Tropical Gastroenterology 2011;Suppl:S27–32

Alcoholic liver disease and antioxidants

Pankaj Tyagi1 and Kaushal Madan2

ABSTRACT

Department of Hepatology, One of the major mechanisms postulated in production of liver injury in alcoholics is the Institute of Liver and Biliary oxidant stress, which is produced by an imbalance between the pro-oxidants and anti-oxidants. 1 Sciences, The major sources of oxidants are the enzyme systems which metabolize ethanol and the New Delhi and enzyme systems operating in the inflammatory cells. Further, alcohol, either directly or by Medanta Institute of digestive and Hepatobiliary Sciences2, means of associated malnutrition also leads to deficiency of anti-oxidants which tips the Gurgaon, balance towards oxidative stress. We have also discussed the various anti-oxidants that have India been used in alcoholic liver disease and their effect on liver necroinflammation and survival in these patients. Correspondence: Dr. Kaushal Madan Email: [email protected] KEYWORDS:Alcoholic hepatitis, antioxidants, oxidative stress.

Introduction

Alcoholic liver disease (ALD), which results from excessive production of oxidative stress, the various pro-oxidants and consumption of alcohol over prolonged periods, is associated what they do to the tissues, the available anti-oxidants and with development of variable combination of liver steatosis, the results of their use in human ALD. inflammation, hepatocyte necrosis and fibrosis. This injury is produced as a result of interplay between the products produced Origin and sources of oxidant stress in ALD during ethanol metabolism, direct ethanol toxicity and the products produced by a number of cells such as hepatocytes, The oxidative stress in ALD is derived from multiple sources inflammatory cells and stellate cells. One of the important (Table 1). Most of it arises as a result of metabolism of alcohol mechanisms involved in producing liver injury in ethanolics is and its metabolites in parenchymal cells, and the other sources oxidant stress.1,2 Normally also the body produces, reactive include the recruited inflammatory cells and their cytokines oxygen and nitrogen species as part of the normal defense and the resident activated stellate cells in the liver. mechanism, but when these species start producing damage of Table 1: Various sources of pro-oxidant production in ALD normal tissues as a result of relative deficiency of anti-oxidant Inflammatory cells Hepatocytes defenses, it leads to a situation called oxidative stress. The NADPH oxidase CYP2E1 oxidative stress associated with ALD most likely is caused not iNOS Mitochondrial dysfunction only by an increase in pro-oxidant formation, but also results Xanthine oxidase Myloperoxidase from a deficiency of anti-oxidants which might be a consequence of poor dietary intake in alcoholics. Therefore it would seem Ethanol is mainly metabolized in the liver through three logical to replenish antioxidants to counteract the detrimental major pathways with different subcellular locations: alcohol effects of pro-oxidants in the system. In the following review, dehydrogenase (ADH) in the cytosol, aldehyde we shall discuss the pertinent mechanisms that lead to the dehydrogenase (ALDH) in the mitochondria, and microsomal S28 Tropical Gastroenterology 2011;Suppl:S27–32 ethanol-oxidizing system (MEOS) in the endoplasmic reticulum. In addition, development of ALD could also be significantly All of the three pathways result in generation of reactive oxygen blunted even in iNOS knockout mice. Further, enteral ethanol species (ROS). However, the MEOS, especially the cytochrome also caused severe fatty accumulation, mild inflammation, and P450 2E1 (CYP2E1), has been shown to play a critical role in necrosis in the liver in wild-type mice but had no effect in iNOS ethanol-induced oxidative stress. Long-term ethanol exposure knockout mice. The accumulation of 4-hydroxynonenal (lipid significantly increases the metabolism of alcohol through the peroxidation) and 3-nitrotyrosine (reactive nitrogen species CYP2E1 pathway accounting for the generation of ROS. The formation) protein adducts caused by alcohol was completely metabolism of alcohol by this enzyme leads to, leakage of blocked in iNOS knockout mice.9 These evidences point towards electrons to the respiratory chain, leading to formation of a key role played by pro-oxidants derived from superoxide anion - 3 superoxide anion O2 with oxygen. However, recent data and NO in initiation and propagation of ALD. The inflammatory suggests that CYP2E1 knockout mice are not protected against cells also contribute to oxidative stress by means of the enzyme alcohol mediated injury.4 If CYP2E1 were an important mediator systems, xanthine oxidase and myeloperoxidase. in alcohol induced oxidative stress related liver injury, then Myeloperoxidase not only produces hypochlorous acid which knocking out CYPE1 should have been protective. It is possible can recruit inflammatory cells, but also leads to production of that other cytochrome P450 enzymes may take up compensatory reactive nitrogen species (RNS).10 role in the absence of CYP2E1 and initiate the oxidative stress induced liver injury.5 Pro-oxidants in ALD Within the hepatocytes mitochondria is another site where pro-oxidants are produced. Normally, about 1-2% of the oxygen There are two major categories of pro-oxidant species: the - consumed in the mitochondria leads to the formation of O2 , reactive oxygen species (ROS; derived from superoxide anion) the production of which gets augmented by chronic alcohol and the reactive nitrogen species (RNS; derived from nitric consumption.6 The free radicals so formed, directly damage oxide) (Table 2). One of the prime pro-oxidant is the superoxide mitochondrial membranes and systems, leading to anion which not only produces direct oxidative damage, but mitochondrial aging and facilitating apoptosis. Besides this, also triggers downstream production of more potent oxidant alcohol also leads to depletion of the key anti-oxidant species during its metabolism, such as the hypochlorous acid mitochondrial glutathione, further aggravating the oxidative and peroxynitrite. Copper-zinc superoxide dismutase (CuZn- stress. A small pool of mitochondrial glutathione is maintained SOD), the enzyme which reduces the superoxide anion to H2O2 by the action of a carrier protein which transfers the glutathione is reduced in experimental ALD (mainly because of consumption from cytosol to the mitochondria. The function of this or deficiency) and correlated inversely with pathological liver glutathione transporter gets impaired during chronic alcohol injury in rats fed on ethanol, favoring the role of the superoxide consumption.7 anion.11 The other important pro-oxidant is derived from nitric Another important source of oxidant production in oxide (NO). Its role is controversial in the pathogenesis of alcoholics is the cytokine mediated inflammatory response. ALD since NO also plays a protective role in patients with There is stimulation of resident inflammatory cells as well as chronic liver diseases. However the peroxynitrite anion can recruitment of neutrophils and lymphocytes. Chronic alcohol induce formation of other species such as 3- nitrotyrosine, consumption produces stimulation of the bactericidal nitrosothiols and hydroxyethyl radicals which augment both processes within these cells which is responsible for producing oxidative and nitrosative injury in ALD.12 pro-oxidants. Within these cells, the important sources of free Table 2: Different pro-oxidants involved in pathogenesis of radical production are the NADPH oxidase and inducible nitric ALD oxide synthase (iNOS) enzyme systems. They lead to the Reactive oxygen species Reactive nitrogen species production of superoxide anion, peroxynitrite and hydroxyethyl (ROS) (RNS) radicals. Studies using NADPH knockout mice and use of .— — Superoxide anion (O2 ) Peroxynitrite radical (ONOO ) inihibitors of NADPH oxidase demonstrated that in enteral Hydrogen peroxide (H2O2) Nitric oxide (NO) — models of ALD, the liver injury could be totally ameliorated.8 Hypochlorous acid (HOCL ) Alcoholic liver disease S29

Effect of oxidant stress on liver TNFα, which drives not only the inflammatory response15 but also leads to hepatocyte apoptosis. The pro-oxidants produced by chronic alcohol exposure by There is also evidence for proteasomal dysfunction in ALD. mechanisms outlined above can either cause a direct damaging Protesome is the prinicipal mechanism responsible for protein effect on biological molecules, or the chemical modification of degradation or clearance of altered proteins, in eukaryotic cells. the biological molecules can interfere with the normal biological Considering that there is an inverse correlation between processes or the modified molecules and incite an immune proteasomal activity and hepatic amounts of lipid peroxides, response, which can augment tissue injury. Oxidative stress as well as the fact that, decrease in proteasomal activity is not causes lipid peroxidation, which can directly damage the detected in ethanol-fed CYP2E1-knockout mice, it can be membranes of cells and organelles leading to release of reactive speculated that CYP2E1-derived oxidative stress plays an aldehydes with potent pro-inflammatory and pro-fibrotic essential role in proteasomal dysfunction in ALD.16 Since properties. The fact that alcoholic hepatitis may persist for CYP2E1 itself is also degraded by proteasomes, ethanol- many months following cessation of alcohol intake, points induced oxidative stress may down-regulate the proteasomal towards an ongoing immune mediated injury. In such patients, degradation pathway and increase CYP2E1 expression, thus auto-antibodies can be demonstrated, directed against further overproducing ROS. phospholipids, alcohol dehydrogenase and heat shock proteins. In addition, there may be antibodies directed against Antioxidants for ALD neo-antigens which are derived from adducts formed between products of alcohol metabolism (acetaldehyde and As has been alluded in the previous section, there is ample hydroxyethyl radicals) and liver proteins.13 Malondialdehyde experimental evidence for the presence of significant oxidative (MDA) adducts trigger a potent immune response, at least in stress in ALD, both during initiation and propagation of hepatic 36% of alcoholics. Although the main source of cytokines are inflammation. Despite this, the available anti-oxidants have not Kupffer cells, activated by endotoxin via toll-like receptors, been able to demonstrate significant clinical efficacy in activated T cells also secrete cytokines, which contribute to ameliorating hepatic necro-inflammation. However we shall liver damage and MDA adducts are responsible, at least in review here the various anti-oxidants which have been tried part, for T-cell activation and cytokine production. In animal for ameliorating the oxidative stress in ALD (Table 3). Some of models, MDA adducted to mouse serum albumin can induce these are direct anti-oxidants and react with free radicals CD4 lymphocytes.14 However, variable immune responses to rendering them harmless, while others replenish the anti-oxidant antigens formed as a result of ethanol metabolism may explain capabilities of the cells. in part the variable inter-individual susceptibility to alcohol and the inter-individual variability of the severity of alcoholic Polyenylphosphatidylcholine liver disease. In addition, oxidants affect several signaling pathways and Polyenylphosphatidylcholine (PPC) or lecithin, derived from the most significant among them, for pathogenesis of ALD, soybeans, is a major active ingredient in essential seems to be NFκB from Kupffer cells. NFκB signaling stimulates phospholipids, has a high bioavailability and affinity for cellular the production of many pro-inflammatory cytokines, especially and sub-cellular membranes and maintains membrane fluidity

Table 3: Studies of anti-oxidants in ALD

Author Anti-oxidant used N Disease Outcome measured Effect Lieber et al18 Polyenylphosphatidylcholine 789 Acoholic hepatitis + cirrhosis Liver fibrosis improvement No effect Mato et al20 S-adenosyl methionine 123 Alcoholic cirrhosis 4 years survival Improved de la Maza et al22 Vitamin E 67 Alcoholic cirrhosis 1 year survival No effect Mezey et al23 Vitamin E × 3months 51 Alcoholic hepatitis 1 year survival No effect Lucena et al25 Silymarin 60 Alcoholic cirrhosis Glutathione levels Increased Pares et al26 Silymarin 200 Alcoholic cirrhosis Survival No effect Ferenci et al27 Silymarin 170 Alcoholic cirrhosis 4 years survival Increased Kaballeria et al29 Metadoxine 136 Alcoholic fatty liver Fatty liver markers Improved S30 Tropical Gastroenterology 2011;Suppl:S27–32 and function. Several experiments have demonstrated the Silymarin hepato-protective effects of PPC by attenuation of hepatic ROS generation by down regulation of CYP2E1 and Silymarin is a complex mixture of four flavonolignan isomers, replenishment of GSH.17,18 In a randomized, prospective, namely silybin, isosilybin, silydianin and silychristin derived double-blind, placebo-controlled clinical trial involving 789 from milk thistle, Silybum marianum. It has anti-oxidant ALD patients, a trend for biochemical (transaminases and properties and protects against lipid peroxidation and exerts bilirubin) improvement was observed in treated patients, but anti-inflammatory and anti-fibrotic properties. In an RCT among without statistically significant differences. There was no 60 patients with alcoholic cirrhosis, silymarin therapy in a dose difference in the main outcome, which was progression of of 150 mg thrice a day resulted in an increase in glutathione liver fibrosis at 2 years.19 levels and reduction in lipid peroxidation.26 However, a large RCT (involving 200 alcoholic cirrhotics, who were given 450 S-adenosyl methionine mg/day of silymarin or placebo) did not show any difference in survival.27 One initial study did show survival advantage in S-adenosyl methionine (SAMe) is the activated form of cirrhotics treated with silymarin. In this double-blind, methionine. It is the major methyl donor in biological reactions prospective, randomized study of 170 cirrhotic patients, and is a precursor in the synthesis of key metabolites such as administration of silymarin 140 mg three times daily for a mean glutathione and polyamines. SAMe is a precursor of cellular duration of 41 months significantly improved the 4-year survival glutathione which is the key cellular antioxidant defense. It rate (p=0.03). Silymarin was significantly effective in the has been demonstrated that long-term ethanol consumption subgroup of patients with alcoholic cirrhosis and in those with in experimental animals is associated with hepatic SAMe milder disease.28 A recent meta-analysis of 18 RCTs (only 28.6% depletion and SAMe deficiency results in glutathione high quality studies) on the use of silymarin in alcoholic and deficiency. Exogenous SAMe corrects hepatic deficiency of non-alcoholic cirrhosis, did not demonstrate any survival both SAM and glutathione.20 Results of a long-term advantage against placebo or no treatment.29 The treatment randomized, placebo controlled, double-blind multicentre was well tolerated. Human studies have shown that silymarin clinical trial of SAMe in 123 patients with alcoholic cirrhosis is generally nontoxic and without side effects when demonstrated that oral SAMe 1200 mg/day improved the 4- administered to adults in a dose range of 240-900 mg/day in year survival marginally and the maximum effect was seen in two or three divided doses. At higher doses of more than 1500 Child’s A or B cirrhotics.21 mg/day, silymarin may produce a laxative effect which may be due to increased bile flow and secretion. Mild allergic reactions Vitamin E have also been noted, but such situations may not necessitate discontinuing the treatment. Vitamin E levels have been demonstrated to be lower in experimental animals consuming alcohol22 and in ALD patients. Metadoxine In one study administration of 500 I.U. of vitamin E for 1 year to patients with alcoholic cirrhosis had no significant influence Metadoxine, a drug that restores hepatic glutathione on clinical status, laboratory parameters or mortality.23 Similarly, concentrations and acts as an anti-fibrogenic agent, proved in another study with 51 patients of alcoholic hepatitis, vitamin efficacious in a double-blind, randomized multicentre trial E supplementation (100mg/day) for 3 months did not show involving 136 patients with alcoholic fatty liver alone. A dosage any improvement in survival. NF-κB binding activity decreased of metadoxine 1500 mg/day administered for 3 months in patients who remained abstinent, regardless of whether accelerated the normalization of liver function tests and the they received vitamin E.24 Another study used a cocktail of ultrasonographic changes of steatosis. This suggests that the antioxidants (N-acetyl cysteine, vitamin A-E, biotin, selenium, drug could be useful in the treatment of the early stages of zinc, manganese, copper, folic acid and co-enzyme Q) for 6 ALD.30 months in a double blind randomized controlled trial but was In addition there was a recent meta-analysis on the role of not able to demonstrate any improvement in survival in anti-oxidant supplementation in liver diseases (both alcoholic alcoholic hepatitis.25 and non-alcoholic). This included 20 randomized controlled Alcoholic liver disease S31 trials and 1225 patients. It demonstrated that there was no 4. Kono H, Bradford BU, Yin M, Sulik KK, Koop DR, Peters JM, effect on all cause mortality or liver related mortality with the et al. CYP2E1 is not involved in early alcohol-induced liver injury. Am J Physiol. 1999;277:G1259–67. use of anti-oxidant supplementation. Even stratification 5. Maher J. The CYP2E1 knockout delivers another punch: first according to the type of liver disease did not affect the ASH, now NASH. Alcoholic steatohepatitis. Nonalcoholic outcomes.31 There are other anti-oxidants which have shown steatohepatitis. Hepatology. 2001;33:311–2. experimental evidence of benefit in reducing hepatic oxidative 6. Cadenas E, Davies KJ. Mitochondrial free radical generation, oxidative stress, and aging. Free Radic Biol Med. stress, such as curcumin and zinc, but there are not enough 2000;29:222–30. human studies, so are not being presented here. Thus most 7. Fernandez-Checa JC, Kaplowitz N, Garcia-Ruiz C, Colell A, studies on the use of anti-oxidants in alcoholic liver disease Miranda M, Mari M, et al. GSH transport in mitochondria: suggest that there is mild improvement in the biochemical defense against TNF-induced oxidative stress and alcohol-induced defect. Am J Physiol. 1997;273:G7–17. parameters with the use of anti-oxidants, but most studies as 8. Kono H, Rusyn I, Yin M, Gabele E, Yamashina S, Dikalova A, et well as meta-analyses have failed to show an improvement in al. NADPH oxidase-derived free radicals are key oxidants in survival with the use of anti-oxidants in ALD patients. alcohol-induced liver disease. J Clin Invest. 2000;106:867–72. In conclusion, even though evidence implicating the role 9. McKim SE, Gabele E, Isayama F, Lambert JC, Tucker LM, Wheeler MD, et al. Inducible nitric oxide synthase is required in of oxidant stress in pathogenesis of alcoholic liver disease is alcohol-induced liver injury: studies with knockout mice. extensive, there is little convincing evidence favoring a Gastroenterology. 2003;125:1834–44. beneficial role of anti-oxidants in reversing the liver necro- 10. Eiserich JP, Hristova M, Cross CE, Jones AD, Freeman BA, inflammation or in improving survival in these patients. One Halliwell B, et al. Formation of nitric oxide-derived inflammatory oxidants by myeloperoxidase in neutrophils. 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