Role of free radicals in failure of fatty liver grafts caused by ethanol

Alcohol 34 (2004) 49–58

Role of free radicals in failure of fatty liver grafts caused by ethanol Zhi Zhong, John J. Lemasters* Department of Cell and Developmental Biology, CB# 7090, 236 Taylor Hall, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA Received 30 March 2004; received in revised form 10 August 2004; accepted 10 August 2004

Abstract Alcohol is associated with accidental deaths and suicides leading to organ donation, and hepatic steatosis is an important risk factor for initial poor function and failure of human liver grafts. Mechanisms of fatty graft failure are not fully understood, but increased oxidative stress may be a major factor. To characterize the role of free radical stress and the efficacy of antioxidant treatments in fatty liver graft injury, donors for orthotopic rat liver transplantation were treated chronically (3 or more weeks) and acutely (single dose) with ethanol. After transplantation, necrosis and alanine aminotransferase release were threefold to fourfold higher in recipients of fatty grafts from donors treated with ethanol either acutely or chronically compared with findings for recipients of grafts from untreated donors. Moreover, graft survival decreased from nearly 100% to less than 20%. Free radical adducts, as measured by electron spin resonance spectroscopy, were detected in the blood and bile of rats receiving fatty grafts caused by ethanol. Markers of lipid peroxidation also increased after transplantation. Destruction of Kupffer cells with gadolinium chloride decreased free radical production and improved graft survival. Leukocyte adhesion increased beginning early after implantation, and adherent white blood cells obtained from transplanted fatty livers produced the same free radical species as were detected in blood. Therefore, Kupffer cells and adherent white blood cells are important sources of free radicals. Free radicals not only damage fatty grafts directly but also lead to enhanced inflammation and disturbed microcirculation. Delivery of superoxide dismutase-1 and superoxide dismutase-2 genes, free radical–scavenging polyphenols, and antioxidant-containing Carolina Rinse solution reduced injury and improved survival of fatty grafts caused by ethanol. Taken together, these findings indicate that free radicals increase in fatty grafts after transplantation and play an important role in injury of fatty grafts obtained from ethanol-exposed donors. Treatment of fatty donor livers with antioxidants and free radical scavengers may thus be an effective clinical therapy to prevent failure of fatty grafts. 쑖 2005 Elsevier Inc. All rights reserved. Keywords: Liver transplantation; Steatosis; Oxidative stress; Free radicals; Alcohol

1. Introduction Because of advances in immunosuppression, organ preservation, and surgical techniques, liver transplantation is performed far more frequently and has become a widely accepted therapy for children and adults with irreversible liver disease (Starzl et al., 1982; Van Thiel et al., 1988). However, use of this lifesaving technique is still limited because of a severe shortage of donor livers (Neuberger, 2000). Despite strong efforts to stimulate organ donation, demand continues to exceed the supply of organs. Only about 5,000 liver transplantations are performed yearly because

* Corresponding author. Tel.: ⫹1-919-966-5507; fax: ⫹1-919-9667197. E-mail address: [email protected] (J.J. Lemasters). Dr. Lemasters and the University of North Carolina hold shares in Transplant Solutions, Inc., a start-up company that was formed to take Carolina Rinse Solution through Food and Drug Administration filings, multicenter trials, and approval for human use. The antioxidant properties of Carolina Rinse Solution are mentioned briefly in this article. Editor: T.R. Jerrells 0741-8329/05/$ – see front matter 쑖 2005 Elsevier Inc. All rights reserved. doi: 10.1016/j.alcohol.2004.08.006

of the shortage of donor organs (http://www.optn.org/data; accessed December 14, 2004). As a result, the number of patients on waiting lists increases rapidly each year (Harper & Rosendale, 1997; Trotter et al., 2002). According to information provided by The Organ Procurement and Transplantation Network (http://www.optn.org/data; accessed December 14, 2004), the number of patients on the waiting list for donor livers in the United States exceeds 17,000 at this writing, and large numbers of patients who could benefit from a new liver never receive one. The problem of livers exposed previously to ethanol as donor organs might be considered insignificant if it were not for the striking fact that organ donors are often victims of accidents involving either chronic or acute consumption of ethanol. Brain-dead accident victims are the major source of donor organs, whereas accidents are overwhelmingly associated with alcohol consumption. For example in 1993, approximately 44% of fatalities due to traffic accidents in the United States were alcohol related (Anonymous, 1994). Another source of organ donation is suicide victims in whom alcohol consumption is also common. Moreover, results of a

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study showed that about half of donors had a blood ethanol concentration of between 4 and 40 mg/dl (Hassanein et al., 1991). Therefore, alcohol use is most likely a frequent characteristic in organ donors. Alcohol frequently induces fatty liver. Even a single inebriating dose of ethanol causes hepatic fatty infiltration (Ylikahri et al., 1972). Hepatic steatosis is a well-established risk factor for orthotopic liver transplantation and liver resection, and the risk of graft malfunction and failure correlates with the severity of fatty infiltration (Adam et al., 1991; D’Alessandro et al., 1991; Markin et al., 1993; Mor et al., 1992; Todo et al., 1989; Uren˜a et al., 1998). Because severely steatotic grafts are associated with primary graft nonfunction, such donor organs are universally declined for use in transplantation. Donor livers with moderate steatosis still show a relatively higher risk and are used only when other known risk factors are absent (Uren˜a et al., 1999). Indeed, alcohol ingestion, both acute (one dose) and chronic (3–5 weeks), causes steatosis in the liver, as well as substantially increases graft injury and decreases graft survival after transplantation in animal models (Gao et al., 1995; Zhong et al., 1996, 1999). During organ recovery, excessive touching, retracting, and moving liver lobes also increase graft injury and decrease graft survival after transplantation (Schemmer et al., 1998). Acute (one dose) and chronic (4–5 weeks) ethanol treatment increases susceptibility to graft injury after implantation caused by organ manipulation during organ recovery (Schemmer et al., 1999). Because of the risk that donor alcohol poses for transplantation, it is important to understand the mechanism of primary nonfunction of ethanolinduced fatty livers, so that effective therapies can be developed to prevent this severe complication, as well as to minimize the huge gap between demand and availability of usable donor livers. In this article, we summarize some of the findings on the role of free radicals in failure of fatty grafts caused by ethanol.

1989; Marzi et al., 1989a), but a series of alterations signifying loss of viability of endothelial cells, such as nuclear membrane vacuolization, mitochondrial swelling, plasma membrane breakdown, cytoplasmic rarefaction, ball-like rounding, and nuclear condensation, occurs in the early stage of reperfusion (Caldwell-Kenkel et al., 1988, 1989; Lemasters et al., 1989; Marzi et al., 1989a). In addition, within minutes after reperfusion, Kupffer cells exhibit structural alterations, including surface ruffling, vacuolization, and formation of wormlike densities, suggesting macrophage activation (Fig. 1) (Caldwell-Kenkel et al., 1989, 1991; Lemasters et al., 1989; Momii et al., 1989). The onset of primary graft failure after transplantation correlates well with the degree of Kupffer cell activation and endothelial cell killing caused by cold storage/reperfusion. Methylpalmitate, a drug that selectively inhibits Kupffer cells, and nisoldipine, a calcium channel blocker that prevents Kupffer cell activation, improve survival after liver transplantation (Marzi et al., 1991; Takei et al., 1990), indicating that Kupffer cell activation plays a role in graft failure. In contrast, parenchymal cells remain viable on reperfusion, even after long-term storage in Euro-Collins solution. Under these conditions, however, liver grafts have a high probability of failing after transplantation (Caldwell-Kenkel et al., 1988). Reactive oxygen species produced on reperfusion play a critical role in the injury caused by ischemia–reperfusion (Farber et al., 1990; McCord, 1987; Parks et al., 1982). Production of oxygen radicals on reperfusion of previously ischemic tissue is due to accumulation of purine derivatives derived from ATP degradation, reduction of mitochondrial ubiquinone, and activation of macrophages and neutrophils (Bellavite, 1988; Boveris & Chance, 1973; Jaeschke &

2. Role of ischemia–reperfusion injury in primary graft nonfunction Primary graft nonfunction is a fatal complication that occurs 1 to 2 days after liver transplantation, whereas immunologic rejection typically begins no less than 1 week after implantation. The cause of primary graft failure is not fully understood but is most likely multifactorial. Although the mechanisms of graft damage remain unclear, evidence supports the suggestion that ischemia–reperfusion injury during liver transplantation plays a key role (Gao et al., 1992; Lemasters et al., 1992). One of the most important factors associated with an increased incidence of primary graft failure experimentally and clinically is long preservation time (Lemasters et al., 1995); however, hepatic injury occurs mainly during reperfusion, rather than in the cold-storage period (Caldwell-Kenkel et al., 1988; Thurman et al., 1988). Changes in nonparenchymal cells are mild during cold storage (Caldwell-Kenkel et al., 1988, 1989; Lemasters et al.,

Fig. 1. Scanning electron micrograph showing Kupffer cell activation and endothelial cell killing after cold storage–reperfusion injury. Note ruffling and rounding of a Kupffer cell (k) and the rough surface of a remnant of a nonviable endothelial cell (e). Hepatocytes (h) show relatively normal features. Bar ⫽ 5 µm. Adapted from J. C. Caldwell-Kenkel, R. T. Currin, Y. Tanaka, R. G. Thurman, and J. J. Lemasters, Kupffer cell activation and endothelial cell damage after storage of rat livers: effects of reperfusion, Hepatology 13(1), pp. 83–95, fig. 3, Copyright 1991, by permission of American Association for the Study of Liver Diseases.

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Mitchell, 1989; McCord, 1987; Parks et al., 1982). During cold storage, xanthine and hypoxanthine accumulate in grafts in a time-dependent manner (Marzi et al., 1989b), and brief perfusion of grafts with nitrogen-saturated media rapidly removes purine derivatives in grafts and decreases cell death after transplantation (Marzi et al., 1989b). Lipid radicals are detected early after implantation by spin trapping and electron spin resonance, and free radical formation after reperfusion is associated with primary graft failure after liver transplantation (Connor et al., 1992). Therefore, primary graft failure is, at least in part, related to free radical production caused by ischemia–reperfusion process.

3. Free radicals are increased in alcohol-induced fatty liver grafts Findings of studies show increased oxidative stress in the liver after alcohol consumption (Shaw et al., 1988). α-Hydroxylethyl radicals are detected in microsomal incubations (Reinke et al., 1987) and in vivo after ethanol treatment (Knecht et al., 1990a, 1990b), indicating that free radicals can be derived directly from ethanol. Moreover, hydrogen peroxide and products of lipid peroxidation increase in the liver after ethanol administration (DiLuzio & Kalish, 1966; Misra et al., 1992; Shaw et al., 1981, 1988; Spencer et al., 1983). Therefore, free radical production might be higher in fatty grafts caused by ethanol, thus exacerbating graft injury. This hypothesis has been tested in a variety of studies by using fatty livers caused by either acute or chronic ethanol treatment. Feeding of rats for 3 to 5 weeks with an ethanol-containing liquid diet causes lipid accumulation in the liver (Gao et al., 1995). Three radical species are detected in the blood immediately after implantation of fatty livers produced by chronic (3–5 weeks) feeding of ethanol and high-fat diet by using alpha-phenyl-N-tert-butylnitrone (PNB) as the spin trap (Gao et al., 1995). Two of these radical species have coupling constants characteristic of lipidderived free radicals, whereas the third radical is most likely oxygen derived (Gao et al., 1995). Two different radical/ alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone (4-POBN) adducts are detected 3 to 5 h after transplantation in bile obtained from steatotic livers produced by chronic (4–5 weeks) ethanol treatment (Lehmann et al., 2000). One species contributing about 60% to the spectrum most likely represents a carbon-centered radical adduct. The other adduct contributing about 40% to the spectrum is most likely an oxygen-centered radical adduct (Lehmann et al., 2000). After transplantation of fatty livers produced by one large dose of ethanol, a treatment that mimics binge drinking, free radical/ 4-POBN adducts detected in bile are twofold to threefold higher than after transplantation of livers from donors not exposed to ethanol (Fig. 2A) (Zhong et al., 1996, 1997). Lipid hydroperoxides, a product of lipid peroxidation, are increased in serum samples obtained from recipients of fatty grafts obtained from ethanol-treated donors (Zhong

Fig. 2. Ethanol increases free radical production and transaminase release after transplantation. Donor rats were given ethanol (5 g/kg, i.g.) or saline 20 h before explantation, and grafts were stored in University of Wisconsin (UW) solution for 42 h before implantation. The spin trapping reagent alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone (1 g/kg) was injected slowly into the tail veins of recipients on opening the vascular clamps after implantation. Bile excreted during the first hour after implantation was collected, and free radical adducts were detected by using a Bruker EMX electron spin resonance spectrometer. Shown are typical spectra (A). Blood was collected from the vena cava at time points indicated in the figure. Serum aspartate aminotranferase (AST) (B) was detected by using an analytic kit obtained from Sigma Co. (St. Louis, MO). Values are means ⫾ S.E.M. (37ºC). *P ⬍ .05 compared with values for the saline group. Adapted from Z. Zhong, H. Connor, R. F. Stachlewitz, M. von Frankenberg, R. P. Mason, J. J. Lemasters, and R. G. Thurman, Role of free radicals in primary nonfunction of marginal fatty grafts from rats treated acutely with ethanol, Molecular Pharmacology 52(5), pp. 912–919, figs. 1 and 2, Copyright 1997, by permission of American Society for Pharmacology and Experimental Therapeutics.

et al., 1997). 4-Hydroxynonenal adducts, another marker of lipid peroxidation, increase markedly after transplantation of fatty grafts obtained from rats treated with one dose of ethanol compared with findings for nonfatty grafts obtained from saline-treated donors (Zhong et al., 2004). Increased free radical formation is associated with severer graft injury

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(transaminase release; Fig. 2B), poorer graft function (decreased bile flow and hyperbilirubinemia), and decreased survival (Gao et al., 1995; Lehmann et al., 2000; Schemmer et al., 1999; Zhong et al., 1996, 1997). These findings indicate that free radical production is increased in ethanolinduced fatty grafts after transplantation and may play an important role in graft failure. 4. Why does free radical production increase in ethanolinduced fatty grafts? Mechanisms by which ethanol increases free radical production after transplantation have not been fully elucidated. α-Hydroxyethyl radicals derived directly from ethanol are not found after liver transplantation (Gao et al., 1995; Lehmann et al., 2000; Zhong et al., 1997). This is not surprising. Ethanol metabolism is rapid, and liver grafts are usually recovered several hours after ethanol treatment and subsequently cold-stored for a substantial period (e.g., 24–48 h). Because recipients are never treated with ethanol, the transplanted fatty liver graft is no longer exposed to ethanol. Therefore, increased free radicals after transplantation are not due to direct free radical formation from ethanol. Activated macrophages and adherent neutrophils in ischemic-reperfused tissue may be important sources of free radicals (Ryan & Aust, 1992). Kupffer cells constitute about 80% of fixed tissue macrophages in the body (Bouwens, 1988). Alcohol may prime and activate these Kupffer cells, thus increasing production of reactive oxygen species on reperfusion. Consistent with this hypothesis, infusion of ethanol stimulates carbon uptake in perfused livers (D’Souza et al., 1993) and increases superoxide production by Kupffer cells (Bautista & Spitzer, 1992). Paradoxically, ethanol decreases endotoxin-induced superoxide production in perfused livers (Bautista & Spitzer, 1996). These different study results are probably due to the temporal effects of ethanol on Kupffer cells. At an early stage (2 h), ethanol blunts endotoxin-induced increases in intracellular calcium in Kupffer cells (Enomoto et al., 1998). However, ethanol enhances elevation of intracellular calcium after 24 h (Enomoto et al., 1998). Pretreatment with gadolinium chloride, which selectively destroys Kupffer cells, blunts free radical production and decreases injury of fatty livers produced by ethanol treatment after low flow–reflow liver perfusion and after transplantation (Zhong et al., 1995, 1997). Destruction of Kupffer cells also improves fatty graft survival after transplantation (Frankenberg et al., 1997, 1999; Zhong et al., 1996, 1997). Moreover, destruction of Kupffer cells decreases injury caused by organ manipulation and ethanol (Schemmer et al., 1999). These results indicate that ethanol increases free radical production, at least in part, by activating Kupffer cells under these conditions (Fig. 3). How does ethanol treatment stimulate Kupffer cells? One possibility is the direct effect of ethanol on Kupffer cells. Pretreatment of Kupffer cells with ethanol increases the probability of calcium channel opening (Goto et al., 1993). An

alternative possibility is priming or activation of Kupffer cells owing to increases in blood endotoxin concentrations in donors caused by ethanol (Thurman et al., 1995). Indeed, pretreatment with nonabsorbable antibiotics, which eliminate bacteria in the gut, largely blunts increases of intracellular calcium in Kupffer cells isolated from rats given one dose of alcohol, supporting the hypothesis that ethanol affects Kupffer cells by increasing endotoxin concentrations in the blood (Enomoto et al., 1998). A third possibility is alcohol-induced hypoxia–reoxygenation in the liver, leading to priming/activation of Kupffer cells (Lemasters et al., 1989; Yuki & Thurman, 1980). Therefore, ethanol may prime Kupffer cells, thus increasing their activation and free radical production when a “second-hit,” transplantation, occurs. Ethanol may also increase free radical formation by causing adhesion and activation of neutrophils. Consistent with this hypothesis, only 30 min after implantation, transplanted fatty livers obtained from rats given ethanol, compared with findings for grafts obtained from control donors, contain twofold more adherent leukocytes (Stachlewitz et al., 1998; Zhong et al., 1997). Moreover, adherent leukocytes obtained from transplanted livers produce three free radical species that are similar to those detected in blood after transplantation (Gao et al., 1995). In addition, production of radical adducts by cells isolated from ethanol-treated grafts is about five times greater than that for cells isolated from salinetreated grafts (Stachlewitz et al., 1998). These findings show that adherent white blood cells produce free radicals that are important in the occurrence of primary graft nonfunction. Expression of cell adhesion molecules [i.e., intercellular adhesion molecule-1 (ICAM-1)] increases more substantially in fatty grafts obtained from ethanol-treated rats than in grafts obtained from saline-treated rats (unpublished observations, M. von Frankenberg, H. K. Bojes, Y. Iimuro, R. Schoonhoven, J. J. Lemasters, and R. G. Thurman, 1995). White blood cell infiltration occurs in alcoholic liver disease (Anggard, 1985), a phenomenon that is probably due to increased formation and decreased degradation of chemotactic mediators such as leukotrienes (Jedlitschky et al., 1990; Murphy & Westcott, 1985). Destruction of Kupffer cells with gadolinium chloride not only decreases leukocyte adhesion in ethanol-induced fatty grafts after transplantation, but also decreases superoxide production by adherent leukocytes (Zhong et al., 1997). These findings support the notion that ethanol increases activation of Kupffer cells after transplantation, as well as the release by Kupffer cells of chemotactic and vasoactive mediators (e.g., leukotrienes, thromboxanes, platelet-activating factor, and cytokines), which increases leukocyte adhesion and activation (Fig. 3). Therefore, destruction of Kupffer cells directly decreases superoxide production by Kupffer cells and indirectly decreases production of reactive oxygen species by blocking leukocyte adhesion and activation. Whether ethanol increases free radical production by other pathways is less conclusive. Ethanol increases conversion of xanthine dehydrogenase to free radical–generating

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Fig. 3. Possible mechanisms of increased free radical production in ethanol-induced fatty grafts. Ethanol may increase free radical production after transplantation by one or more mechanisms: (1) production of alpha-hydroxylethyl radicals, (2) ethanol-/endotoxemia-mediated activation of Kupffer cells (KC) and superoxide (O2⫺) formation by means of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, (3) increased adhesion and superoxide production of leukocytes by Kupffer cell–released proinflammatory cytokines and eicosanoids, (4) increased activation of xanthine oxidase (XO), (5) increased lipid content that amplifies lipid peroxidation reaction, (6) induction of cytochrome P450 2E1 (P450 2E1), and (7) increased superoxide production from mitochondria due to depletion of antioxidants. EC ⫽ Endothelial cells; PAF ⫽ platelet-activating factor; PC ⫽ parenchymal cells; PMN ⫽ polymorphonuclear leukocytes (neutrophils); TNFα ⫽ tumor necrosis factor-alpha; L· ⫽ lipid radicals; LO· ⫽ lipid alkoxyl radicals; LOO· ⫽ lipid peroxyl radicals.

xanthine oxidase (Kato et al., 1990; Oei et al., 1986). Hence, ethanol could increase superoxide production by means of xanthine oxidase after reperfusion. Pretreatment of donors with allopurinol, an xanthine oxidase inhibitor, decreases free radicals after transplantation of fatty grafts (Zhong et al., 1997). Although allopurinol is also a free radical scavenger at high concentrations (Cohen, 1992), allopurinol administered to recipients and added to a solution for rinsing grafts just before implantation did not alter free radical formation by fatty liver grafts, indicating that allopurinol most likely does not act directly as a free radical scavenger per se under these conditions (Zhong et al., 1997). Thus, xanthine oxidase may also play a role in fatty graft failure (Fig. 3). Xanthine dehydrogenase converts to xanthine oxidase after cold storage much more rapidly in Kupffer cells than in hepatocytes (Wiezorek et al., 1994). This observation seems to indicate that Kupffer cells may also produce superoxide by means of xanthine oxidase (Fig. 3). However, the specificity of allopurinol on xanthine oxidase is not very high. Therefore, any conclusions on the role of xanthine oxidase should be made with caution. Mitochondria are another potential source of free radicals. Ethanol treatment decreases mitochondrial antioxidants, such as glutathione and α-tocopherol (Ferna´ndez-Checa et al., 1993; Nordmann et al., 1988; Speisky et al., 1985; Videla & Valenzuela, 1982), and disorganizes mitochondrial

membranes (Nordmann et al., 1988). These alterations might promote mitochondrial formation of reactive oxygen species. However, malondialdehyde and lipid hydroperoxides in mitochondria isolated from ethanol-induced fatty grafts are not significantly different from those isolated from nonfatty grafts obtained from saline-treated rats (Zhong et al., 1997). Gene delivery of manganese–SOD-2, a mitochondrial antioxidant enzyme, has only a moderate protective effect on injury of ethanol-induced fatty grafts after transplantation (Lehmann et al., 2003). The protective effect of SOD-2 is less than with Cu/Zn–SOD-1, a cytosolic antioxidant enzyme (Lehmann et al., 2003). Therefore, it is likely that mitochondria do not play a major role in free radical production in ethanol-induced fatty grafts (Fig. 3). Another attractive hypothesis is that fat accumulated in steatotic livers increases lipid peroxidation. Because lipid peroxidation is a chain reaction, accumulated fat might provide more substrate amplifying free radical formation (Fig. 3). Thiobarbituric acid reactive substances increase in livers of mice fed a high-fat diet, supporting the suggestion that fat accumulation increases susceptibility to free radical attack (Ahotupa et al., 1993). However, ethanol increases free radicals after hypoxia–reoxygenation independent of fat content in the liver (Zhong et al., 1998). Destruction of Kupffer cells with gadolinium chloride, rinsing grafts with the antioxidant catechin, and gene delivery of SOD do not reduce fatty

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infiltration in the liver, but they do significantly decrease free radical formation and blunt injury and graft failure after transplantation (Lehmann et al., 2000; Zhong et al., 1997, 1999). These findings indicate that fat accumulation per se is not responsible for free radical production. In fact, increased fat droplets in parenchymal cells do not necessarily mean that substrates for free radical formation are actually more available because lipid-soluble antioxidants also accumulate in fat droplets (Gibbons et al., 1994; Zammit, 1996). Many enzymatic and nonenzymatic defense systems against oxidative stress occur in hepatocytes where fat droplets are located (Jaeschke, 1995). Lipid-soluble antioxidants may accumulate in fat droplets in hepatocytes, thus protecting lipids from peroxidation. Therefore, the presence of fat droplets in hepatocytes might not increase free radical production. In contrast, cell membranes exposed to the extracellular space, in which concentrations of antioxidants, SOD, and catalase are very low (Freeman & Crapo, 1982; Jaeschke, 1995; Recknagel et al., 1982), may be more susceptible to oxidative stress. Ethanol treatment induces cytochrome P450 2E1 in hepatocytes, which represents another source of superoxide radicals (Nordmann et al., 1988; Teschke & Gellert, 1986). However, destruction of Kupffer cells does not alter expression of cytochrome P450 2E1, but it does block production of reactive oxygen species (Niemela¨ et al., 2002). In addition, survival and transaminase release are not different in ethanolinduced fatty grafts obtained from wild-type and P450 2E1 knockout mice (unpublished observations, M. Lehnert, L. O. Conzelmann, Z. Zhong, F. J. Gonzalez, H. Bunzendahl, and R. G. Thurman, 2001). Therefore, cytochrome P450 2E1 probably does not play an important role in free radical formation after transplantation of fatty grafts caused by ethanol. In conclusion, ethanol treatment enhances free radical formation after transplantation by mechanisms involving activation of Kupffer cells and increases in white blood cell adhesion. Other mechanisms remain to be evaluated more carefully.

5. Pathophysiologic effects of free radicals in fatty livers Increased free radicals can cause graft injury by directly damaging cell components, such as cell membranes, proteins, and DNA (Ryan & Aust, 1992). In addition, free radicals can cause graft injury indirectly. Reactive oxygen and nitrogen species are important signaling molecules regulating expression of cytokines and enzymes (Wolin, 2000). In ethanol-induced fatty livers, activation of nuclear factorkappa B and c-Jun N-terminal kinase, a stress kinase, increases (Lehmann et al., 2000). Activation of the c-Jun N-terminal kinase pathway is also related to occurrence of apoptosis (Dunn et al., 2002). Activation of nuclear factorkappa B leads to synthesis of proinflammatory cytokines and cell adhesion molecules (Collart et al., 1990; Jobin et al.,

1998; Schreck et al., 1991; Shakhov et al., 1990). Reactive oxygen production also mediates endotoxin-induced production of nuclear factor-kappa B activation and tumor necrosis factor-alpha (Han et al., 2001; Torrie et al., 2001). Production of tumor necrosis factor-alpha is about 3.5-fold higher in Kupffer cells isolated from fatty liver grafts obtained from ethanol-treated rats than in those isolated from grafts obtained from untreated donors (Thurman et al., 1995). Proinflammatory cytokines induce adhesion molecules, such as E-selectin, ICAM-1 and vascular cell adhesion molecule-1, in endothelial cells, leading to increased leukocyte adhesion (Rosales & Juliano, 1995). Indeed, ethanol increases ICAM-1 expression, white blood cell adhesion, and superoxide production after transplantation [unpublished observations, M. von Frankenberg, H. K. Bojes, Y. Iimuro, R. Schoonhoven, J. J. Lemasters, and R. G. Thurman (1995); Stachlewitz et al. (1998); Zhong et al. (1997)]. In addition, oxidative stress activates phospholipase A2 (Goldman et al., 1997), which increases production of lipid-derived vasoactive and chemotactic mediators, such as eicosanoids and platelet-activating factor. Therefore, oxidative stress not only produces direct tissue damage (necrosis, aspartate aminotranferase release) but also modulates production of toxic cytokines, leading to inflammation and multiple organ failure after fatty liver transplantation. Hepatic microcirculation is pivotal for graft survival. Disturbances of hepatic microcirculation increase graft injury and failure after transplantation (Marzi et al., 1990; Takei et al., 1991), and cold storage–reperfusion causes overt injury to sinusoidal lining cells, which could lead to disturbances of microcirculation (Caldwell-Kenkel et al., 1988, 1989; Marzi et al., 1989a). Infusion of ethanol into perfused livers causes hepatic vasoconstriction, disturbs hepatic microcirculation, and results in hepatic hypoxia (Hijioka et al., 1991; Oshita et al., 1992, 1993). Liver surface oxygen tension decreases dramatically and portal pressure increases in fatty grafts obtained from ethanol-treated rats, indicating that ethanol disturbs the microcirculation after transplantation (Zhong et al., 1999). Pimonidazole, a 2-nitroimidazole hypoxia marker, increases massively after transplantation of ethanol-treated grafts, indicating significant tissue hypoxia (Schemmer et al., 1999; Zhong et al., 1999). It is important to note that free radical scavengers (catechin), destruction of Kupffer cells, and inhibition of biosynthesis of leukotrienes with MK-886 improve graft microcirculation and decrease injury (Zhong et al., 1997, 1999). These findings are consistent with the hypothesis that ethanol increases oxidative stress, leading to production of vasoactive and chemotactic mediators and disturbances of the hepatic microcirculation after transplantation. 6. Antioxidants improve the outcomes of fatty liver transplantation from ethanol-treated donors Because ethanol increases free radical formation and graft failure after transplantation, antioxidant strategies to diminish graft injury after transplantation are under active investigation. Gene delivery of SOD-1, a cytosolic antioxidant

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enzyme, substantially decreases graft injury and improves survival of fatty grafts obtained from ethanol-treated donors. Superoxide dismutase-2, a mitochondrial enzyme, has a moderate protective effect (Lehmann et al., 2003). In contrast, SOD-3, an extracellular enzyme, shows no protection on fatty grafts (Lehmann et al., 2003). Although gene delivery of SOD and gadolinium chloride improves outcomes of fatty liver transplantation, these treatments may produce adverse effects and require a relatively long time of pretreatment (24–72 h), which is usually not available for clinical transplantation. Therefore, strategies that can be applied readily to livers already recovered for donation are desirable. When fatty grafts are rinsed before implantation with Carolina Rinse solution, which contains antioxidants and several compounds that inhibit the generation of free radicals (glutathione, desferrioxamine, and allopurinol), free radical adduct formation is decreased by about 50% (Zhong et al., 1997). Camellia sinenesis (green tea) contains high concentrations of polyphenols, including catechin, epicatechin, gallocatechin, epigallocatechin, epicatechin gallate, and gallocatechin gallate (Frankel, 1999; Hara, 1994), which are efficient free radical and singlet oxygen scavengers (Slater, 1981; Zhao et al., 1989). Rinsing of grafts with polyphenols before implantation decreases transaminase release by 66% and improves survival (Zhong et al., 2004). Protective effects of polyphenols are associated with decreased free radicals and 4-hydroxynonenal adducts. Treatment of recipients 45 min before implantation with ebselen, an organoselenium compound and glutathione peroxidase mimic, also decreases transaminase release, suppresses leukocyte infiltration, and improves graft function (unpublished observations, Z. Zhong, M. Froh, M. Lehnert, L. O. Conzelmann, G. Arteel, H. Bunzendahl, J. J. Lemasters, and R. G. Thurman, 2002). Taken together, treatment with antioxidants improves the outcome of fatty liver transplants and therefore represents a promising therapy for clinical settings. 7. Future directions Mechanisms of fatty graft failure caused by ethanol are not fully elucidated, and further studies are needed, especially the signal transduction processes leading to graft failure. Studies to date have been dependent primarily on pharmacologic approaches, which are sometimes not highly specific. New developments in gene manipulation with knockin and knockout techniques, gene therapy, and siRNA provide highly specific and powerful tools to study the mechanisms of fatty graft failure. Although significant progress has been achieved on the relation of reactive oxygen species and fatty graft failure caused by ethanol, the role of reactive nitrogen species is unclear. Nitric oxide has many protective effects in a variety of physiologic and pathophysiologic conditions (Moncada et al., 1991). However, nitric oxide reacts rapidly with superoxide to produce peroxynitrite, which decomposes to highly

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toxic hydroxyl-like radicals. Study findings have shown that mice deficient of inducible nitric-oxide synthase have attenuated liver injury after chronic (4 weeks) ethanol treatment (McKim et al., 2003). Studies should be performed to investigate the role of reactive nitrogen species on fatty liver grafts. In conclusion, free radical production plays an important role in fatty graft failure, and antioxidants are promising therapies to prevent graft injury. Further studies are needed to elucidate the mechanisms.

Acknowledgments This work was supported, in part, by grants AA09156, K01 DK62089, and DK37034 from the National Institutes of Health. Imaging facilities were supported, in part, by NIH center grants 5-P30-DK34987 and 1-P50-AA11605.

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