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,