Dangerous byproducts of alcohol breakdown--focus on adducts.

1Most of the alcohol a person ingests is eliminated from the body via a series of chemical reactions in the liver that are collectively referred to as oxidative metabolism. Because the liver is one of the organs that most commonly exhibits alcohol-induced damage, researchers have attributed many of the disturbances in liver structure and function frequently seen in alcoholics to the products of alcohol metabolism. The most important enzyme involved in the breakdown of alcohol is called alcohol dehydrogenase, which converts alcohol into acetaldehyde, a highly reactive and toxic molecule that may play a crucial role in alcohol-related liver damage.

2Another enzyme that can mediate the initial step of alcohol metabolism is cytochrome P450 2E1. The chemical reaction promoted by this enzyme also results in the formation of acetaldehyde, as well as in the production of highly reactive oxygen-containing molecules called oxygen radicals, including the hydroxyethyl radical (HER). Excessive production of oxygen radicals and/or a concurrent deficiency of molecules that can eliminate these radicals (i.e., antioxidants) creates a condition in the cell known as oxidative stress, which can lead to cell death. (For more information on the role of oxygen radicals and oxidative stress in liver disease, see the article in this issue by Wu and Cederbaum.) Furthermore, oxygen radicals can interact with fat (i.e., lipid) molecules in the cell membranes in a process called lipid peroxidation, which in turn results in the generation of additional reactive molecules similar to acetaldehyde, especially malondialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE).

3Because acetaldehyde and the lipid peroxide–derived aldehydes (i.e., MDA and HNE) are chemically reactive, they can interact with proteins and other complex molecules to form modified molecules known as adducts. The formation of these aldehyde–protein adducts is believed to be a key event in the development of alcohol-induced liver injury.

4This article describes the types of adducts formed in the liver, reviews evidence that such adducts are generated during alcohol consumption, and discusses the possible role that adducts may play in liver injury.

5As mentioned above, alcohol degradation using both alcohol dehydrogenase and cytochrome P450 2E1 generates reactive compounds that can bind to proteins and form adducts (see the accompanying figure). Numerous studies have reported that a variety of protein adducts are formed in the liver as a result of alcohol consumption and degradation. The major reactive molecules participating in adduct formation appear to be those that are chemically known as aldehydes, specifically acetaldehyde, MDA, and HNE.

6The formation of acetaldehyde–protein adducts has received the most attention from researchers to date. Acetaldehyde can react with proteins in the body to form both unstable adducts, which are immediately converted into other compounds, and stable adducts, which remain in the cell for a certain length of time. Proteins are made up of approximately 20 different building blocks (i.e., amino acids). Researchers have found that acetaldehyde interacts with specific amino acids, particularly lysine, during adduct formation. Although investigators also have identified the chemical group—a so-called amino group1—with which acetaldehyde interacts, the exact chemical structures of the stable adducts have not yet been resolved.

7Hydroxyethyl radical, a highly reactive oxygen radical that participates in adduct formation

84-hydroxy-2-nonenal, a reactive molecule that is generated during lipid peroxidation and participates in adduct formation

9Mixed MDA–acetaldehyde–protein adducts; compounds that contain MDA plus acetaldehyde plus protein components

10Malondialdehyde, a reactive molecule that is generated during lipid peroxidation and participates in adduct formation.

11The lipid peroxide–derived aldehydes, MDA and HNE, also can react with proteins, generating a variety of adducts. MDA, like acetaldehyde, reacts mainly with amino groups found in proteins and forms a variety of diverse adducts, complicating the analysis of the exact processes occurring during MDA–protein adduct formation. HNE can react with various amino acids in proteins but appears to interact primarily with the amino acids lysine, cysteine, and histidine to form relatively stable HNE–protein adducts.

12Each of the aldehydes discussed here can form adducts with proteins on its own. During alcohol metabolism in the liver, however, the aldehydes coexist in the cells and may influence each other’s reactivity with proteins. For example, researchers have found that MDA and acetaldehyde react with proteins in a synergistic manner. This means that the presence of both aldehydes strikingly increases each aldehyde’s binding to proteins, generating mixed (i.e., hybrid) adducts that clearly differ from the adducts formed by either aldehyde alone. These composite MDA–acetaldehyde–protein adducts, called MAA adducts, consist of two major components whose structures have been determined and that contain the amino acid lysine: (1) a relatively stable compound containing two molecules of MDA and one molecule of acetaldehyde; and (2) a compound containing one molecule of MDA and one molecule of acetaldehyde, which appears to serve as a precursor for the more stable adduct.

13Although many proteins contain the amino acids—primarily lysine—that interact with the aldehydes, not all of those proteins are equally likely to form adducts with aldehydes. Proteins that appear to be preferentially modified by aldehydes include, among others, the following:

14Hemoglobin, which is crucial for the transport of oxygen in the red blood cells.

15Albumin, a protein found in the blood.

16Tubulin, a component of cell structures called microtubules that are essential for cell division as well as for the secretion and transport of proteins within the cells.

17Lipoproteins, which consist of proteins and fat molecules and influence the risk of heart disease.

18Collagen, the major protein in the connective tissues, which is involved in scar tissue formation.

19Cytochrome P450 2E1, which plays a role in the breakdown of alcohol and other toxic substances.

20Ketosteroid reductase, a key enzyme involved in proper digestion (i.e., it influences the production of bile acid biosynthesis).

21Researchers have not yet determined the basis for this preferential binding of aldehydes to certain proteins. It seems, however, that some lysine residues in proteins are particularly reactive with aldehydes and preferentially form adducts because of their local environments (i.e., the other amino acids surrounding them within the protein).

22Aldehyde–protein adducts are not the only class of adducts formed during alcohol metabolism; another class of adducts involves oxygen radicals generated during alcohol breakdown. For example, cytochrome P450 2E1–mediated oxidation of alcohol can result not only in the formation of acetaldehyde but also in the formation of the HER oxygen radical that can readily bind to proteins and form HER–protein adducts. Because it is highly reactive, HER is likely to react with a variety of different sites on proteins, generating a range of adducts.

23Adducts created with proteins are the most widely studied and probably the most important adducts resulting from alcohol metabolism. Nevertheless, the aldehydes and oxygen radicals generated during alcohol metabolism also can form adducts with other complex molecules, such as DNA or lipids. Further study of the roles and consequences of these adducts is needed.

24The fact that it is possible to create adducts through chemical reactions in test tubes (i.e., in vitro) does not prove that adduct formation actually occurs in the liver (i.e., in vivo) and contributes to the development of alcoholic liver disease (ALD). Therefore, it is important to verify that adducts are generated in the liver during alcohol consumption. Numerous studies over the years have indicated that chronic alcohol consumption in both animals and humans results in the formation of various protein adducts in the liver; the findings of these studies are summarized in this section.

25The first evidence indicating that adducts are generated in vivo as a result of alcohol intake came from studies demonstrating that after proteins have been modified by reactive molecules such as aldehydes, the adducts that are produced can elicit an immune response. This means that the body recognizes these adducts as something “foreign” (i.e., not normally belonging to the body) and in response generates antibodies that recognize and bind to these foreign molecules. The interaction between a foreign molecule and the antibodies produced by the body marks the foreign molecule for destruction by other immune cells. In most cases, however, the antibodies recognize the modified protein adducts but not the original proteins; therefore, the original proteins can remain in the body and exert their influence without being destroyed by the immune system.

26Researchers subsequently have detected antibodies against acetaldehyde, MDA, HNE, MAA, and HER adducts in the blood of animals and humans who chronically consumed alcohol, implying that adducts are formed in the body and then stimulate the production of antibodies. These findings provide indirect evidence that alcohol metabolism in vivo does indeed result in the formation of aldehyde– protein and HER–protein adducts.

27Direct evidence that adducts form in the livers of animals and humans consuming alcohol was derived from studies using antibodies against specific adducts. For example, several studies have reported the presence of acetaldehyde–protein adducts in the livers of alcohol-fed animals and humans. These adducts were detected mainly in the principal liver cells (i.e., the hepatocytes), although they may occur in other types of liver cells as well. Various other types of adducts also have been observed, sometimes at specific locations in the liver or liver cells, as follows:

28Most of the proteins that are modified by acetaldehyde appear to be located in the fluid filling the cells (i.e., the cytosol); however, adducts also form with proteins embedded in the membrane surrounding the cells and in other membrane-enclosed cell structures. Acetaldehyde adducts are found predominantly in a certain region of the liver lobules2 (i.e., the perivenous region, which is located around the small vein through which the blood exits the lobule).

29MDA and HNE adducts have been observed in the livers of alcohol-consuming animals; like acetaldehyde adducts, MDA adducts are found at sites in the liver where tissue damage can be detected.

30MAA adducts are generated in vivo and appear to involve primarily proteins in the cytosol of liver cells.

31HER adducts have been detected in the cell membrane and in certain membrane-enclosed cell components (i.e., microsomes) of the hepatocytes of alcohol-fed animals.

32Although the formation of adducts in the liver during alcohol consumption has been well established, more information is needed concerning the effects of these adducts on liver cell function and the role they play in liver injury. Recent research in this area has provided some interesting and exciting information on the link between adduct formation and liver dysfunction and injury. These findings may represent a first step in the development of therapeutic interventions that can interfere with adduct formation and its consequences and thereby help reduce the risk of ALD.

33One line of evidence concerns the locations of adduct formation and alcohol-related tissue damage in the liver. As mentioned in the previous section, acetaldehyde adducts form primarily in the perivenous region of the liver, which is also the region where alcoholic liver injury starts and predominates, thus supporting the hypothesis that acetaldehyde adducts may contribute to alcoholic liver disease. Moreover, acetaldehyde adducts are evident in the early phase of ALD, and in advanced liver disease they are found in the same areas that show evidence of inflammation and scar tissue formation (i.e., fibrosis). MDA adducts are found at the same sites as (i.e., colocalize with) acetaldehyde adducts in areas where changes in tissue structure (i.e., histological changes) occur in alcoholic liver disease. Additional studies using a specially bred type of small pig (i.e., micropigs) as a model system for the development of alcoholic liver disease reported a progressive accumulation of acetaldehyde, MDA, and HNE adducts after prolonged alcohol intake, which coincided with progressive liver injury. Moreover, acetaldehyde and MDA adducts, which increased after alcohol feeding, colocalized with the sites of collagen deposits, a characteristic step in scar tissue formation that occurs prior to fibrosis. Thus, these findings indicate a link between acetaldehyde and MDA adducts and the subsequent development of fibrosis in the perivenous region.

34Researchers have identified several mechanisms through which various adducts could contribute to liver damage. As mentioned earlier, aldehydes interact primarily with the amino acid lysine. Thus aldehydes particularly interfere with the functions of those proteins that carry a lysine residue at a location in the protein that is critical to the protein’s function. Examples of such proteins include the lysine-dependent enzymes, the regulatory protein calmodulin, and the cytoskeletal protein tubulin. Acetaldehyde–tubulin adducts appear to be especially important and relevant to alcohol-induced liver injury. Studies have shown that modification of only 5 percent of the individual molecules of a certain type of tubulin (i.e., α-tubulin) by acetaldehyde leads to complete inhibition of tubulin assembly into microtubules. Impaired microtubule function likely accounts for the observed defects in protein secretion and other protein transport pathways in the liver that result from chronic alcohol consumption. This altered microtubule function also could lead to a considerable disorganization of the hepatocytes that is characterized by various structural changes and which could progress to more severe liver damage in alcohol abusers. Investigators have described impaired function of numerous proteins other than tubulin by adduct formation; however, the role of these adducts in liver dysfunction and injury remains to be established.

35Another important process in the liver that is affected when adduct formation interferes with the functions of proteins is extracellular matrix production. The extracellular matrix is a set of proteins (e.g., collagen, fibronectin, and laminin) that form a kind of scaffolding in which the cells are embedded. Disturbances in extracellular matrix production could lead to the formation of scar tissue in the liver (i.e., hepatic fibrosis)—the abnormal accumulation of extracellular matrix components that is characteristic of stages of alcoholic liver disease prior to cirrhosis. Many studies have shown that acetaldehyde as well as HNE and MDA increase collagen production in various types of liver cells, such as fibroblasts and activated stellate cells3. The intriguing possibility that adduct-stimulated collagen production may contribute to the overall process of alcoholic hepatic fibrosis has not been completely evaluated.

36Another interesting mechanism by which adducts could induce liver injury involves the immune system. Numerous studies have shown that aldehyde–protein and HER–protein adducts elicit a distinct immune response, and additional analyses have demonstrated that antibodies against such adducts are present in both humans and animals following chronic alcohol exposure. These findings have led to the hypothesis that adducts formed as a result of alcohol consumption may be recognized by the immune system and may trigger harmful immune responses that could lead to liver damage. This hypothesis is supported by findings that hepatitis can be induced in guinea pigs by feeding alcohol to animals previously immunized with acetaldehyde adducts. Further studies are necessary to evaluate the exact roles of adducts and the immune system in the development of alcohol-induced liver injury.

37Recently, researchers also have devoted considerable attention to the role of the two main MAA adducts in liver injury.4 Acetaldehyde and MDA can interact with each other and then modify other proteins to form MAA adducts, and conditions in the liver during chronic alcohol metabolism should actually favor the formation of those hybrid MAA adducts over the formation of the individual acetaldehyde and MDA adducts. Therefore, MAA adducts may well represent the most prevalent aldehyde adducts formed in the liver after alcohol consumption.

38MAA adducts elicit a potent immune response that may represent an important event in the development of alcoholic liver injury. Several lines of evidence are consistent with this hypothesis:

39MAA adducts induce strong antibody responses against certain components of the MAA adducts. Unlike most antibody responses, however, these antibodies sometimes also recognize certain regions of the original proteins, even protein regions that normally identify the protein as belonging to the body and which therefore should not cause an immune response. An immune response to proteins that belong to the body resulting in the destruction of those normal proteins, however, can be highly detrimental to the organism.

40Antibodies against MAA adducts circulating throughout the body have been observed in alcohol-fed animals and in humans with alcoholic liver disease; moreover, the levels of these antibodies correlated with the severity of liver injury.

41Circulating antibodies to “self ” proteins, which probably were generated when these proteins were involved in adduct formation, have been reported in alcohol-fed animals.

42MAA modification of a certain protein found in the liver elicited a specific type of immune response.

43Taken together, these findings suggest that the formation of MAA–protein adducts generates harmful compounds that could trigger destructive immune responses targeting liver cells; however, further studies are needed to elucidate this proposed mechanism.

44Other investigators have demonstrated that MAA adducts can directly induce processes that promote inflammation and fibrosis in two types of liver cells—the previously mentioned stellate cells and the endothelial cells that line the walls of the small channels that allow blood to flow through the liver. Exposure of these two cell types to MAA adducts in vitro caused:

45Increased secretion of messenger molecules—cytokines and chemokines—that mediate communication among cells and promote inflammation, including, for example, a cytokine called tumor necrosis factor.

46Increased production of molecules that allow cells to adhere to each other (i.e., cellular adhesion molecules).

47Increased production of a molecule (i.e., fibronectin) that is part of the extracellular matrix and is involved in fibrosis.

48Enhanced secretion of a protein5 that leads to the activation of growth factors which promote the formation of tissue fibers (e.g., during scar tissue formation).

49All these effects appear to be mediated by MAA adducts that bind to specific molecules (i.e., receptors) on the surface of the liver cells and initiate a cascade of chemical signals within the cells, resulting in altered cellular functions.

50To establish the importance of MAA adducts in the development and/or progression of alcoholic liver injury, researchers must conduct similar work in intact organisms (i.e., in vivo) to confirm the results of these in vitro studies indicating that MAA adducts can induce cellular responses that favor inflammation and fibrosis.

51Excessive alcohol consumption results in the production of several types of protein adducts in the liver. Some plausible and intriguing mechanisms have been proposed to explain the role of adducts in the development of alcoholic liver injury; however, innovative approaches to better identify the mechanisms through which adducts cause liver injury remain challenging goals. The development of new therapeutic interventions for patients with alcoholic liver disease aimed at modifying or preventing adduct formation also poses a challenge for investigators. Both of these endeavors represent fertile areas for future research and should provide valuable information concerning alcohol’s toxic effects on the liver and the treatment of alcoholic liver disease.