Dean J. Tuma

Acetaldehyde adducts with proteins: Binding of [14C]acetaldehyde to serum albumin

Acetaldehyde, the immediate oxidation product of ethanol metabolism, was assessed for its ability to bind covalently to a purified protein in solution. Bovine serum albumin (BSA) was used as the model protein incubated in the presence of 0.2 mM [14C]acetaldehyde at pH 7.4 and at 37 degrees C. Acetaldehyde formed both stable and unstable adducts with serum albumin. Unstable adducts were identified following stabilization with the reducing agent sodium borohydride. We examined both types of binding using trichloroacetic acid precipitation, gel filtration, and dialysis as means to separate bound from free acetaldehyde. All three methods of analysis gave comparable results except that the number of stable acetaldehyde adducts with albumin were significantly lower following separation by dialysis. The effects of L-cysteine, L-lysine, and reduced glutathione were assessed for their abilities as competitive reagents to decrease binding of [14C]acetaldehyde to BSA. Addition of cysteine caused a rather dramatic concentration-dependent reduction in [14C]acetaldehyde binding to BSA when compared to that caused by lysine which displayed a relatively mild effect on covalent binding. The presence of glutathione caused a concentration-dependent decrease in protein-bound radioactivity that was stronger than that by lysine but not as effective as cysteine. The ability of each reagent to reverse the formation of preformed acetaldehyde adducts with BSA was also examined. Only L-cysteine effectively decreased the number of unstable acetaldehyde adducts with BSA while stable binding of acetaldehyde remained essentially unaffected by any of the three reagents. These results indicate that acetaldehyde can covalently bind to protein and form unstable as well as stable adducts.

Role of malondialdehyde-acetaldehyde adducts in liver injury.

Malondialdehyde and acetaldehyde react together with proteins in a synergistic manner and form hybrid protein adducts, designated as MAA adducts. MAA-protein adducts are composed of two major products whose structures and mechanism of formation have been elucidated. MAA adduct formation, especially in the liver, has been demonstrated in vivo during ethanol consumption. These protein adducts are capable of inducing a potent immune response, resulting in the generation of antibodies against both MAA epitopes, as well as against epitopes on the carrier protein. Chronic ethanol administration to rats results in significant circulating antibody titers against MAA-adducted proteins, and high anti-MAA titers have been associated with the severity of liver damage in humans with alcoholic liver disease. In vitro exposure of liver endothelial or hepatic stellate cells to MAA adducts induces a proinflammatory and profibrogenic response in these cells. Thus, during excessive ethanol consumption, ethanol oxidation and ethanol-induced oxidative stress result in the formation of acetaldehyde and malondialdehyde, respectively. These aldehydes can react together synergistically with proteins and generate MAA adducts, which are very immunogenic and possess proinflammatory and profibrogenic properties. By virtue of these potentially toxic effects, MAA adducts may play an important role in the pathogenesis of alcoholic liver injury.

Betaine, ethanol, and the liver: a review.

Two of the most important biochemical hepatic pathways in the liver are those that synthesize methionine and S-adenosylmethionine (SAM) through the methylation of homocysteine. This article reviews some recent findings in this laboratory, which demonstrate that ethanol feeding to rats impairs one of these pathways involving the enzyme methionine synthetase (MS), but by way of compensation increases the activity of the enzyme betaine:homocysteine methyl transferase (BHMT), which catalyzes the second pathway in methionine and SAM biosynthesis. It has been shown that despite the inhibition of MS, the enhanced BHMT pathway utilizes hepatic betaine pools to maintain levels of SAM. Subsequent to the above findings, it has been shown that minimal supplemental dietary betaine at the 0.5% level generates SAM twofold in control animals and fivefold in ethanol-fed rats. Concomitant with the betaine-generated SAM, ethanol-induced hepatic fatty infiltration was ameliorated. In view of the fact that SAM has already been used successfully in the treatment of human maladies, including liver dysfunction, betaine, shown to protect against the early stages of alcoholic liver injury as well as being a SAM generator, may become a promising therapeutic agent and a possible alternative to expensive SAM in the treatment of liver disease and other human maladies.

The Functional Implications of Acetaldehyde Binding to Cell Constituents

Chronic ethanol consumption is a major cause of liver disease. Although the underlying pathogenic mechanisms are unclear, present evidence indicates that ethanol and/or its metabolites are directly injurious to the liver. However, genetic, environmental. and nutritional factors may also modulate the hepatotoxicity of ethanol.’-’ Since ethanol is primarily metabolized in the liver, which is a major target organ of ethanol-induced toxicity, many of the functional and structural alterations in the liver produced by ethanol consumption have been attributed to the products of ethanol 0xidati0n.l~~ I n this regard, formation of acetaldehyde, the first metabolite of ethanol, and the altered redox state (increased NADH/NAD ratios in the cytosolic and mitochondria1 compartments) have been implicated in many of the ethanol-induced alterations of hepatic structure and function.’-5 Recently, we have formulated a hypothesis, postulating that acetaldehyde via its covalent binding to hepatic proteins may be a critical event leading to liver inj~ry.~*’Such a ypothesis is consistent with the evidence implicating ethanol as a direct hepatotoxin and, in addition, takes into account the possible role of metabolic, nutritional, and environmental factors in the pathogenesis of alcoholic liver injury. In this report, we will briefly describe some of the characteristics of acetaldehyde binding to proteins, but will mostly focus on potential consequences of such binding.

Detection of circulating antibodies to malondialdehyde-acetaldehyde adducts in ethanol-fed rats

BACKGROUND & AIMS Malondialdehyde and acetaldehyde react together with proteins and form hybrid protein conjugates designated as MAA adducts, which have been detected in livers of ethanol-fed rats. The aim of this study was to examine the immune response to MAA adducts and other aldehyde adducts during long-term ethanol exposure. METHODS Rats were pair-fed for 7 months with a liquid diet containing either ethanol or isocaloric carbohydrate. Circulating antibody titers against MAA adducts and acetaldehyde adducts were measured and characterized in these animals. RESULTS A significant increase in antibody titers against MAA-adducted proteins was observed in the ethanol-fed animals. Competitive inhibitions of antibody binding indicated that the circulating antibodies against MAA-modified proteins in the ethanol-fed rats recognized mainly a specific, chemically defined MAA epitope. Antibody titers to reduced and nonreduced acetaldehyde adducts were very low, and no significant differences were observed between ethanol-fed and control animals. Significant plasma immunoreactivity to not only MAA-adducted but also unmodified rat liver proteins (cytosol, microsomes, and especially plasma membrane) were also observed in the ethanol-fed rats. CONCLUSIONS Long-term ethanol feeding generates circulating antibodies not only against MAA epitopes but possibly also against unmodified, native (self) protein epitopes, suggesting that MAA adducts could trigger harmful autoimmune responses.

Association of malondialdehyde-acetaldehyde (MAA) adducted proteins with atherosclerotic-induced vascular inflammatory injury

Atherosclerosis is a vascular injury characterized by elevated tissue levels of tumor necrosis factor-alpha (TNF-alpha), increased expression of endothelial cell adhesion molecules, and vascular wall inflammatory cell infiltration. Foam cells are associated with atherosclerotic plaque material, and low density lipoprotein (LDL) is a lipid component of foam cells. Malondialdehyde (MDA) is an oxidative product of unsaturated fatty acids and is also present in atherosclerotic lesions. MDA-modified (adducted) proteins, including MDA-modified LDL, are present in atherosclerotic human vascular tissue. Acetaldehyde (AA) is the major metabolic product of ethanol oxidation. Both MDA and AA are highly reactive aldehydes and will combine with proteins to produce an antigenically distinct protein adduct, termed the MAA adduct. This study demonstrates that proteins modified in the presence of high concentrations of MDA can produce MAA-modified proteins in vitro. In addition, MAA adducted proteins are capable of inducing rat heart endothelial cell cultures (rHEC) to produce and release TNF-alpha, and cause rHEC upregulation of endothelial adhesion molecule expression, including ICAM-1. These adhesion molecules are required for circulating inflammatory cells to adhere to endothelium which allows inflammatory cell tissue infiltration. Additionally, MAA modified proteins were defected in human atherosclerotic aortic vascular tissue but not in normal aortic tissue. Since atherosclerosis is associated with an inflammatory vascular injury characterized by elevated tissue TNF-alpha concentrations and inflammatory cell infiltration, these data suggest that MAA-adducted proteins may be formed in atherosclerotic plaque material and may be involved in the inflammatory reaction that occurs in atherosclerosis. These data further suggest that previous studies demonstrating MDA modified protein in atherosclerotic plaque may in fact have MAA modified proteins associated with them.

The interaction of acetaldehyde with tubulin.

Acetaldehyde covalently binds to purified tubulin in vitro to form both stable and unstable adducts. The formation of stable adducts can be greatly facilitated by the inclusion of the relatively gentle and Schiff base specific reducing agent, sodium cyanoborohydride. Although the tubulin molecule has multiple lysine resides available to react with acetaldehyde, certain key lysine residues on the alpha-chain appear to be selective targets for adduct formation. The formation of alpha-chain specific stable acetaldehyde-tubulin adducts results in functional impairment of the ability of tubulin to polymerize. Under relatively physiologic conditions where acetaldehyde-to-protein ratios are low, alpha-chain specific binding is prominent. These results, coupled with the studies presented in another report in this volume, raise the possibility that low levels of adduct formation may be detrimental to the structure or function of certain proteins (e.g. tubulin) in the liver. The alteration of this or other biologically important proteins by sustained low levels of adduct formation may contribute to the pathogenesis of alcoholic liver injury.

Acetaldehyde and Microtubules

Acetaldehyde covalently binds to tubulin to form stable and unstable adducts. Although tubulin has numerous lysine residues available to react with acetaldehyde, a key highly reactive lysine (HRL) on the alpha chain appears to be a preferential target for stable binding. The HRL residue is available for selective binding when tubulin is in the free (dimer) state but not when it is in the polymerized (microtubule) state. Stable binding of acetaldehyde to the HRL residue markedly inhibits tubulin assembly into microtubules, whereas stable binding to other residues (bulk adducts) has little influence on assembly. Substoichiometric stable binding of acetaldehyde to the HRL is sufficient to inhibit polymerization, via direct interference of tubulin dimer-dimer interactions, and an HRL adduct on only one out of 20 tubulin molecules can totally inhibit polymerization. These findings, along with our previous studies demonstrating impaired microtubule-dependent protein trafficking pathways in livers of ethanol-fed animals, indicate that low acetaldehyde concentrations, formed during ethanol oxidation in vivo, could generate sufficient amounts of HRL adducts on the alpha chain of tubulin in cellular systems to alter microtubule formation and function. In addition to alpha-tubulin, calmodulin and actin have also been found to have enhanced reactivity toward acetaldehyde. Thus, a general hypothesis to describe cellular injury induced by acetaldehyde adducts can be formulated: during ethanol oxidation, acetaldehyde forms stable adducts via binding to reactive lysine residues of preferential target proteins, resulting in selective functional impairment of these proteins and ultimately leading to cellular injury.

Enhancement of acetaldehyde−protein adduct formation by L-ascorbate

The effect of L-ascorbate on the binding of [14C]acetaldehyde to bovine serum albumin was examined. In the absence of ascorbate, acetaldehyde reacted with albumin to form both unstable (Schiff bases) and stable adducts. Ascorbate (5 mM) caused a time-dependent increase in the formation of total acetaldehyde-albumin adducts, which were comprised mainly of stable adducts. Significant enhancement of adduct formation by ascorbate was observed at acetaldehyde concentrations as low as 5 microM. An ascorbate concentration as low as 0.5 mM was still effective in stimulating stable adduct formation. The electron acceptor, 2,6 dichlorophenolindophenol, prevented the ascorbate-induced increase in albumin-adduct formation. Ascorbate also caused enhanced acetaldehyde adduct formation with other purified proteins, including cytochrome c and histones, as well as the polyamino acid, poly-L-lysine. These results indicate that ascorbate, acting as a reducing agent, can convert unstable acetaldehyde adducts to stable adducts, and can thereby increase and stabilize the binding of acetaldehyde to proteins.

Inhibition of markers of hepatic stellate cell activation by the hormone relaxin.

Hepatic fibrosis results from excess extracellular matrix produced primarily by hepatic stellate cells (HSC). In response to injury, HSC differentiate to a myofibroblastic phenotype expressing smooth muscle actin and fibrillar collagens. Relaxin is a polypeptide hormone shown to have antifibrotic effects in fibrosis models. In this study, activated HSC from rat liver were treated with relaxin to determine if relaxin can reverse markers of HSC activation. Relaxin treatment resulted in a decrease in the expression of smooth muscle actin, but had no effect on cell proliferation rate. The levels of total collagen and type I collagen were reduced, while the synthesis of new collagen was inhibited. Furthermore, relaxin caused an increase in the expression and secretion of rodent interstitial collagenase (MMP-13), but there was no effect on the gelatinases MMP-2 or MMP-9. Relaxin also increased secretion of TIMP-1 and TIMP-2. The effective concentration of relaxin to induce these effects was consistent with action through the relaxin receptor. In conclusion, relaxin reversed markers of the activated phenotype of HSC including the production of fibrillar collagen. At the same time, the activity of a fibrillar collagenase was increased. These data suggest that relaxin not only inhibits HSC properties that contribute to the progression of hepatic fibrosis, but also promotes the clearance of fibrillar collagen. Therefore, relaxin may be a useful approach in the treatment of hepatic fibrosis.