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Mitochondrial glutathione depletion in alcoholic liver disease
Alcohol, Vol. 10, pp. 469-475, 1993 Printed in the U.S.A. All riots reserved.
0741-8329/93 $6.00 + .00 Copyright © 1993Pergamon Press Ltd.
Mitochondrial Glutathione Depletion in Alcoholic Liver Disease JOSI~ C. F E R N A N D E Z - C H E C A , * t I T A K E S H I H I R A N O , * H I D E K A Z U TSUKAMOTO~: A N D N E l L K A P L O W I T Z *
*Division of Gastrointestinal and Liver Diseases, University of Southern California School of Medicine, The Department of Veterans Affairs Outpatient Clinic, Los Angeles, CA 90033 t Unidad de H[gado, Hospital Clinic i Provincial, Universidad de Barcelona, Barcelona 08036, Spain ~Vletro Health Medical Center, Department of Veterans Affairs Medical Center, Case Western Reserve University, Cleveland, OH 44109 FERNANDEZ-CHECA, J. C., T. HIRANO, H. TSUKAMOTO, AND N. KAPLOWITZ. Mitochondrial glutathione depletion in alcoholic liver disease. ALCOHOL 10(6) 469-475, 1993.-Alcoholic liver disease (ALD) is one the most serious consequences of chronic alcohol abuse. Liver cirrhosis, the culmination of the illness, is one of the leading causes of death in Western countries. Mitochondria are a target of ethanol intoxication mainly due to the toxic effects of acetaldehyde, a byproduct of ethanol metabolism. Morphological and functional changes in mitochondria are one of the key hallmarks of chronic ethanol exposure in both chronic alcoholics and experimental models of alcoholism. The functional changes observed in mitochondria from ethanol-treated animals are translated in an overall decrease in ATP levels resulting from a lower rate of ATP synthesis as a consequence of impaired processing at the translational level of some components of oxidative phosphorylation encoded by mitochondrial DNA genome. Mitochondrial glutathione (GSI-I) plays a critical role in the maintenance of cell functions and viability and in mitochondrial physiology by metabolism of oxygen free radicals generated in the respiratory chain. GSH in mitochondria originates from cytosol by a transport system which translocates GSH into the matrix. This transport system is impaired in chronic ethanol-fed rats, which translates in a selective and significant depletion of the mitochondrial GSH content resulting in the development of an increased susceptibility to oxidant stress. Using the intragastric infusion model of experimental ALD in rats, the profound and selective mitochondrial GSH depletion precedes the onset of alcoholic fiver disease, mitochondrial lipid peroxidation, and progression of liver damage. These results suggest
that depletion of mitochondrial GSH by impairment of its transport from cytosol into mitochondria could be an additional contributing factor in the development of ALD by favoringpro-oxidant productionand oxidant stress in mitochoodria. Oxidant stress Mitochondrial GSH transport Mitochondrial physiology
Oxygen free radicals
LIVER cirrhosis is the culmination of alcoholic liver disease (ALD) and is still one of the leading causes of death in developed countries. However, the pathogenesis of the alcoholinduced liver disease is not yet completely understood. The causes that lead to the development of this human disease state are multifactorial, so that the hypotheses and factors which might cause or contribute to the ethiology of ALD are numerous. Despite the possible role that malnutrition might play in the overall development of the disease, it is likely that ALD is mainly a consequence of the toxic effects of ethanol (1,17,34). As a consequence of chronic ethanol intake, biochemical changes in the hepatocyte occur due to the metabolism of ethanol resulting in cellular redox potential shift (NAD÷/NADH decrease) and production of acetaldehyde, a potent toxicant. Additional factors resulting from ethanol metabolism have been documented in animal models of ALD, including autoimmune-induced injury by antibodies to protein-acetaldehyde adducts, hemodynamic alterations of
Experimental alcoholicliver disease
the hepatic blood supply along sinusoids, and peroxidation of membrane lipids and oxidant stress (3,24). The purpose of the present paper is to emphasize the functional alterations of mitochondria induced by ethanol feeding and to focus our attention on the mitochondrial pool of glutathione in the physiology of this organelle and the role that its selective depletion by chronic ethanol feeding might play in the Tsukamoto-French animal model of ALD (48-50). MITOCHONDRIA: A TARGET OF ETHANOL INTOXICATION
Most of the ethanol that enters the circulation undergoes an oxidative metabolism in the liver. Ethanol is converted into acetaldehyde by different routes: alcohol dehydrogenase, a cytosolic enzyme; the microsomal ethanol-oxidizing system (MEOS), and the peroxisomal catalase. Acetaldehyde produced from ethanol is a very reactive substance which accounts for most of the toxic effects of ethanol. This aldehyde
Requests for reprints should be addressed to Dr. Jos6 C. Fermindez-Checa, Unidad de Htgado, Hospital Clinic i Provincial, Villarroel, 170,
08036-Barcelona, Spain.
469
470 is mainly metabolized in mitochondria by the l o w - K m acetaldehyde dehydrogenase, and its activity decreases in advanced states of chronic ethanol intoxication. Its distribution is not homogeneously localized in the liver acinus, displaying an increased gradient from the perivenous to periportal areas of the liver (13). This could be one of the factors that contribute to the selective centrilobular necrosis observed in chronic alcoholics. Functional and morphological changes in mitochondria are key features of chronic ethanol exposure. In chronic alcoholics as well as in animal models of alcoholism, mitochondria are enlarged, appearing as swollen or elongated structures with cristae disrupted or without normal organization (8, 25,37). This suggests the possibility that hepatic energy metabolism is compromised by chronic ethanol intake, given the key role of mitocbondria in energy conservation. This will result in impaired bepatocellular ATP production, which in turn might have important functional consequences. The lower ATP levels observed in chronic ethanol-fed animals maintalned on the diet for three weeks results from a lower state 3 respiration with NADH-linked substrates (complex I) (9-11). Slightly longer feeding periods also decrease the succinatedriven state 3 respiration (complex II) in ethanol-fed mitochondria. The ADP-stimulated electron transport is sustained during extended periods of time and affects all segments of the electron transport chain. Thus, the state 3 respiration through the cytochrome oxidase portion of the electron transport chain is depressed by chronic ethanol feeding (44,46). The magnitude of this depressed effect by ethanol feeding ranges between 20 and 40% compared to control mitochondria, and there is lifle variation at longer feeding times (16). The lower oxygen consumption in the presence of ADP and Pi (state 3 respiration) demonstrates that the rate at which ATP is synthesized via the oxidative phosphorylation process is lower in ethanol-fed mitochondria. The rate of oxygen consumption in the absence of ADP (state 4 respiration) is not altered by chronic ethanol feeding to the same degree as is state 3 respiration. An increase in the state 4 respiration would indicate that the mitochondria have less capacity to conserve the proton gradient generated by electron transport and the mitocbondria would be less well coupled. This resting state of respiration is an indication of the functional viability of isolated mitochondria. Feeding ethanol for three to four weeks results in either no loss or a slight decrease (10-15% vs. control values) in state 4 respiration when either NADH-linked substrates or succinate were utilized as oxidizable substrates (10,43). At longer feeding periods, significant increases in state 4 respiration are observed, indicating that the mitochondria had become more uncoupled. However, state 3 respiration through cytochrome oxidase is significantly depressed by chronic ethanol, pointing to the dramatic alteration in this portion of the energy conservation system (10,16,44). Thus, the overall decreased ATP levels observed in animals treated chronically with ethanol result from a lower rate of ATP synthesis rather than from a decreased efficiency in the synthesis of ATP, since there is little or no change in the resting state respiration, which indicates a lack of uncoupling by ethanol feeding.
FERNANDEZ-CHECA ET AL. ations in several of the respiratory chain components by ethanol. The activity and haem content of cytochrome oxidase is decreased 50-600/0 in ethanol mitochondria compared to control organelles. In addition, cytochrome b and some of the iron sulphur centres associated with NADH-ubiquinone reductase complex are also decreased by ethanol (4,40,41,43, 47). Electron transport and proton translocation through the NADH-ubiquinone reductase portion of the electron transport chain are decreased by 30 and 40°70, respectively (4,7). The mitochondrial ATP synthetase is a very complex structure with the catalytic subunits (Ft) projecting into the matrix of the mitochondria and the proton channel portion (F0) formed by other polypeptides attached to the catalytic portion that traverses the inner membrane [for review, see (22)]. It has been shown in both inner membrane preparations and submitochondrial particles capable of synthesizing ATP that the ATPase is significantly lowered by chronic ethanol feeding (23,46). The same results have recently been shown in intact mitochondria (7). In addition, ethanol feeding lowers the oligomycin sensitivity of ATP synthetase and renders it less tightly attached to the inner membrane. The decrease in oligomycin sensitivity and the structural changes of the ATP synthase induced by ethanol are associated exclusively in the F0 subunits of the complex (35). The molecular basis for the ethanol-induced lesion in the oxidative phosphorylation system of mitochondria is at the level of translation of several components. Mitochondria have a circular DNA that encodes only 13 open reading frames in addition to two rRNAs and a complete set of tRNAs with a high degree of direct sequence homology (2,5,51). The mitochondrial DNA gene products have been identified as members of the oxidative phosphorylation system (Table 1). Those portions of the oxidative phosphoryiation system that are decreased by ethanol feeding are encoded by the mitochondrial DNA, whereas those components not affected by ethanol have a nuclear gene expression and are subsequently imported from the cytoplasm into mitochondria. These results suggest that
TABLE 1 EFFECTS OF ETHANOLON THE MITOCHONDRIAL DNA PRODUCTSOF COMPONENTSOF THE OXIDATIVEPHOSPHORYLATIONSYSTEM MitochondrialDNAGeneExpression GeneProduct
Protein Levels
mRNALevels
MOLECULARBASISFOR THE ETHANOL-INDUCEDALTERATION OF THE OXIDATIVEPHOSPHORYLATIONSYSTEM
ND5* COIt ND4* Cyt b:~ ND2* NDI* COIIt COIIIt ATPase 6§ ND6* ND3* ATPase 8§ NIML*
Decreased Decreased Decreased Decreased Decreased Decreased Decreased Decreased Decreased Decreased Decreased Decreased Decreased
ND Unchanged Unchanged ND ND ND Unchanged Unchanged Unchanged ND Unchanged Unchanged Unchanged
Since the mitochondrial oxidative phosphorylation system is multicomponent, changes in any single component of the system would result in the rate limiting synthesis of ATP and decreased oxygen consumption. There are dramatic alter-
ND = not determined. *Components of the NADH-ubiquinone reductase complex. ,Components of the cytochrome oxidase subunits. $Cytochromeb. §Twoof the membrane subunits (F0)of ATPase synthase.
MITOCHONDRIAL GLUTATHIONE DEPLETION IN ALD chronic ethanol consumption could interfere with mitochondrial gene expression. The rate of incorporation of radiolabeled methionine into all polypeptides was lower for all 13 polypeptides, establishing a correlation between loss of catalytic activity and decrease in the rate of synthesis of those components affected by chronic ethanol feeding (14). However, ethanol feeding does not affect the DNA, total RNA levels, or the RNA polymerase activity. With slot blot analyses, eight of the mRNA and ribosomal RNA were present in normal amounts in ethanol mitochondria (26), indicating that the defect caused by ethanol is in the processing of the mRNA of the mitochondrial gene products. Thus, the amount of ribosomes from ethanol mitocbondria are decreased by 60-70% compared to control mitochondria, which could be the result of decreased synthesis in the endoplasmic reticulum or a decrease in transport across the mitochondrial membranes into the matrix (15). MITOCHONDRIAL GLUTATHIONE
Intracellular GSH is synthesized exclusively in cytosol, but it is also found in mitoehondria and the nucleus. The mitochondHal pool of GSH is derived from cytosol by a mitochondrial transporter which translocates GSH into the mitochondrial matrix. The concentration of GSH in mitochondria is thought to be as high as in cytosol (10-15 raM). The exact mechanism of this transport system is presently unknown, although recent studies have suggested that GSH diffuses into mitochondria through a channel (27) or is transported by a primary active, high affinity transport system (31). Our recent studies have shown a rapid exchange between cytosol and mitochondria with a t~/2 of mitochondrial GSH of 18 rain (18). The mitochondrial GSH participates in a redox cycle through the GSH peroxidase/reductase activities (38,42). Reduction of endogenous hydroperoxides by GSH peroxidase may be a key function of mitochondrial GSH. As a consequence of aerobic metabolism, cells are under an endogenous oxidative stress challenge. Even under physiological conditions, the reduction of molecular oxygen to water in the respiratory chain is incomplete and involves the formation of toxic oxygen intermediates (12). Hydrogen peroxide, if not reduced to water, can lend to the formation of the very reactive hydroxyl radical through the Fenton-HaberWeiss reaction resulting in the formation of lipid peroxides that can damage mitochondrial membranes and their functions (6). Although mitochondria have a Mnsuperoxide dismutase (Mn-SOD) isoenzyme, it would afford only partial protection because its action would lead to formation of hydrogen peroxide. Since mitochondria do not contain catalase (36), their detoxification of hydroperoxides, formed either under physiological conditions or as a consequence of bioreduction reactions to activate a drug to an unstable intermediate(s), depends exclusively on GSH peroxidase. This enzyme utilizes the reducing equivalents of GSI-I which, in turn, is converted to oxidized giutathione (GSSG). The GSH redox cycle uses NADPH- and, indirectly, NADH-reducing equivalents in the mitochondrial matrix to provide a recycling supply of GSH by the GSSG-reductase catalyzed reduction of GSSG. Thus, GSH is critical in protecting unsaturated fatty acids in membrane phospholipids from peroxidation and mitochondrial DNA from strand scission by attack of oxygen free radicads generated either endogenously or as a result of ethanol metabolism. The fact that mitochondrial GSH depletion below critical levels is associated with cell injury in a variety of organs and that its selective repletion affords protection emphasizes the
471 importance of this small pool of GSH in maintaining vital cell functions (30,32,33). It is thought that mitochondrial GSH plays this critical role through regulation of mitochondrial inner membrane permeability by maintaining Ca2+ homeostasis. GSH and pyridine nucleotide oxidation in mitochondria precede an increase in the permeability of the inner membrane to Ca2+ (28,29). This suggests that oxidative stress or severe GSH depletion affects the redox status in the mitochondria. Although mitochondrial regulation of cytosolic Ca2+ seems unlikely physiologically, under pathologic conditions, mitochondria may be a major regulator of cytosolic Ca2+ concentrations (39). Several groups have noted a correlation between the depletion of mitochondrial GSH and the inability of mitochondria to sequester Ca2+ [for review, see (19)]. In agreement with this, it has been shown that acetaminophen depletes mitochondrial GSH to a greater extent than its regioisomer 3'hydroxyacetanilide, which correlates with the formation of mitochondrial protein adducts and disruption of cellular Ca2+ homeostasis by inhibiting the plasma membrane calciumATPase. Alterations in the Ca2+ cycling in the inner mitochondrial membrane may be an important regulator of cytosolic concentrations of Ca2+, which leads to cell injury, since ruthenium red, an inhibitor of the mitochondrial Ca2+ uniport system, can prevent oxidative stress and the associated injury under certain conditions (19). E T H A N O L SELECTIVELY IMPAIRS T H E M I T O C H O N D R I A L TRANSPORT OF GSH IN A L D
In view of the striking structural and functional changes of mitochondria induced by ethanol consumption, as detailed previously, and in light of the key function that mitochondrial GSH plays in maintaining the integrity of this organelle and cell viability, it would be important to determine if depletion of the mitochondrial GSH pool by ethanol feeding determines the development or contributes to the progression of ALD. A suitable model of experimental alcoholic liver disease which reproduces different stages of the ALD lesion observed in humans was not available until recently (48-50). However, the use of other models of experimental alcoholism such as the Lieber-DeCarli liquid diet has allowed the determination of the effect of chronic ethanol feeding in rats on the homeostasis of mitochondrial GSH. Until recently, no specific data were available in the literature on the effect of ethanol on the mitochondrial GSH pool. Using the Lieber-DeCarli model in wellnourished rats, it has been shown that chronic ethanol feeding decreases rather substantially (50-60%) the mitochondrial pool of GSH in isolated hepatocytes from ethanol-fed rats compared to pair-fed animals (20,21). This effect was accompanied by a modest decrease of cytosol GSH in ethanol-fed rats, an effect that was variable and did not result from impaired GSH synthetic capacity by ethanol. The effect of chronic ethanol feeding on the depletion of mitochondHal GSH is rather selective, since after two weeks of feeding the depletion of the mitochondrial GSH pool is the only change observed in chronic ethanol-fed hepatocytes. The selective effect of ethanol on the mitochondrial pool of GSH requires the in vivo administration of ethanol, since in vitro incubation of hepatocytes with ethanol (up to 80 mM) up to 4 h does not affect this pool of GSH. Since the mitochondrial pool of GSH is derived from the uptake of cytosol GSH, we tried to increase the mitochondrial pool of GSH in ethanol-fed hepatocytes by increasing the concentration of cytosol GSH using methionine as precursor of GSH (Fig. l). In contrast to control mitochondria which increase their pool of GSH in parallel to the increase of the cytosol GSH pool, in ethanol-fed rats
472
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mitochondria do not increase their pool of GSH despite a significant increment of the cytosol one (21). These data demonstrate a lower relationship of cytosol to mitochondriai GSH induced by ethanol feeding, suggesting an impaired capacity for repletion of GSH into mitochondria. To rule out the possibility of an artifactual effect on the depletion of mitochondrial GSH by ethanol seen in isolated hepatocytes, and to determine if such selective depletion results from an impaired uptake of GSH from cytosol to mitochondria rather than an increased release from mitochondria, we have performed tracer kinetic analyses on the transport of radiolabeled cytosol GSH into mitochondria from ethanol- and pair-fed liver biopsies in vivo
(18). A rapid fractionation of the liver specimens into cytosol and mitochondria allowed us to determine the relationship of isotopic equilibrium between labeled cytosol (precursor) and mitochondria (product). Modelling of these data have shown that the mass fractional rate of transport of GSH from cytosol to mitochondria is decreased by 35% in ethanol-fed livers, which is translated into a lower steady-state pool size of GSH. Thus, the depleted mitochondrial pool of GSH by ethanol results from an impaired capacity of the mitochondriai transport system which translocates GSH. Recently we have examined the homeostasis of the mitochondrial GSH pool (48-50) using the Tsukamoto-French ex-
MITOCHONDRIAL GLUTATHIONE DEPLETION IN ALD perimental model of ALD to determine the temporal relationship of mitochondrial GSH depletion and progression of ALD at different times of ethanol feeding. Intragastric infusion of ethanol to rats leads to a selective depletion of GSH in the mitochondrial fraction of the rat liver compared to control liver, with no changes seen in the cytosol GSH pool at any time of infusion. This decrease ranges from 40, 61, and 85% after infusion of ethanol at 3, 6, and 16 weeks, respectively (Fig. 2) (45). This effect is absolutely specific for liver, since renal mitochondria show unchanged GSH pool sizes. Serum alanine transferase (ALT) levels increased significantly at 6 and 16 weeks of infusion, and mitochondrial lipid peroxidation was observed only when the mitochondrial pool of GSH was severely depleted at 16 weeks of feeding. Since this model of experimental ALD reproduces various histological stages of the human disease depending on the time of infusion (from liver steatosis to necrosis and fibrosis at 3, 6, and 16 weeks of feeding), these results suggest that the selective depletion of mitochondrial GSH by ethanol precedes the onset of alcoholic liver disease which is associated with mitochondrial lipid peroxidation and progression of liver damage. In the LieberDeCarli model of ethanol toxicity in rats, depletion of mitochondrial GSH pool size renders the hepatocytes more susceptible to cell injury by oxidant stress, which is then reversed when the mitochondrial pool of GSH is increased by GSHethyl ester (18). Thus, results obtained in both animal models indicate that depletion of mitochondrial GSH by ethanol could be an additional contributing factor in the development of ALD by decreasing antioxidant capacity, thus disturbing the balance of pro-oxidants and antioxidants in favor of oxidant stress in mitochondria. The mechanisms by which ethanol perturbs the capacity of mitochondria to transport GSH from cytosol are presently unknown and are the subject of ongoing research. Lower levels of the functional mature protein due to a decreased nuclear gene expression of the protein, posttranslational modification changes, or alterations in the mechanism of import of nuclear proteins into mitochondria as well as qualitative changes in the physicocbemical properties of mitochondrial membrane by ethanol or any combination of those factors could result in an organelle devoid of GSH to protect it from endogenous or exogenous oxidant stress. It is unclear whether impaired transport capacity in mitochondria is a cause or effect of mitochondrial dysfunction by ethanol feeding. However, it is worth emphasizing that this impaired mitochondrial transport capacity is not the result of an "unspecific" or generalized effect by ethanol, since other transport systems of the mitochondria including the A D P / P i exchanger, the dicarboxylate and tricarboxylate exchanger, the A D P / A T P translocator, and the NADPH shuttles are not affected by ethanol metabolism (15). It remains to be seen in the near future if correcting this selectively depleted GSH pool size would alter the progression of ALD in the intragastric infusion model, and if this mitochondrial GSH depletion is also present in chronic alcoholics at different stages of the disease.
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FIG. 2. Effect of time course of chronic ethanol infusion on hepatic GSH in cytosol and raitochondrial fractions. *p < 0.01 vs. control by unpaired t test. From Hepatology 16:1423-1428; 1992. Reprinted with permission. ACKNOWLEDGEMENTS The work presented here has been supported by grants from the Department of Veterans Affairs (N.K. and H.T.), NIAAA AA 09526 (N.K., J.C.F.-C., and H.T.), and DIGICYT, Spain (PB92-1110 to J.C.F.-C.).
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rats after prolonged alcohol consumption. Exp. Cell Res. 33:350354; 1964. Kobayashi, M.; Seki, T.; Yaginuma, K.; Knike, K. Nucleotide sequences of small ribosomal RNA and adjacent transfer RNA genes in rat mitochondrial DNA. Gene 16:297-307; 1981. Kurosawa, K.; Hayashi, N.; Sato, N.; Kamada, T.; Tagawa, N. Transport of GSH across mitochondrial membranes. Biochem. Biophys. Res. Commun. 167:367-372; 1991. Lehninger, A. L.; Vercesi, A.; Bababumni, E. Regulation of Ca 2+ release from mitochondria by the oxidation-reduction state of pyridine nuclcotides. Proc. Natl. Acad. Sci. U. S. A. 75:16901694; 1980. Lotscher, H. R.; Winterhaiter, K.; Carafoli, E.; Richter, C. Hydroperoxide-induced loss of pyridine nucleotide and release of calcium from rat liver mitochondria. J. Biol. Chem. 255:93259330; 1980. Martensson, J.; Jain, A.; Frayer, W.; Meister, A. Glutathione metabolism in the lung: Inhibition of its synthesis leads to lamellar body and mitocbondrial defects. Proc. Natl. Acad. Sci. U. S. A. 86:5296-5300; 1989. Martensson, J.; Lai, C. K.; Meister, A. High affinity transport of GSH is part of a multicomponent system essential for mitochondrial function. Proc. Natl. Acad. Sci. U. S. A. 87:71857189; 1990. Martensson, J.; Meister, A. Mitochondrial damage in muscle occurs after marked depletion of GSH and is prevented by giving GSH-ester. Proc. Natl. Acad. Sci. U. S. A. 86:471-475; 1989. Meredith, M.; Reed, D. J. Depletion in vitro of mitoehondrial GSH in rat hepatocytes and enhancement of lipid peroxidation by adriamycin and BCNU. Biochem. Pharmacol. 32:1383-1388; 1988. Mitchell, M. C.; Herlong, H. F. Alcohol and nutrition: Caloric value bioeoergetics and relationship to liver damage. Annu. Rev. Nutr. 6:457-474; 1986. Montgomery, R. I.; Coleman, W. B.; Eble, K. S.; Cunningham, C. C. Ethanol-elicited alterations in the oligomycin sensitivity and structural stability of the mitochondrial FoFI ATPase. J. Biol. Chem. 262:13281-13289; 1987. Nenbert, D.; Wojtszak, A. B.; Lehninger, A. L. Purification and enzymatic identity of mitochondrial contraction factors I and II. Proc. Natl. Acad. Sci. U. S. A. 48:1651-1658; 1962. Porta, E. A.; Hartroft, W. S.; de la Iglesia, F. A. Hepatic changes associated with chronic alcoholism in rats. Lab. Invest. 14:1437-1455; 1965. Reed, D. J. Regulation of reductive processes by giutathione. Biochem. Pharmacol. 35:7-13; 1986. Reed, D. J. Chemical mechanisms in drug-induced liver injury. In: Zakim, D.; Boyer, T. D., eds. Hepatology: A textbook of liver diseases. Philadelphia: W.B. Saunders; 1990:737-754. Rubin, E.; Beattie, D. S.; Toth, A.; Lieber, C. S. Structural and functional effects of ethanol on hepatic mitoehondria. Fed. Proc. 3h131-140; 1972. Schilling, R. J.; Reitz, R. C. A mechanism for ethanol-induced damage to liver mitoehondrial structure and function. Biochim. Biophys. Acta 603:266-277; 1980. Sies, H.; Mosskim, A. Role of mitochondrial glutathione peroxidase in modulating mitochondrial oxidations in liver. Eur. J. Biochem. 84:377-383; 1978. Spach, P. I.; Bottenus, R. E.; Cunningham, C. C. Control of adenine nucleotide metabolisn hepatic mitochondria from rats with ethanol-induced fatty liver. Biochem. J. 202:445-453; 1982. Spach, P. I.; Cunningham, C. C. Control of state 3 respiration in liver mitochondria from rats subjected to chronic ethanol consumption. Biochim. Biophys. Acta 894:461-467; 1987. Takeshi, H.; Kaplowitz, N.; Kamimura, T.; Tsukamoto, H.; Fermtndez-Checa, J. C. Hepatic mitochondrial GSH depletion and progression of experimental alcoholic liver disease in rats. Hepatology 16:1423-1428; 1992. Thayer, W. S.; Rubin, E. Effects of chronic ethanol intoxication on oxidative phosphorylation in rat liver submitochondrial particles. J. Biol. Chem. 254:7717-7723; 1979. Thayer, W. S.; Rubin, E. Molecular alterations in the respiratory
M I T O C H O N D R I A L G L U T A T H I O N E D E P L E T I O N IN A L D chain of rat liver after chronic ethanol feeding. J. Biol. Chem. 256:6090--6097; 1981. 48. Tsukamoto, H.; French, S. W.; Benson, N. Severe and progressive steatosis and focal necrosis in rat liver induced by continuous intragastric infusion of ethanol and low fat diet. Hepatology 5: 224-232; 1985. 49. Tsukamoto, H.; Gaal, K.; French, S. W. Insights into the patho-
475 genesis of alcohol liver necrosis and fibrosis: Status report. Hepatology 12:599-608; 1990. 50. Tsukamoto, H.; Towner, S. J.; Ciofalo, L. H.; French, S. W. Ethanol-induced liver fibrosis in rats fed high fat diet. Hepatology 6:814-822; 1986. 51. Tzagoloff, A.; Myers, A. Genetics of mitochondrial biogenesis. Annu. Rev. Biochem. 55:249-285; 1986.
0741-8329/93 $6.00 + .00 Copyright © 1993Pergamon Press Ltd.
Mitochondrial Glutathione Depletion in Alcoholic Liver Disease JOSI~ C. F E R N A N D E Z - C H E C A , * t I T A K E S H I H I R A N O , * H I D E K A Z U TSUKAMOTO~: A N D N E l L K A P L O W I T Z *
*Division of Gastrointestinal and Liver Diseases, University of Southern California School of Medicine, The Department of Veterans Affairs Outpatient Clinic, Los Angeles, CA 90033 t Unidad de H[gado, Hospital Clinic i Provincial, Universidad de Barcelona, Barcelona 08036, Spain ~Vletro Health Medical Center, Department of Veterans Affairs Medical Center, Case Western Reserve University, Cleveland, OH 44109 FERNANDEZ-CHECA, J. C., T. HIRANO, H. TSUKAMOTO, AND N. KAPLOWITZ. Mitochondrial glutathione depletion in alcoholic liver disease. ALCOHOL 10(6) 469-475, 1993.-Alcoholic liver disease (ALD) is one the most serious consequences of chronic alcohol abuse. Liver cirrhosis, the culmination of the illness, is one of the leading causes of death in Western countries. Mitochondria are a target of ethanol intoxication mainly due to the toxic effects of acetaldehyde, a byproduct of ethanol metabolism. Morphological and functional changes in mitochondria are one of the key hallmarks of chronic ethanol exposure in both chronic alcoholics and experimental models of alcoholism. The functional changes observed in mitochondria from ethanol-treated animals are translated in an overall decrease in ATP levels resulting from a lower rate of ATP synthesis as a consequence of impaired processing at the translational level of some components of oxidative phosphorylation encoded by mitochondrial DNA genome. Mitochondrial glutathione (GSI-I) plays a critical role in the maintenance of cell functions and viability and in mitochondrial physiology by metabolism of oxygen free radicals generated in the respiratory chain. GSH in mitochondria originates from cytosol by a transport system which translocates GSH into the matrix. This transport system is impaired in chronic ethanol-fed rats, which translates in a selective and significant depletion of the mitochondrial GSH content resulting in the development of an increased susceptibility to oxidant stress. Using the intragastric infusion model of experimental ALD in rats, the profound and selective mitochondrial GSH depletion precedes the onset of alcoholic fiver disease, mitochondrial lipid peroxidation, and progression of liver damage. These results suggest
that depletion of mitochondrial GSH by impairment of its transport from cytosol into mitochondria could be an additional contributing factor in the development of ALD by favoringpro-oxidant productionand oxidant stress in mitochoodria. Oxidant stress Mitochondrial GSH transport Mitochondrial physiology
Oxygen free radicals
LIVER cirrhosis is the culmination of alcoholic liver disease (ALD) and is still one of the leading causes of death in developed countries. However, the pathogenesis of the alcoholinduced liver disease is not yet completely understood. The causes that lead to the development of this human disease state are multifactorial, so that the hypotheses and factors which might cause or contribute to the ethiology of ALD are numerous. Despite the possible role that malnutrition might play in the overall development of the disease, it is likely that ALD is mainly a consequence of the toxic effects of ethanol (1,17,34). As a consequence of chronic ethanol intake, biochemical changes in the hepatocyte occur due to the metabolism of ethanol resulting in cellular redox potential shift (NAD÷/NADH decrease) and production of acetaldehyde, a potent toxicant. Additional factors resulting from ethanol metabolism have been documented in animal models of ALD, including autoimmune-induced injury by antibodies to protein-acetaldehyde adducts, hemodynamic alterations of
Experimental alcoholicliver disease
the hepatic blood supply along sinusoids, and peroxidation of membrane lipids and oxidant stress (3,24). The purpose of the present paper is to emphasize the functional alterations of mitochondria induced by ethanol feeding and to focus our attention on the mitochondrial pool of glutathione in the physiology of this organelle and the role that its selective depletion by chronic ethanol feeding might play in the Tsukamoto-French animal model of ALD (48-50). MITOCHONDRIA: A TARGET OF ETHANOL INTOXICATION
Most of the ethanol that enters the circulation undergoes an oxidative metabolism in the liver. Ethanol is converted into acetaldehyde by different routes: alcohol dehydrogenase, a cytosolic enzyme; the microsomal ethanol-oxidizing system (MEOS), and the peroxisomal catalase. Acetaldehyde produced from ethanol is a very reactive substance which accounts for most of the toxic effects of ethanol. This aldehyde
Requests for reprints should be addressed to Dr. Jos6 C. Fermindez-Checa, Unidad de Htgado, Hospital Clinic i Provincial, Villarroel, 170,
08036-Barcelona, Spain.
469
470 is mainly metabolized in mitochondria by the l o w - K m acetaldehyde dehydrogenase, and its activity decreases in advanced states of chronic ethanol intoxication. Its distribution is not homogeneously localized in the liver acinus, displaying an increased gradient from the perivenous to periportal areas of the liver (13). This could be one of the factors that contribute to the selective centrilobular necrosis observed in chronic alcoholics. Functional and morphological changes in mitochondria are key features of chronic ethanol exposure. In chronic alcoholics as well as in animal models of alcoholism, mitochondria are enlarged, appearing as swollen or elongated structures with cristae disrupted or without normal organization (8, 25,37). This suggests the possibility that hepatic energy metabolism is compromised by chronic ethanol intake, given the key role of mitocbondria in energy conservation. This will result in impaired bepatocellular ATP production, which in turn might have important functional consequences. The lower ATP levels observed in chronic ethanol-fed animals maintalned on the diet for three weeks results from a lower state 3 respiration with NADH-linked substrates (complex I) (9-11). Slightly longer feeding periods also decrease the succinatedriven state 3 respiration (complex II) in ethanol-fed mitochondria. The ADP-stimulated electron transport is sustained during extended periods of time and affects all segments of the electron transport chain. Thus, the state 3 respiration through the cytochrome oxidase portion of the electron transport chain is depressed by chronic ethanol feeding (44,46). The magnitude of this depressed effect by ethanol feeding ranges between 20 and 40% compared to control mitochondria, and there is lifle variation at longer feeding times (16). The lower oxygen consumption in the presence of ADP and Pi (state 3 respiration) demonstrates that the rate at which ATP is synthesized via the oxidative phosphorylation process is lower in ethanol-fed mitochondria. The rate of oxygen consumption in the absence of ADP (state 4 respiration) is not altered by chronic ethanol feeding to the same degree as is state 3 respiration. An increase in the state 4 respiration would indicate that the mitochondria have less capacity to conserve the proton gradient generated by electron transport and the mitocbondria would be less well coupled. This resting state of respiration is an indication of the functional viability of isolated mitochondria. Feeding ethanol for three to four weeks results in either no loss or a slight decrease (10-15% vs. control values) in state 4 respiration when either NADH-linked substrates or succinate were utilized as oxidizable substrates (10,43). At longer feeding periods, significant increases in state 4 respiration are observed, indicating that the mitochondria had become more uncoupled. However, state 3 respiration through cytochrome oxidase is significantly depressed by chronic ethanol, pointing to the dramatic alteration in this portion of the energy conservation system (10,16,44). Thus, the overall decreased ATP levels observed in animals treated chronically with ethanol result from a lower rate of ATP synthesis rather than from a decreased efficiency in the synthesis of ATP, since there is little or no change in the resting state respiration, which indicates a lack of uncoupling by ethanol feeding.
FERNANDEZ-CHECA ET AL. ations in several of the respiratory chain components by ethanol. The activity and haem content of cytochrome oxidase is decreased 50-600/0 in ethanol mitochondria compared to control organelles. In addition, cytochrome b and some of the iron sulphur centres associated with NADH-ubiquinone reductase complex are also decreased by ethanol (4,40,41,43, 47). Electron transport and proton translocation through the NADH-ubiquinone reductase portion of the electron transport chain are decreased by 30 and 40°70, respectively (4,7). The mitochondrial ATP synthetase is a very complex structure with the catalytic subunits (Ft) projecting into the matrix of the mitochondria and the proton channel portion (F0) formed by other polypeptides attached to the catalytic portion that traverses the inner membrane [for review, see (22)]. It has been shown in both inner membrane preparations and submitochondrial particles capable of synthesizing ATP that the ATPase is significantly lowered by chronic ethanol feeding (23,46). The same results have recently been shown in intact mitochondria (7). In addition, ethanol feeding lowers the oligomycin sensitivity of ATP synthetase and renders it less tightly attached to the inner membrane. The decrease in oligomycin sensitivity and the structural changes of the ATP synthase induced by ethanol are associated exclusively in the F0 subunits of the complex (35). The molecular basis for the ethanol-induced lesion in the oxidative phosphorylation system of mitochondria is at the level of translation of several components. Mitochondria have a circular DNA that encodes only 13 open reading frames in addition to two rRNAs and a complete set of tRNAs with a high degree of direct sequence homology (2,5,51). The mitochondrial DNA gene products have been identified as members of the oxidative phosphorylation system (Table 1). Those portions of the oxidative phosphoryiation system that are decreased by ethanol feeding are encoded by the mitochondrial DNA, whereas those components not affected by ethanol have a nuclear gene expression and are subsequently imported from the cytoplasm into mitochondria. These results suggest that
TABLE 1 EFFECTS OF ETHANOLON THE MITOCHONDRIAL DNA PRODUCTSOF COMPONENTSOF THE OXIDATIVEPHOSPHORYLATIONSYSTEM MitochondrialDNAGeneExpression GeneProduct
Protein Levels
mRNALevels
MOLECULARBASISFOR THE ETHANOL-INDUCEDALTERATION OF THE OXIDATIVEPHOSPHORYLATIONSYSTEM
ND5* COIt ND4* Cyt b:~ ND2* NDI* COIIt COIIIt ATPase 6§ ND6* ND3* ATPase 8§ NIML*
Decreased Decreased Decreased Decreased Decreased Decreased Decreased Decreased Decreased Decreased Decreased Decreased Decreased
ND Unchanged Unchanged ND ND ND Unchanged Unchanged Unchanged ND Unchanged Unchanged Unchanged
Since the mitochondrial oxidative phosphorylation system is multicomponent, changes in any single component of the system would result in the rate limiting synthesis of ATP and decreased oxygen consumption. There are dramatic alter-
ND = not determined. *Components of the NADH-ubiquinone reductase complex. ,Components of the cytochrome oxidase subunits. $Cytochromeb. §Twoof the membrane subunits (F0)of ATPase synthase.
MITOCHONDRIAL GLUTATHIONE DEPLETION IN ALD chronic ethanol consumption could interfere with mitochondrial gene expression. The rate of incorporation of radiolabeled methionine into all polypeptides was lower for all 13 polypeptides, establishing a correlation between loss of catalytic activity and decrease in the rate of synthesis of those components affected by chronic ethanol feeding (14). However, ethanol feeding does not affect the DNA, total RNA levels, or the RNA polymerase activity. With slot blot analyses, eight of the mRNA and ribosomal RNA were present in normal amounts in ethanol mitochondria (26), indicating that the defect caused by ethanol is in the processing of the mRNA of the mitochondrial gene products. Thus, the amount of ribosomes from ethanol mitocbondria are decreased by 60-70% compared to control mitochondria, which could be the result of decreased synthesis in the endoplasmic reticulum or a decrease in transport across the mitochondrial membranes into the matrix (15). MITOCHONDRIAL GLUTATHIONE
Intracellular GSH is synthesized exclusively in cytosol, but it is also found in mitoehondria and the nucleus. The mitochondHal pool of GSH is derived from cytosol by a mitochondrial transporter which translocates GSH into the mitochondrial matrix. The concentration of GSH in mitochondria is thought to be as high as in cytosol (10-15 raM). The exact mechanism of this transport system is presently unknown, although recent studies have suggested that GSH diffuses into mitochondria through a channel (27) or is transported by a primary active, high affinity transport system (31). Our recent studies have shown a rapid exchange between cytosol and mitochondria with a t~/2 of mitochondrial GSH of 18 rain (18). The mitochondrial GSH participates in a redox cycle through the GSH peroxidase/reductase activities (38,42). Reduction of endogenous hydroperoxides by GSH peroxidase may be a key function of mitochondrial GSH. As a consequence of aerobic metabolism, cells are under an endogenous oxidative stress challenge. Even under physiological conditions, the reduction of molecular oxygen to water in the respiratory chain is incomplete and involves the formation of toxic oxygen intermediates (12). Hydrogen peroxide, if not reduced to water, can lend to the formation of the very reactive hydroxyl radical through the Fenton-HaberWeiss reaction resulting in the formation of lipid peroxides that can damage mitochondrial membranes and their functions (6). Although mitochondria have a Mnsuperoxide dismutase (Mn-SOD) isoenzyme, it would afford only partial protection because its action would lead to formation of hydrogen peroxide. Since mitochondria do not contain catalase (36), their detoxification of hydroperoxides, formed either under physiological conditions or as a consequence of bioreduction reactions to activate a drug to an unstable intermediate(s), depends exclusively on GSH peroxidase. This enzyme utilizes the reducing equivalents of GSI-I which, in turn, is converted to oxidized giutathione (GSSG). The GSH redox cycle uses NADPH- and, indirectly, NADH-reducing equivalents in the mitochondrial matrix to provide a recycling supply of GSH by the GSSG-reductase catalyzed reduction of GSSG. Thus, GSH is critical in protecting unsaturated fatty acids in membrane phospholipids from peroxidation and mitochondrial DNA from strand scission by attack of oxygen free radicads generated either endogenously or as a result of ethanol metabolism. The fact that mitochondrial GSH depletion below critical levels is associated with cell injury in a variety of organs and that its selective repletion affords protection emphasizes the
471 importance of this small pool of GSH in maintaining vital cell functions (30,32,33). It is thought that mitochondrial GSH plays this critical role through regulation of mitochondrial inner membrane permeability by maintaining Ca2+ homeostasis. GSH and pyridine nucleotide oxidation in mitochondria precede an increase in the permeability of the inner membrane to Ca2+ (28,29). This suggests that oxidative stress or severe GSH depletion affects the redox status in the mitochondria. Although mitochondrial regulation of cytosolic Ca2+ seems unlikely physiologically, under pathologic conditions, mitochondria may be a major regulator of cytosolic Ca2+ concentrations (39). Several groups have noted a correlation between the depletion of mitochondrial GSH and the inability of mitochondria to sequester Ca2+ [for review, see (19)]. In agreement with this, it has been shown that acetaminophen depletes mitochondrial GSH to a greater extent than its regioisomer 3'hydroxyacetanilide, which correlates with the formation of mitochondrial protein adducts and disruption of cellular Ca2+ homeostasis by inhibiting the plasma membrane calciumATPase. Alterations in the Ca2+ cycling in the inner mitochondrial membrane may be an important regulator of cytosolic concentrations of Ca2+, which leads to cell injury, since ruthenium red, an inhibitor of the mitochondrial Ca2+ uniport system, can prevent oxidative stress and the associated injury under certain conditions (19). E T H A N O L SELECTIVELY IMPAIRS T H E M I T O C H O N D R I A L TRANSPORT OF GSH IN A L D
In view of the striking structural and functional changes of mitochondria induced by ethanol consumption, as detailed previously, and in light of the key function that mitochondrial GSH plays in maintaining the integrity of this organelle and cell viability, it would be important to determine if depletion of the mitochondrial GSH pool by ethanol feeding determines the development or contributes to the progression of ALD. A suitable model of experimental alcoholic liver disease which reproduces different stages of the ALD lesion observed in humans was not available until recently (48-50). However, the use of other models of experimental alcoholism such as the Lieber-DeCarli liquid diet has allowed the determination of the effect of chronic ethanol feeding in rats on the homeostasis of mitochondrial GSH. Until recently, no specific data were available in the literature on the effect of ethanol on the mitochondrial GSH pool. Using the Lieber-DeCarli model in wellnourished rats, it has been shown that chronic ethanol feeding decreases rather substantially (50-60%) the mitochondrial pool of GSH in isolated hepatocytes from ethanol-fed rats compared to pair-fed animals (20,21). This effect was accompanied by a modest decrease of cytosol GSH in ethanol-fed rats, an effect that was variable and did not result from impaired GSH synthetic capacity by ethanol. The effect of chronic ethanol feeding on the depletion of mitochondHal GSH is rather selective, since after two weeks of feeding the depletion of the mitochondrial GSH pool is the only change observed in chronic ethanol-fed hepatocytes. The selective effect of ethanol on the mitochondrial pool of GSH requires the in vivo administration of ethanol, since in vitro incubation of hepatocytes with ethanol (up to 80 mM) up to 4 h does not affect this pool of GSH. Since the mitochondrial pool of GSH is derived from the uptake of cytosol GSH, we tried to increase the mitochondrial pool of GSH in ethanol-fed hepatocytes by increasing the concentration of cytosol GSH using methionine as precursor of GSH (Fig. l). In contrast to control mitochondria which increase their pool of GSH in parallel to the increase of the cytosol GSH pool, in ethanol-fed rats
472
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mitochondria do not increase their pool of GSH despite a significant increment of the cytosol one (21). These data demonstrate a lower relationship of cytosol to mitochondriai GSH induced by ethanol feeding, suggesting an impaired capacity for repletion of GSH into mitochondria. To rule out the possibility of an artifactual effect on the depletion of mitochondrial GSH by ethanol seen in isolated hepatocytes, and to determine if such selective depletion results from an impaired uptake of GSH from cytosol to mitochondria rather than an increased release from mitochondria, we have performed tracer kinetic analyses on the transport of radiolabeled cytosol GSH into mitochondria from ethanol- and pair-fed liver biopsies in vivo
(18). A rapid fractionation of the liver specimens into cytosol and mitochondria allowed us to determine the relationship of isotopic equilibrium between labeled cytosol (precursor) and mitochondria (product). Modelling of these data have shown that the mass fractional rate of transport of GSH from cytosol to mitochondria is decreased by 35% in ethanol-fed livers, which is translated into a lower steady-state pool size of GSH. Thus, the depleted mitochondrial pool of GSH by ethanol results from an impaired capacity of the mitochondriai transport system which translocates GSH. Recently we have examined the homeostasis of the mitochondrial GSH pool (48-50) using the Tsukamoto-French ex-
MITOCHONDRIAL GLUTATHIONE DEPLETION IN ALD perimental model of ALD to determine the temporal relationship of mitochondrial GSH depletion and progression of ALD at different times of ethanol feeding. Intragastric infusion of ethanol to rats leads to a selective depletion of GSH in the mitochondrial fraction of the rat liver compared to control liver, with no changes seen in the cytosol GSH pool at any time of infusion. This decrease ranges from 40, 61, and 85% after infusion of ethanol at 3, 6, and 16 weeks, respectively (Fig. 2) (45). This effect is absolutely specific for liver, since renal mitochondria show unchanged GSH pool sizes. Serum alanine transferase (ALT) levels increased significantly at 6 and 16 weeks of infusion, and mitochondrial lipid peroxidation was observed only when the mitochondrial pool of GSH was severely depleted at 16 weeks of feeding. Since this model of experimental ALD reproduces various histological stages of the human disease depending on the time of infusion (from liver steatosis to necrosis and fibrosis at 3, 6, and 16 weeks of feeding), these results suggest that the selective depletion of mitochondrial GSH by ethanol precedes the onset of alcoholic liver disease which is associated with mitochondrial lipid peroxidation and progression of liver damage. In the LieberDeCarli model of ethanol toxicity in rats, depletion of mitochondrial GSH pool size renders the hepatocytes more susceptible to cell injury by oxidant stress, which is then reversed when the mitochondrial pool of GSH is increased by GSHethyl ester (18). Thus, results obtained in both animal models indicate that depletion of mitochondrial GSH by ethanol could be an additional contributing factor in the development of ALD by decreasing antioxidant capacity, thus disturbing the balance of pro-oxidants and antioxidants in favor of oxidant stress in mitochondria. The mechanisms by which ethanol perturbs the capacity of mitochondria to transport GSH from cytosol are presently unknown and are the subject of ongoing research. Lower levels of the functional mature protein due to a decreased nuclear gene expression of the protein, posttranslational modification changes, or alterations in the mechanism of import of nuclear proteins into mitochondria as well as qualitative changes in the physicocbemical properties of mitochondrial membrane by ethanol or any combination of those factors could result in an organelle devoid of GSH to protect it from endogenous or exogenous oxidant stress. It is unclear whether impaired transport capacity in mitochondria is a cause or effect of mitochondrial dysfunction by ethanol feeding. However, it is worth emphasizing that this impaired mitochondrial transport capacity is not the result of an "unspecific" or generalized effect by ethanol, since other transport systems of the mitochondria including the A D P / P i exchanger, the dicarboxylate and tricarboxylate exchanger, the A D P / A T P translocator, and the NADPH shuttles are not affected by ethanol metabolism (15). It remains to be seen in the near future if correcting this selectively depleted GSH pool size would alter the progression of ALD in the intragastric infusion model, and if this mitochondrial GSH depletion is also present in chronic alcoholics at different stages of the disease.
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FIG. 2. Effect of time course of chronic ethanol infusion on hepatic GSH in cytosol and raitochondrial fractions. *p < 0.01 vs. control by unpaired t test. From Hepatology 16:1423-1428; 1992. Reprinted with permission. ACKNOWLEDGEMENTS The work presented here has been supported by grants from the Department of Veterans Affairs (N.K. and H.T.), NIAAA AA 09526 (N.K., J.C.F.-C., and H.T.), and DIGICYT, Spain (PB92-1110 to J.C.F.-C.).
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