis. Quantitative determination of each compound was accomplished by a modification of the HPLC method described by Noack et al. 14 Samples were injected onto a 25 cm C-18 reversed phase column (Partisil 5 0 D S 3 with WVS cartridge, Whatman Inc., Clifton, N J) with a Brownlee RP-18 guard column cartridge (Rainin Instrument Co.). The mobile phase was a binary step gradient of potassium phosphate (0.2 M, pH 5.95 ) and methanol. The total flow rate was 1.3 ml/min as follows: 0% methanol, 0 to 5 min, 4% methanol, 5 to 15 min, 8% methanol, 15 to 25 min, 40% methanol, 25 to 26 min. Individual nucleotides were detected at 254 nm. Standard curves were generated for NAD, NADH, NADP, and NADPH by extracting a mixture containing known amounts of all four pyridine nucleotides using the same procedures as for a mitochondrial sample. The peak areas were plotted against the corresponding amounts. The recovery of each analyte standard was measured and ranged from 93% to 104%.

Peroxide assay The PeroXOquant T M assay was used for the quantitative determination of t-BOOH. The mitochondrial incubation ( 100/zl) was mixed with the assay reagents. This mixture was incubated for 20 min at room temperature and the absorbance at 560 nm determined. The micromolar extinction coefficient of the xylenol orange-Fe 3+ complex is 1.5 × 10 : in 25 mM H2SO4. Measurements of a t-BOOH standard indicated a linear relationship between peroxide concentration and absorbance at 560 nm (R 2 = 0.9997).

Statistics Data are expressed as means ___ standard error. All data were analyzed by analysis of variance and, where required, post hoc analyses were performed using the S t u d e n t - N e w m a n - K e u l ' s test. A p-value of less than 0.05 was considered significant.

436

H. Ltu and J. P. KEHRER Table 1. Effect of t-BOOH on the Production of GSSG in Isolated Rat Hepatic Mitochondria~' t-BOOH (nmol/mg protein) Control 6 15 24 30

GSSG (% total glutathione) 2.5 ± 1.2 4 2.6 92 ± 3 85 ± 6.3

(3) (1) (1) (3)* (3)*

GSSG levels were determined 4 min after the addition of t-BOOH. Data are expressed as mean + SE. Values in parentheses = n. * Significantly different from control (p < 0.001).

RESULTS Total G S H in freshly isolated rat liver m i t o c h o n d r i a was 6.3 ___ 0.4 n m o l / m g protein with 2.5% in the form o f G S S G . The oxidation o f mitochondrial G S H by tB O O H s h o w e d a threshold response ( T a b l e 1). At doses up to 15 n m o l t - B O O H / m g protein, no significant G S S G f o r m a t i o n was o b s e r v e d while there was a 91% oxidation o f total m i t o c h o n d r i a l G S H 4 min after treatment with 24 n m o l / m g protein ( 6 0 # M ) . Further increases in the dose o f t - B O O H did not result in significantly m o r e G S S G production. The threshold effect was p r o b a b l y due to the need to c o n s u m e all available m i t o c h o n d r i a l reducing equivalents prior to the accum u l a t i o n o f G S S G . W i t h the dose o f 24 n m o l / m g protein, the P e r o X O q u a n f f M assay indicated that the concentration o f t - B O O H d e c r e a s e d rapidly and no peroxide could be detected at the end o f the 4 rain incubation ( < 1 # M ) . Thus, a dose o f t - B O O H at 24 n m o l / m g protein was chosen for all further studies e x a m i n i n g the reduction o f G S S G by mitochondria.

Effect of t-BOOH on mitochondrial function W h e n m a i n t a i n e d on ice, m i t o c h o n d r i a l function was well p r e s e r v e d during the 4 to 5 h e x p e r i m e n t a l period. The low doses o f t - B O O H used in these studies significantly d e c r e a s e d the respiratory control ratio ( T a b l e 2 ) . This was due to an increase in state 4 respiration rates, while state 3 rates r e m a i n e d unchanged.

The effect of different respiratory states and substrates on the reduction of GSSG A 4 - m i n incubation with g l u t a m a t e / m a l a t e , succinate, or a s c o r b a t e / T M P D supported the reduction o f 84, 84, and 28% o f the G S S G f o r m e d by t - B O O H

treatment, respectively (Fig. 1 A ) . W i t h o u t the addition o f a reducing cosubstrate there was no reduction o f G S S G during this 4 min period. A l t h o u g h the extent o f G S S G reduction during state 3 (with 1.5 m M A D P ) respiration with g l u t a m a t e / m a l a t e was identical to that seen under state 4 conditions, no statistically significant G S S G reduction was o b s e r v e d during state 3 respiration with either succinate or a s c o r b a t e / T M P D as substrates (Fig, 1B).

Effect of an uncoupling agent or inhibitor of ATP synthesis on the reduction of GSSG The reduction o f mitochondrial G S S G , supported either by g l u t a m a t e / m a l a t e or succinate, was decreased b y the uncoupler F C C P from 84% to 7% and 2% for g l u t a m a t e / m a l a t e (Fig. 2 A ) and succinate (Fig. 2 B ) , respectively. Treatment of rat liver mitochondria with o l i g o m y c i n , an A T P synthase inhibitor, greatly enhanced the m i n i m a l reduction o f G S S G supported by succinate during state 3 respiration (Fig. 1B vs. Fig. 3 A ) . T w o minutes after initiation o f state 3 respiration, the reduction o f G S S G in the o l i g o m y c i n treatment group was significantly different from control (Fig. 3 A ) . In the presence o f g l u t a m a t e / m a l a t e , which supports G S S G reduction both in the presence and absence o f A D P , o l i g o m y c i n had no effect (Fig. 3 B ) .

Effect of respiratory chain inhibitors on the reduction of mitochondrial GSSG W i t h succinate as substrate under state 3 conditions, there was no significant reduction o f G S S G either with or without rotenone (Fig. 4 A ) . W h e n g l u t a m a t e / m a late was used as substrate, only 45% o f the mitochondrial G S S G f o r m e d during 4 min t - B O O H treatment was reduced in rotenone-treated m i t o c h o n d r i a (Fig. 4 A ) c o m p a r e d with 83% reduction obtained in the corresponding group without rotenone. U n d e r state 4 conditions, rotenone decreased the succinate-supported

Table 2. The Effect of t-BOOH on the Function of Isolated Rat Hepatic Mitochondria~ RCR" Treatment

Glutamate/Malate

Succinate

Control t-BOOH Treatment

9.8 ± 1.2 3.7 _+ 0.3*

7.1 + 1 2.7 ± 0.3*

~ Mitochondria were incubated with 24 nmol t-BOOH/ mg protein for 4 min at 30°C before measuring respiratory function. Data are expressed as mean -+ SE (n = 3). b State 3/State 4 respiration. *'* Significantly different from RCR obtained before tBOOH treatment (control) (p < 0.01 and 0.05, respectively).

Mitochondrial reduction of glutathione

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study, mitochondria were incubated with 0.1 mM for 10 min at room temperature in order to minimize mitochondrial damage that occurs with longer incubation times. Under these conditions, mitochondrial glutathione reductase activity was inhibited 19 _+ 0.6%. Activity of this enzyme was determined by measuring the oxidation of NADPH at 340 nm in the presence of an excess of GSSG at 30°C ~6 following lysis of the mitochondria. It is possible that reactivation of enzyme activity occurred during this process. Although the extent of measured inhibition was relatively small, the ability of the mitochondria given succinate under state 4 conditions to reduce GSSG formed during t-BOOH treatment was inhibited by 90% (data not shown).

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Fig. 1. Reduction of GSSG by respiring mitochondria. Rat hepatic mitochondria (2.5 m g protein/ml ) were incubated with t-BOOH (24 nmol/mg protein) for 4 min. State 4 respiration ( A ) was then initiated by adding either glutamate/malate (20 m M / 2 m M ) , succinate (30 m M ) , or ascorbate/TMPD (30 mM/0.3 m M ) . State 3 respiration (B) was initiated with ADP (1.5 mM, 600 n m o l / m g protein). Glutathione levels were determined at the indicated time points after addition of t-BOOH. Data are expressed as means _+ SE (n = 3 5). *Significantly different from control (p < 0.05). *Significantly different from ascorbate treatment group (p < 0.01 ). ~Significantly different from all other groups (p < 0.01 ).

reduction of mitochondrial GSSG from 84 to 8% after 4 min (Fig. 4B). Antimycin A, an inhibitor of succinate dehydrogenase, reportedly prevents the degradation of t-BOOH by mitochondria. 8 In the current study, the 84% reduction of mitochondrial G S S G supported by succinate was fully prevented by antimycin A (Fig. 5A). Antimycin A also blocked the minimal reduction of G S S G supported by succinate during state 3 respiration (Fig. 5B).

Effect of BCNU BCNU inhibits glutathione reductase in vitro at concentrations from 0.05 to 0.5 mM. "~'j5 In the current

There was a pronounced decrease in NADPH, 4 min after treatment with t-BOOH (Table 3). Conversely, both NAD + and NADP + levels were elevated at this time. The addition of reducing cosubstrates (state 4 respiration) rapidly restored the levels of N A D P H (4 vs. 6 min). There was a tendency for N A D H to reach supranormal levels 2 min after the addition of substrate. Glutamate/malate appeared to be somewhat more effective than succinate in reversing t - B O O H - i n d u c e d changes in the pyridine nucleotide profile. Total mitochondrial pyridine nucleotides were not altered significantly by any of these treatments.

The redox status of mitochondrial pyridine nucleotides under various conditions The N A D H / ( N A D + N A D H ) ratio, an indicator of the reduction state of this nucleotide, increased fourfold 2 min after glutamate/malate was added (6 min after tB O O H ) (Fig. 6A). The N A D H / ( N A D + N A D H ) ratio was also increased by succinate, but to a significantly lesser extent than by glutamate/malate (Fig. 6B ). FCCP prevented the glutamate/malate supported increase in the N A D H / ( N A D + N A D H ) ratio while rotenone enhanced this increase. In contrast, rotenone prevented the increase supported by succinate. Treatment with t-BOOH significantly lowered the mitochondrial N A D P H / ( N A D P + N A D P H ) ratio (Fig. 7A). The addition of glutamate/malate rapidly reversed this effect. Both FCCP and rotenone prevented the reduction of NADP by glutamate/malate, even though rotenone increased N A D H levels. Succinate also supported an increase in the N A D P H / ( N A D P + N A D P H ) ratio (Fig. 7B). This effect was again blocked by rotenone, which also inhibited the reduction of GSSG (Fig. 4). Treatment of mitochondria with BCNU prevented the utilization of NADPH by

438

H. Llu and J. P. KEHRER 100-

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At the level of an intact cell, the supply of NADPH involves multiple pathways including the hexosemonophosphate shunt, the malic enzyme, the NADP + dependent isocitrate and glutamate dehydrogenases, and the NAD + and N A D H kinases. ~s Because mitochondria lack glucose 6-phosphate dehydrogenase, transhydrogenation is believed to be an important mechanism by which the NADP + redox state is maintained.S.;9.20 It has been estimated that the NADPH generated by membrane potential-dependent transhydrogenase is responsible for about half of the t-BOOH degraded during an oxidative stress in rat liver mitochondria, and NADP + -dependent isocitrate dehy-

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Fig. 2. Effect of FCCP on reduction of mitochondrial GSSG during state 4 respiration. Rat hepatic mitochondria (2.5 mg protein/ml) were incubated with t-BOOH (24 nmol/mg protein) for 4 min. State 4 respiration was then initiated by adding ( A ) glutamate/malate 20 m M / 2 mM or (B) succinate (30 mM). FCCP was added at a concentration of 0.4/zM. Glutathione levels were determined at the indicated time points after addition of t-BOOH. Data are expressed as means _+ SE (n = 3). *Significantly different from its corresponding group without FCCP treatment (p < 0.01 ).

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DISCUSSION

The present study examined in detail the supply of reducing equivalents for mitochondrial GSSG reduction. Within mitochondria, electrons are captured from a variety of oxidative reactions and then transfered as pairs into the respiratory chain. These dehydrogenases use either pyridine or flavin nucleotides as electron acceptors. N A D H cannot directly be used by glutathione reductase under physiological conditions. 16 Thus, N A D P H is the primary nucleotide that channels reducing equivalents to glutathione and/or to other antioxidants. ~7

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Fig. 3. Effect of oligomycin on the reduction of mitochondrial GSSG during state 3 respiration. Rat hepatic mitochondria (2.5 mg protein/ ml) were incubated with t-BOOH (24 nmol/mg protein) for 4 min. State 3 respiration was initiated by adding (A) succinate (30 mM) or (B) glutamate/malate (20 raM/2 mM) followed by ADP (1.5 raM, 600 nmol/mg protein). Oligomycin was added at a concentration of 5 /.tM. Glutathione levels were determined at the indicated time points after addition of t-BOOH. Data are expressed as mean _+ SE (n = 3). *Significantly different from control (p < 0.05). ~Significantly different from the corresponding group without oligomycin treatment (p < 0.05). ~Significantly different from control (p < 0.001) but not the corresponding group without oligomycin treatment (p > 0.05).

Mitochondrial reduction of glutathione 100 A

N A D P H w e r e c o n s u m e d , and there w a s no r e d u c t i o n o f the G S S G f o r m e d w i t h o u t a d d i n g a r e d u c i n g c o s u b strate. S e v e r a l studies h a v e c o m p a r e d the ability o f d i f f e r e n t substrates to s u p p l y the r e d u c i n g e q u i v a l e n t s f o r G S S G r e d u c t i o n in m i t o c h o n d r i a . 5'8"1°'24'25 In the

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c u r r e n t study, r e d u c t i o n o f G S S G o c c u r r e d d u r i n g b o t h state 3 and state 4 m i t o c h o n d r i a l r e s p i r a t i o n w h e n g l u t a m a t e / m a l a t e , that f e e d s e l e c t r o n s into site I and, thus, has the m o s t n e g a t i v e r e d u c t i o n potential, w a s u s e d as substrate. S u c c i n a t e , w h o s e d e h y d r o g e n a t i o n r e q u i r e s F A D as c o f a c t o r ( t h a t f e e d s e l e c t r o n s into site II and has a r e l a t i v e l y less n e g a t i v e r e d u c t i o n p o t e n t i a l ) , prov i d e d m i t o c h o n d r i a w i t h f e w r e d u c i n g e q u i v a l e n t s to r e d u c e G S S G d u r i n g state 3 respiration, but w a s e q u i v alent to m a l a t e / g l u t a m a t e u n d e r state 4 c o n d i t i o n s . T h e

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3

t-BOtH

Fig. 4. Effect of rotenone on the reduction of mitochondrial GSSG. Rat hepatic mitochondria (2.5 mg protein/ml) were incubated with t-BOtH (24 nmol/mg protein) for 4 min. (A) State 3 respiration was initiated by adding either glutamate/malate (20 mM/2 mM) or succinate (30 mM ) and ADP (1.5 raM, 600 nmol/mg protein). (B) State 4 respiration was initiated by adding succinate (30 mM). Rotenone was added at a concentration of 0.1/zM. Glutathione levels were determined at the indicated time points after addition of tBOTH. Data are expressed as means _+ SE (A) n = 3 for glutamate/ malate, n = 4 for succinate (B) n = 3 for succinate, n = 4 for succinate with rotenone. *Significantly different from control (p < 0.01 ). ~Significantly different from the respective group without rotenone (p < 0.001 ).

5

6

7

i 6

7

8

t-BOtH

100-

80Succinate +

f

~60-

ADP

-+ Antimycin A

40

20-

d r o g e n a s e t a k e s c a r e o f the o t h e r half. 8 H o w e v e r , the p r o d u c t i o n o f N A D P H c a n i n c r e a s e d r a m a t i c a l l y and b e m a i n t a i n e d at a h i g h l e v e l a c c o r d i n g to the d e m a n d t9 and all N A D P H - p r o d u c i n g e n z y m e s a p p e a r to res p o n d to an o x i d a t i v e stress. 2t It s e e m s l i k e l y that NADPH production will be influenced by competing p a t h w a y s and the t i s s u e i n v o l v e d , a n d u n d e r s o m e c o n d i t i o n s the s u p p l y o f N A D P H m a y b e c o m e l i m i t i n g f o r G S S G r e d u c t i o n . 22 S i m i l a r to p r e v i o u s reports, ~0,23 m i t o c h o n d r i a c o n t a i n e d a h i g h l e v e l o f g l u t a t h i o n e that w a s o n l y 2 . 5 % o x i d i z e d . T h e o x i d a t i o n o f m i t o c h o n d r i a l G S H b y tBOtH o c c u r r e d r a p i d l y after e n d o g e n o u s l e v e l s o f

4

TIME (rain)

0 0

i 1

i 2

---I--

Control

--D-"

Succinate

i 3

i 4

+ ADP

i 5

TIME (min) t-BOtH

Fig. 5. Effect of antimycin A on the mitochondrial reduction of GSSG supported by succinate. Rat hepatic mitochondria (2.5 mg protein/ml) were incubated with t-BOtH (24 nmol/mg protein) for 4 min. Respiration was initiated with succinate (30 mM) without (A) or with (B) ADP ( 1.5 mM, 600 nmol/mg protein). Antimycin A was added at a concentration of 7 #M. Glutathione levels were determined at the indicated time points after addition of t-BOtH. Data are expressed as means ___ SE (n = 4 for succinate state 3, n = 3 for other groups). *Significantly different from corresponding group without antimycin A treatment (p < 0.001 ), but not control group (p > 0.05).

440

H. LIu and J. P. KEHRER Table 3. Levels of Pyridine Nucleotides in Isolated Rat Hepatic Mitochondria" Substrate:

None

Time (min)

Glutamate/Malate

0

NAD NADH NADP NADPH

1.07 0.07 0.47 1.04

4

± 0.24 ± 0.01 _+ 0.07 ± 0.25

1.74 0.07 1.43 0.17

6

_+ 0.46 ± 0.01 ± 0.22* _+ 0.03*

1.05 0.51 0.60 1.87

Succinate 8

± 0.32 ± 0.16 ± 0.08* _+ 0.62*

1.29 0.42 0.52 1.24

± 0.26 + 0.24 _+ 0.061 ± 0.27 +

6 1.43 0.22 0.46 1.19

± 0.64 ± 0.13 _+ 0.08* +_ 0.43*

8 1.39 0.26 0.53 1.17

_+ 0.63 _+ 0.18 _+ 0.09* _+ 0.43 '~

Rat hepatic mitochondria (2.5 mg protein/ml) were incubated with t-BOOH (24 nmol/mg protein) for 4 min (control). Respiration was then initiated by adding glutamate/malate (20 mM/2 mM) or succinate (30 mM). The levels of individual mitochondrial pyridine nucleotides are expressed as nmol/mg protein ± SE (n = 5 for 0 and 4 min, n = 3 for all other time points). * Significantly different from 0 min (p < 0.05). + Significantly different from 4 min (p < 0.05).

5O

45~

A

I*+

-l40

35-

=

Glutamate/Malate

A

Glutamate/Malate +

f

R~:~o2~:e/Malale+

I

/

dL

30252015 lOS1

II

o

.

.

4

o

. 5

.

.

6 TIME (min)

7

8

7

8

substrate ± inhibitor

tBOOH

B

25-

-l--

~ 20_

Succinate

--O-- Succinate + Rot. . . . . ~ Succinate+ BCNU

~

1

ol

II

0

.

0

4

+

+

tBOOH

[

~

.

~

. 5

. 6

.

TIME (min)

substrate + inhibitor

Fig. 6. The redox status of NAD ( H ) in isolated rat hepatic mitochondria (2.5 m g protein/ml) incubated with t-BOOH (24 nmol/mg protein) for 4 min. State 4 respiration was then initiated by adding ( A ) glutamate/malate (20 m M / 2 m M ) or (B) succinate (30 m M ). FCCP (0.4/~M) or rotenone (0.1 # M ) were added to the incubation system at the same time as substrate. Individual reduced and oxidized mitochondrial pyridine nucleotides were determined at the indicated time points after addition of t-BOOH. The redox status of NAD (H) was calculated and expressed as N A D H / ( N A D + N A D H ) × 100%. Data are expressed as means ± SE (n = 3 except for BCNU where n = 2). *Significantly different from 0 and 4 rain after t-BOOH treatment (p < 0.001). *Significantly different from the corresponding time point in FCCP treatment group (p < 0.05).

greatly decreased rate of GSSG reduction supported by succinate during state 3 respiration is likely due to the presence of competing reactions that channel the electron flow to other metabolic pathways such as oxidative phosphorylation. Ascorbate/TMPD was much less effective in supporting the reduction of GSSG, presumably because it inserts even fewer electrons into the respiratory chain at site III. Together, these results indicated that the reduction potential of the substrate is important with regard to the reduction of GSSG. Both transhydrogenation and glutamate dehydrogenase may play a role in supplying NADPH when glutamate/malate is used as substrate. J8 In addition, reversed electron transport 26'27is probably involved with succinate- and ascorbate/TMPD-supported GSSG reduction. This conclusion is supported by the ability of rotenone, which inhibits the reversed electron transport, to also inhibit the reduction of GSSG. The uncoupling agent FCCP, which abolishes the mitochondrial proton gradient, prevented the production of NADPH, indicating a role for the transmembrane potential, although the consumption of NADH may also have been a factor. Consistent with these findings, lubrol, which can disrupt the membrane potential, inhibits the reduction of t-BOOH in isolated rat mitochondria 9 while the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) blocked succinate- and pyruvate-supported GSSG reduction in brain mitochondria. 25 The inhibition of ATP production by oligomycin did not block the reduction of mitochondrial GSSG. In fact, the reduction of GSSG was potentiated when succinate was used to energize the mitochondria. Because oligomycin can inhibit ATP synthase activity while leaving the transmembrane proton gradient unaltered (or even enhanced), it appears that NADPH can be generated in the absence of ATP. Whether the proton gradient is required, or simply the absence of a drain on reducing equivalents as occurs during uncoupling, thereby increasing FADH2 and initiating reversed electron transport to reduce NAD + to NADH, remains unclear.

Mitochondrial reduction of glutathione

,00/ ---I-- Glutamate/Malate + ~- s0]_+_ GlutamatefMalate .o,..... '~

~-Z+ 6 0

z

Glutamate/Malate + FCCP

~

~

,

/ j-

"[" -

/

I

40-

t

gh Z

2oo

• I

0

tBOOH

nm=

4 +

t

5 TIME

8

* * q [ ~ ~

---ll--- Succinate

/~

Succinate +

!60-'-.ot .....

Succinate +

~/

/

/

/3-

°~ 2o

o±

+

t tBOOH

7

substrate _+ inhibitor

B

80 -

6 (rain)

TIME

(min)

substrate _+ inhibitor

Fig. 7. The redox status of NADP(H) in isolated rat hepatic mitochondria (2.5 mg protein/ml) incubated with t-BOOH (24 nmol/ mg protein) for 4 rain. State 4 respiration was then initiated by adding (A) glutamate/malate (20 mM/2 mM) or (B) succinate (30 mM). FCCP (0.4 #M) or rotenone (0.1 #M) were added to the incubation system at the same time as substrate. Individual reduced and oxidized mitochondrial pyridine nucleotides were determined at the indicated time points after addition of t-BOOH. The redox status of NADP(H) was calculated and expressed as NADPH/ (NADP+NADPH) × 100%. Data are expressed as means _+ SE (n = 3). *Significantly different from time 0 (p < 0.001 ). **Significantly different from 4 min after t-BOOH treatment (p < 0.01 ). • Significantly different from the corresponding time points in glutamate/malate only group (p < 0.05). ~Significantly different from 0 and 4 min (p < 0.05). ~Significantly different from the corresponding time points in succinate and BCNU treatment groups (p < 0.05).

T h e p r e d o m i n a n t effect o f other m i t o c h o n d r i a l respiratory chain inhibitors on m i t o c h o n d r i a l G S S G reduction was inhibition. Rotenone, a site I inhibitor, leads to the a c c u m u l a t i o n o f N A D H in the m i t o c h o n drial matrix thereby preventing further oxidation o f reducing cosubstrate. It has been reported that rotenone also blocks the reversed electron transport. 2s This agent does not inhibit e n e r g y - l i n k e d transhydrogenation, but it does inhibit the n o n e n e r g y - l i n k e d reaction c a t a l y z e d by the b e e f heart e n z y m e . 29 In the presence o f rotenone, the activity o f rat liver m i t o c h o n d r i a l t r a n s h y d r o g e n a s e

441

tends to be l o w e r ( u n p u b l i s h e d d a t a ) . Together, these effects can explain the decreased formation o f N A D P H and, thus, decreased reduction o f G S S G in the presence o f rotenone. H o w e v e r , rotenone was only able to partially prevent g l u t a m a t e / m a l a t e - s u p p o r t e d G S S G reduction. This m a y be attributed to: ( a ) an incomplete b l o c k a d e o f electron transfer; ( b ) high concentrations o f N A D H driving transhydrogenation; or ( c ) glutamate d e h y d r o g e n a s e directly y i e l d i n g N A D P H . A n t i m y c i n A c o m p l e t e l y b l o c k e d the succinate-supported G S S G reduction, p r e s u m a b l y by stopping succinate oxidation, but also perhaps by b l o c k i n g energylinked transhydrogenation reactions. 3° Alternately, G S S G production m a y have been increased because a n t i m y c i n A enhances m i t o c h o n d r i a l oxidant production. 3j R e d u c e d p y r i d i n e nucleotide levels after antim y c i n A were not measured. H o w e v e r , it has been reported that N A D P H is o x i d i z e d to N A D P in the presence o f a n t i m y c i n A. 8 The steady-state concentrations o f various redox pairs within cells are generally well controlled. O u r previous w o r k in intact heart tissue indicated that N A D P H is particularly well maintained, even in the presence o f severe stresses, while N A D H levels are m o r e variable. 32 T h e maintenance o f N A D P H levels in hepatocytes has been studied and m a y be related to the interconversion o f N A D ( H ) to N A D P ( H ) . 2°'33 This process is b e l i e v e d to be c a t a l y z e d b y N A D + or N A D H kinase. 2° N A D + kinase activity is increased during o x i d a t i v e stress, suggesting that this is an important transhydrogenase pathway. 34 H o w e v e r , the c o m p l e t e a b s e n c e o f any role for A T P 32 and current data indicate that only the e n e r g y - i n d e p e n d e n t p a t h w a y can be involved. A previous report that t - B O O H leads to a r a p i d and extensive oxidation o f N A D H and a slow N A D P H oxidation 35 differs from the current study, most likely due to the use o f much higher levels o f p e r o x i d e (3 m M ) and the presence o f rotenone in the earlier study. Overall, e n e r g y - l i n k e d transhydrogenase c a t a l y z e d direct transfer o f h y d r o g e n from N A D H to N A D P + appears to be important as a redox buffer mechanism. s'19'36 The current data are consistent with this pathway. H o w e v e r , multiple N A D P H - p r o d u c i n g pathw a y s including isocitrate d e h y d r o g e n a s e and glutamate d e h y d r o g e n a s e m a y be responsible for a portion o f the reduction o f mitochondrial G S S G in the presence o f glutamate/malate.2 Acknowledgements--This work was supported by NIH grant

HL51005. J.P.K. is the Gustavus and Louise Pfeiffer Professor of Toxicology. REFERENCES

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442

H. Ltu and J. P. KEHRER

2. Boveris, A. Mitochondrial production of superoxide radical and hydrogen peroxide. Adv. Exp. Med. Biol. 78:67-82; 1976. 3. Turrens, J. F.; Boveris, A. Generation of superoxide anion by the NADH dehydrogenase of bovine heart mitochondria. Biochem. J. 191:421-427; 1980. 4. Kehrer, J. P. Free radical as mediators of tissue injury and disease. Crit. Rev. Toxicol. 23:21-48; 1993. 5. Jocelyn, P. C. Some property of mitochondrial glutathione. Biochim. Biophys. Acta 396:427-436; 1975. 6. Wahllander, A.; Soboll, S.; Sies, H. Hepatic mitochondrial and cytosolic glutathione content and the subcellular distribution of GSH-S-transferases. FEBS Lett. 97:138-140; 1979. 7. Olafsdottir, K.; Reed, D. J. Retention of oxidized glutathione by isolated rat liver mitochondria during hydroperoxide treatment. Biochim. Biophys. Acta 964: 377-382; 1988. 8. Kurosawa, K.; Shibata, H.; Hayashi, N.; Sato, N.; Kamada, T.; Tagawa, K. Kinetics of hydroperoxide degradation by NADPglutathione system in mitochondria. J. Biochem. 108:9-16; 1990. 9. Jocelyn, P. C.; Dickson, J. Glutathione and the mitochondrial reduction of hydroperoxides. Biochim. Biophys. Acta 590:1 - 12; 1980. 10. Brodie, A. E.; Reed, D. J. Glutathione disulfide reduction in tumor mitochondria after t-butyl hydroperoxide treatment. Chem.-Biol. Interact. 84:125-132; 1992. 11. Jocelyn, P. C. The reduction of diamide by rat liver mitochondria and the role of glutathione. Biochem. J. 176:649-664; 1978. 12. ltzhaki, R. F.; Gill, D. M. A micro-biuret method for estimating proteins. Anal. Biochem. 9:401-410; 1964. 13. Neuschwander-Tetri, B. A.; Roll, F. J. Glutathione measurement by high-performance liquid chromatography separation and fluorometric detection of the glutathione-ortho-phthalaldehyde adduct. Anal. Biochem. 179:236-241; 1989. 14. Noack, H.; Kunz, W. S.; Augustin W. Evaluation of a procedure for the simultaneous determination of oxidized and reduced pyridine nucleotides and adenylates in organic phenol extracts from mitochondria. Anal. Biochem. 202:162-165; 1992. 15. Eklow, L.; Moldeus, P.; Orrenius, S. Oxidation of glutathione during hydroperoxide metabolism. Eur. J. Biochem. 138:459463; 1984. 16. Carlberg, 1.; Mannaervik, B. Purification and characterization of the flavoenzyme glutathione reductase from rat liver. J. Biol. Chem. 250:5475-5480; 1975. 17. Reed, D. J. Glutathione: Toxicological implications. Annu. Rev. Pharmacol. Toxicol. 30:603-631; 1990. 18. Sies, H. Nicotinamide nucleotide compartmentation. In: Sies, H., ed. Metabolic compartmentation. London: Academic Press; 1982:205-231. 19. Hoek, J. B.; Rydstr6m, J. Physiological role of nicotinamide nucleotide transhydrogenase. Biochem. J. 254:1-10; 1988. 20. Stubberfield, C. R.; Cohen, G. M. Interconversion of NAD(H) to NADP(H): A cellular response to quinone-induced oxidative stress in isolated hepatocytes. Biochem Pharmacol. 38:26312637; 1989. 21. Winkler, B. S.; DeSantis, N.; Solomon, F. Multiple NADPHproducing pathways control glutathione (GSH) content in retina. Exp. Eye Res. 43:829-847; 1986. 22. Tribble, D. L.; Jones, D. P. Oxygen dependence of oxidative stress. Rate of NADPH supply for maintaining the GSH pool during hypoxia. Biochem. Pharmacol. 39:729-736; 1990.

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ABBREVIATIONS:

BCNU--

1,3-bis ( 2 - c h l o r o e t h y l ) - 1 - n i t r o s o u r e a

t-BOOH--t butyl hydroperoxide FCCP--p-trifluoromethoxyphenylhydrazone MOPS--3-(N-morpholino)propane NEM--N-ethylmaleimide OPA-- o-phthalaldehyde

sulfonic acid

R C R - - r e s p i r a t o r y control ratio ROS--reactive oxygen species TMPD-N,N,N',N'-tetramethyl-p-phenylenediamine