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Chronic ethanol consumption is associated with serious and potentially fatal alcohol-related liver injuries such as hepatomegaly, alcoholic hepatitis and cirrhosis. Moreover, it has been documented that the clinical progression of alcohol-induced liver damage may be associated with an increase in hepatocellular death that involves apoptotic mechanisms. Although much information has been learned about the clinical manifestations associated with alcohol-related diseases, the search continues for a better understanding of the molecular and/or cellular mechanisms by which ethanol exerts its deleterious effects such as the induction of pro-apoptotic mechanisms and related cell damaging events. As part of the effort to enhance our understanding of those particular cellular pathways and mechanisms associated with ethanol toxicity, researchers over the years have utilized a variety of model systems. Recently, work has come forth demonstrating the utility of a hybrid cell line (WIF-B) as a cell culture model system for the study of alcohol-associated alterations in hepatocellular mechanisms. Success with such emerging model systems could aid in the development of potential therapeutic treatments for the prevention of alcohol-induced apoptotic cell death that may ultimately serve as a significant target in delaying the onset and/or progression of clinical symptoms of alcohol-mediated liver disease. This review article summarizes the current understanding of ethanol-mediated modifications in cell survival and thus the promotion of pro-apoptotic events with emphasis on analyses made in various experimental model systems, particularly the more recently characterized WIF-B cell system.

Background/aims: Protein-L-isoaspartyl methyltransferase (PIMT) is a methyltransferase that plays a crucial role in the repair of damaged proteins. In this study, we investigated whether ethanol exposure causes an accumulation of modified proteins bearing atypical isoaspartyl residues that may be related to impaired PIMT activity. We further sought to determine whether betaine administration could prevent the accumulation of these types of damaged proteins., Methods: Livers of male Wistar rats, fed the Lieber DeCarli control, ethanol or 1% betaine-supplemented diets for 4 weeks, were processed for PIMT-related analyses., Results: We observed a significant increase in the accumulation of modified proteins bearing isoaspartyl residues, i.e. the substrates for PIMT, in homogenate samples and various subcellular fractions of livers from ethanol-fed rats. Betaine supplementation prevented this accumulation of damaged proteins. In contrast, ethanol exposure induced no changes in the PIMT enzyme activity levels as compared to controls. The accumulation of damaged proteins negatively correlated with hepatic S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH) ratios., Conclusions: Ethanol consumption results in the accumulation of modified proteins bearing atypical isoaspartyl residues via impaired in vivo PIMT activity. Betaine administration prevents the ethanol-induced accumulation of isoaspartyl-containing proteins by restoring the PIMT-catalyzed protein repair reaction through normalizing the hepatocellular SAM:SAH ratios.

The polypeptide hormone relaxin has antifibrotic effects on a number of tissues, including the liver. Central to the progression of hepatic fibrosis is the transdifferentiation of hepatic stellate cells (HSC) from a quiescent state to an activated, myofibroblastic phenotype that secretes fibrillar collagen. Relaxin inhibits markers of HSC activation, but relaxin receptor expression in the liver is unclear. The purpose of this study was to determine the expression of the relaxin receptors LGR7 and LGR8 in activated HSC. Production of cAMP was induced by treatment of HSC with relaxin, or the relaxin-related peptides InsL3 or relaxin-3, selective activators of LGR8 and LGR7, respectively. Quiescent HSC expressed low levels of LGR7 but not LGR8. During progression to the activated phenotype, expression of both receptors increased markedly. Immunocytochemistry confirmed the presence of both receptors in activated HSC. In normal rat liver, LGR7, but not LGR8, was expressed at low levels. In cirrhotic liver, expression of both receptors significantly increased. Neither receptor was detectable in normal liver by immunohistochemistry, but both LGR7 and LGR8 were readily detectable in cirrhosis. These results were confirmed in human cirrhotic tissue, with the additional finding of occasional perisinusoidal LGR7 immunoreactivity in non-cirrhotic tissue. In conclusion, the expression of LGR7 and LGR8 is increased with activation of HSC in culture. Cirrhosis also caused increased expression of both receptors. Therefore, agents that stimulate LGR8 and LGR7 may be therapeutically useful to limit the activation of hepatic stellate cells in liver injury.

Background/aims: Previous studies in our laboratory implicated ethanol-induced decreases in hepatocellular S-adenosylmethionine to S-adenosylhomocysteine (SAM:SAH) ratios in lowering the activity of phosphatidylethanolamine methyltransferase (PEMT), which is associated with the generation of steatosis. Further in vitro studies showed that betaine supplementation could correct these alterations in the ratio as well as attenuate alcoholic steatosis. Therefore, we sought to determine whether the protective effect of betaine is via its effect on PEMT activity., Methods: Male Wistar rats were fed the Lieber DeCarli control or ethanol diet with or without 1% betaine supplementation for 4 weeks., Results: We observed that ethanol feeding resulted in decreased phosphatidylcholine (PC) production by a PEMT-catalyzed reaction. Betaine supplementation corrected the ethanol-induced decrease in hepatic SAM:SAH ratios and by normalizing PC production via the PEMT-mediated pathway, significantly reduced fatty infiltration associated with ethanol consumption. This restoration of hepatocellular SAM:SAH ratio by betaine supplementation was associated with increases in the activity, enzyme mass and gene expression of the enzyme, betaine homocysteine methyltransferase (BHMT), that remethylates homocysteine., Conclusions: Betaine, by virtue of promoting an alternate remethylation pathway, restores SAM:SAH ratios that, in turn, correct the defective cellular methylation reaction catalyzed by PEMT resulting in protection against the generation of alcoholic steatosis.

Background: Chronic ethanol consumption can lead to a variety of pathological consequences by as yet undefined mechanisms. Recently, it has been noted that alcohol-associated liver disease is often accompanied by morphological liver changes that include the increased production of apoptotic cells. Additionally, it has been demonstrated that hepatocellular uptake and removal of potentially damaging apoptotic cells is impaired after ethanol treatment. The aim of the present study was to determine whether the presence of apoptotic cells leads to Kupffer cell (KC) production and release of proinflammatory cytokines that have been linked to hepatocyte damage, such as interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha)., Methods: Kupffer cells were isolated from female rats after an 8-week oral administration of a dextrose control or ethanol-containing fish-oil diet. The isolated KCs were cultured for up to 24 hours in the absence or presence of apoptotic or nonapoptotic hepatoma cells, or lipopolysaccharide. After incubation, media from the cultures were assayed for the presence of TNF-alpha and IL-6 by immunoassay detection. Also, the expression of these cytokines was measured in KC lysates by a quantitative real-time polymerase chain reaction., Results: Kupffer cells cultured for up to 24 hours in the presence of apoptotic cells produced significantly more TNF-alpha and IL-6 (80 and 60%, respectively, p<0.05) when the cells were isolated from ethanol-fed animals compared with controls. Additionally, after as early as 4 hours in culture with apoptotic cells, mRNA levels of both cytokines were increased (2-5-fold) in KCs isolated from ethanol-fed animals compared with controls., Conclusions: The presence of apoptotic cells results in the in vitro activation of KCs. Additionally, chronic ethanol administration results in an enhanced responsiveness of KCs to produce proinflammatory cytokines indicated by the increased production of inflammatory mediators from KCs obtained from ethanol-fed animals.

Unlabelled: Processing of peptides for antigen presentation is catalyzed by antigen-trimming enzymes, including the proteasome and leucine aminopeptidase. Oxidative stress suppresses proteasome function. We hypothesized that in liver cells, processing of antigenic peptides is altered by ethanol metabolism. To address this issue, soluble extracts of ethanol-metabolizing VL-17A cells treated with 100 mM ethanol or left untreated were incubated with C-extended or N-extended 18-27 HBV core peptides. Peptide cleavage was measured by recovery after HPLC. Ethanol exposure to VL-17A cells increased CYP2E1 and decreased proteasome peptidase activities. The latter effect was prevented by treatment of cells with inhibitors, 4-methylpyrazole and diallyl sulfide. Ethanol treatment of VL-17A cells also reduced the activity of leucine aminopeptidase (LAP). Consequently, cleavage of both C-extended and N-extended peptides by cytosolic extracts was suppressed by pretreatment of cells with ethanol. Treatment of cells with interferon gamma, which enhances proteasome activity, did not reverse the effects of ethanol. Ethanol exerted similar effects on WIFB cells, indicating that its effects are not unique to one cell type., Conclusion: Ethanol metabolism suppresses activities of antigen-trimming enzymes, thereby decreasing the cleavage of C-extended and N-extended peptides. This defect may potentially result in decreased MHC class I-restricted antigen presentation on virally infected liver cells.

Background: It has been noted that alcohol-related liver diseases can be associated with an increase in apoptotic hepatocellular death. Moreover, the promotion of hepatocyte apoptosis may be linked to signals emanating from death receptors, particularly Fas [CD95/apoptosis-inducing protein 1 (APO-1)]. In the present study, we utilized an in vitro hepatic culture model [hybrid of human fibroblast (WI 38) and rat hepatoma (Fao) cells, WIF-B cells] to study potential contributing mechanisms involved in hepatocellular apoptosis following ethanol administration., Methods: WIF-B cultures (differentiated hepatic cells that efficiently metabolize alcohol) were treated with or without ethanol and specific inhibitors of alcohol metabolism and cysteine protease activity, followed by morphological and biochemical examination of proapoptotic parameters., Results: The results of this work demonstrated that ethanol administration leads to an increase (45%-60%) in caspase-3 activity and that the induction of apoptosis was found to be linked to the metabolism of alcohol. Additionally, increases were observed in the activity of upstream initiator caspases (caspase-2 and caspase-8) that are directly related to membrane signaling events of death receptors such as Fas. Moreover, it was determined that the activation of caspase-3 could be blocked by the presence of a specific caspase-8 inhibitor, again linking death receptor-associated proteases to downstream effector caspase activity in alcohol-related death. Finally, ethanol administration was found to result in an increase in the amount of Fas protein present in the membrane fraction of the cell. The increase in membrane Fas protein indicates ligand-independent membrane targeting of Fas in the alcohol-treated cells that could potentially be a key signaling event in the induction of the proapoptotic caspase cascade., Conclusions: The data presented here indicate that alcohol metabolism induces apoptosis in WIF-B cells that occurs, in part, by mechanisms involving signals emanating from death receptors.

Background: The effects of chronic ethanol feeding on rat hepatocytes have been shown to include impaired cell-extracellular matrix (ECM) adhesion events, such as decreased attachment and spreading as well as increased integrin-actin cytoskeleton association. These results, observed previously by this laboratory, are highly suggestive of impaired actin cytoskeleton reorganization, an event mediated by differential activation of the Rho family GTPases Rac, Cdc42, and RhoA. Therefore, the purpose of this study was to examine the effects of chronic ethanol administration on these GTPases., Methods: Male Wistar rats were pair-fed 4 to 5 weeks with a liquid diet containing either ethanol (as 36% of total calories) or isocaloric carbohydrate. Hepatocytes were isolated and plated on collagen IV up to 24 hours. At specific times, the hepatocytes were lysed and these lysates were analyzed for RhoA, Cdc42, and Rac activation., Results: In freshly isolated hepatocytes from ethanol-fed rats, the GTP-bound (active) forms of Rac and Cdc42 were significantly decreased compared with pair-fed control rats, while the GTP-bound form of RhoA was not significantly altered. These ethanol-induced impairments in Rac and Cdc42 activation persisted even after plating the hepatocytes on collagen IV. Additionally, chronic ethanol treatment did not directly affect GTP binding of Cdc42 and Rac, as incorporation of GTPgammaS was not affected., Conclusions: Chronic ethanol administration selectively impairs Rac and Cdc42 activation in rat hepatocytes. As activation of these 2 GTPases is crucial for efficient cell attachment and spreading on ECM substrates, the results from this study suggest that the ethanol-induced impairments in Rac and Cdc42 activation are responsible for the impaired hepatocyte-ECM adhesion events observed previously by our laboratory. Furthermore, these results raise the intriguing possibility that these GTPases are involved in other ethanol-induced functional impairments, such as protein trafficking and receptor-mediated endocytosis.

Background/aims: Chronic alcohol consumption can lead to serious liver disease. Although the disease progression is clinically well-described, the molecular basis for alcohol-induced hepatotoxicity is not understood., Methods: We examined hepatocyte-specific, alcohol-induced alterations in microtubule dynamics in WIF-B cells. These cells provide an excellent model for studying alcohol-induced hepatotoxicity; they remain differentiated in culture and metabolize alcohol., Results: Consistent with reports in other hepatic systems, microtubule polymerization in ethanol-treated WIF-B cells was impaired. However, when viewed by epifluorescence, the microtubules in ethanol-treated cells resembled stable polymers. Antibodies to acetylated alpha-tubulin confirmed their identity morphologically and revealed biochemically that ethanol-treated cells had approximately three-fold more acetylated alpha-tubulin than control cells. Livers from ethanol-fed rats also contained increased levels of acetylated alpha-tubulin. Consistent with increased acetylated alpha-tubulin levels, microtubules in ethanol-treated WIF-B cells were more stable. Because stability increased with increased time of ethanol exposure or concentration, was prevented by 4-methylpyrazole and was potentiated by cyanamide, we conclude that increased acetylation requires alcohol metabolism and is likely to be mediated by acetaldehyde., Conclusions: Ethanol metabolism impairs tubulin polymerization, but once microtubules are formed they are hyperstabilized. These ethanol-induced alterations in microtubule integrity likely have profound effects on hepatocyte function.

Previous studies from our laboratory have shown that ethanol consumption results in an increase in hepatocellular S-adenosylhomocysteine levels. Because S-adenosylhomocysteine is a potent inhibitor of methylation reactions, we propose that increased intracellular S-adenosylhomocysteine levels could be a major contributor to ethanol-induced pathologies. To test this hypothesis, hepatocytes isolated from rat livers were grown on collagen-coated plates in Williams' medium E containing 5% FCS and exposed to varying concentrations of adenosine in order to increase intracellular S-adenosylhomocysteine levels. We observed increases in caspase-3 activity following exposure to adenosine. This increase in caspase activity correlated with increases in intracellular S-adenosylhomocysteine levels and DNA hypoploidy. The adenosine-induced changes could be significantly attenuated by betaine administration. The mechanism of betaine action appeared to be via the methylation reaction catalyzed by betaine-homocysteine-methyltransferase. To conclude, our results indicate that the elevation of S-adenosylhomocysteine levels in the liver by ethanol is a major factor in altering methylation reactions and in increasing apoptosis in the liver. We conclude that ethanol-induced alteration in methionine metabolic pathways may play a crucial role in the pathologies associated with alcoholic liver injury and that betaine administration may have beneficial therapeutic effects.

Previous studies with alcohol-associated malondialdehyde-acetaldehyde (MAA)-modified proteins have demonstrated an increase in the expression of adhesion molecules, and the secretion of pro-inflammatory cytokines/chemokines by rat sinusoidal liver endothelial cells (SECs). However, no studies have been initiated to examine the effects of MAA-modified proteins on the expression of the extracellular matrix (ECM) protein, fibronectin and its isoforms. For these studies, SECs were isolated from the liver of normal rats, and exposed to MAA-modified bovine serum albumin (MAA-Alb). At selected time points, the total plasma and cellular fibronectin were determined by Western blot. Injection of rat liver via the mesenteric vein with MAA-Alb was performed in an effort to evaluate the potential in vivo role of MAA-modified proteins in the development of fibrosis. Expression of both plasma and cellular fibronectin was significantly increased over controls in the MAA-Alb stimulated SECs (>3-fold). Importantly, the isotype of fibronectin secreted was determined to be of the EIIIA variant and not EIIIB. These data were confirmed using RT-PCR procedures on liver tissue from; isolated SECs, and from an in vivo animal model wherein MAA-Alb was administered via the mesenteric vein. Thus, these studies demonstrate that MAA-modified proteins initiate a pro-fibrogenic response by initiating the expression of the fibronectin EIIIA isoform by SECs.

Scavenger receptors on sinusoidal liver endothelial cells (SECs) eliminate potentially harmful modified proteins circulating through the liver. It was shown recently that aldehyde-modified proteins bind to scavenger receptors and are associated with the development/progression of alcoholic liver diseases. For these studies, rat livers were perfused in situ with 125I-formaldehyde-bovine serum albumin (f-Alb) or 125I-malondialdehyde-acetaldehyde-bovine serum albumin (MAA-Alb) in the presence of known scavenger receptor ligands as inhibitors. Reverse transcription-polymerase chain reaction (RT-PCR) analysis and scavenger receptor Type A (SRA) knock-out mice were used to assess the role of these receptors in mediating immune responses. The degradation of 125I-f-Alb or 125I-MAA-Alb in whole livers and isolated SECs can be inhibited by known scavenger receptor ligands, including f-Alb, maleylated bovine albumin, and fucoidan. 125I-f-Alb could not be completely inhibited by MAA-Alb. In contrast, 125I-MAA-Alb was only partially inhibited with advanced glycosylated endproduct albumin. RT-PCR data show the presence of a number of scavenger receptors on SECs that may be responsible for the binding of MAA-modified proteins. SRA seems to be one of these receptors involved in the effects mediated by MAA-modified proteins. In a study using SRA knockout mice, it was shown that a decreased antibody response to MAA-Alb resulted. By RT-PCR, CD36, LOX-1, and SR-AI are the scavenger receptors most likely involved in the degradation of MAA-Alb.

Most people who abuse alcohol are cigarette smokers. Previously, we have shown that malondialdehyde, an inflammation product of lipid peroxidation, and acetaldehyde, a component of both ethanol metabolism and cigarette smoke, form protein adducts that stimulate protein kinase C (PKC) activation in bronchial epithelial cells. We have also shown that PKC can regulate bronchial epithelial cell wound repair. We hypothesize that bovine serum albumin adducted with malondialdehyde and acetaldehyde (BSA-MAA) decreases bronchial epithelial cell wound repair via binding to scavenger receptors on bronchial epithelial cells. To test this, confluent monolayers of bovine bronchial epithelial cells were grown in serum-free media prior to wounding the cells. Bronchial epithelial cell wound closure was inhibited in a dose-dependent manner (up to 60%) in the presence of BSA-MAA than in media treated cells (Laboratory of Human Carcinogenesis [LHC]-9-Roswell Park Memorial Institute [RPMI]). The specific scavenger receptor ligand, fucoidan, also stimulated PKC activation and decreased wound repair. Pretreatment with fucoidan blocked malondialdehyde-acetaldehyde binding to bronchial epithelial cells. When bronchial epithelial cells were preincubated with a PKC alpha inhibitor, Gö 6976, the inhibition of wound closure by fucoidan and BSA-MAA was blocked. Western blot demonstrated the presence of several scavenger receptors on bronchial epithelial cell membranes, including SRA, SRBI, SRBII, and CD36. Scavenger receptor-mediated activation of PKC alpha may function to reduce wound healing under conditions of alcohol and cigarette smoke exposure where malondialdehyde-acetaldehyde adducts may be present.

Relaxin has antifibrotic effects on the hepatic stellate cells (HSCs) responsible for collagen deposition in cirrhosis. The expression of relaxin receptors LGR7 and LGR8 in HSCs and liver disease was examined. Activated and quiescent HSCs expressed LGR7, whereas only activated HSCs expressed LGR8. Relaxin, relaxin-3, or InsL3 treatment increased cAMP, suggesting activation of both receptors. LGR8 and LGR7 were present in cirrhotic rat liver, but were undetectable in normal liver. In conclusion, both LGR7 and LGR8 are expressed in activated HSCs and cirrhotic liver, suggesting that relaxin, InsL3, or relaxin-3 may be useful in the treatment of hepatic fibrosis.

Cigarette smoke is a risk factor for the development of several diseases, but the exact mechanism responsible has not been well-characterized. Because modification, or adducting, of biomolecules is thought to mediate the toxic effects observed from exposure to a wide variety of harmful chemicals, this study investigated the ability of cigarette smoke to produce specific adducts on a peptide to gain insight into the likely effect on cellular proteins. We describe the modification of the epsilon-amino group of lysine contained in a test peptide with stable fluorescent adducts derived from monofunctional aldehydes occurring in cigarette smoke and malonaldehyde, a product of lipid peroxidation. Utilizing high-performance liquid chromatography, fluorescent measurements, and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy, the 1,4-dihydropyridine-3,5-dicarbaldehyde and 4-methyl-1,4-dihydropyridine-3,5-dicarbaldehyde derivatives of lysine were identified as products of exposure to cigarette smoke extract and malonaldehyde. These data suggest that cigarette smoke may promote the modification of proteins, like those associated with oxidized low-density lipoprotein, and may contribute to smoking-related disease.

BACKGROUND: The possibility exists for major complications to occur when individuals are intoxicated with alcohol prior to anesthetization. Halothane is an anesthetic that can be metabolized by the liver into a highly reactive product, trifluoroacetyl chloride, which reacts with endogenous proteins to form a trifluoroacetyl-adduct (TFA-adduct). The MAA-adduct which is formed by acetaldehyde (AA) and malondialdehyde reacting with endogenous proteins, has been found in both patients and animals chronically consuming alcohol. These TFA and MAA-adducts have been shown to cause the release of inflammatory products by various cell types. If both adducts share a similar mechanism of cell activation, receiving halothane anesthesia while intoxicated with alcohol could exacerbate the inflammatory response and lead to cardiovascular injury. METHODS: We have recently demonstrated that the MAA-adduct induces tumor necrosis factor-alpha (TNF-alpha) release by heart endothelial cells (HECs). In this study, pair and alcohol-fed rats were randomized to receive halothane pretreatments intra peritoneal. Following the pretreatments, the intact heart was removed, HECs were isolated and stimulated with unmodified bovine serum albumin (Alb), MAA-modified Alb (MAA-Alb), Hexyl-MAA, or lipopolysaccharide (LPS), and supernatant concentrations of TNF-alpha were measured by ELISA. RESULTS: Halothane pre-treated rat HECs released significantly greater TNF-alpha concentration following MAA-adduct and LPS stimulation than the non-halothane pre-treated in both pair and alcohol-fed rats, but was significantly greater in the alcohol-fed rats. CONCLUSION: These results demonstrate that halothane and MAA-adduct pre-treatment increases the inflammatory response (TNF-alpha release). Also, these results suggest that halothane exposure may increase the risk of alcohol-induced heart injury, since halothane pre-treatment potentiates the HEC TNF-alpha release measured following both MAA-Alb and LPS stimulation.

Previous studies showed that chronic ethanol administration alters methionine metabolism in the liver, resulting in increased intracellular S-adenosylhomocysteine (SAH) levels and increased homocysteine release into the plasma. We showed further that these changes appear to be reversed by betaine administration. This study compared the effects of betaine and S-adenosylmethionine (SAM), another methylating agent, on ethanol-induced changes of methionine metabolism and hepatic steatosis. Wistar rats were fed ethanol or control Lieber-Decarli liquid diet for 4 wk and metabolites of the methionine cycle were measured in isolated hepatocytes. Hepatocytes from ethanol-fed rats had a 50% lower intracellular SAM:SAH ratio and almost 2-fold greater homocysteine release into the media compared with controls. Supplementation of betaine or SAM in the incubation media increased this ratio in hepatocytes from both control and ethanol-fed rats and attenuated the ethanol-induced increased hepatocellular triglyceride levels by approximately 20%. On the other hand, only betaine prevented the increase in generation of homocysteine in the incubation media under basal and methionine-loaded conditions. SAM can correct only the ratio and the methylation defects and may in fact be detrimental after prolonged use because of its propensity to increase homocysteine release. Both SAM and betaine are effective in increasing the SAM:SAH ratio in hepatocytes and in attenuating hepatic steatosis; however, only betaine can effectively methylate homocysteine and prevent increased homocysteine release by the liver.

Background: The nonparenchymal cells of the liver have been suggested to play a significant role in the inflammatory processes observed in the development and/or progression of alcoholic liver disease. Our laboratories have shown that malondialdehyde-acetaldehyde (MAA)-modified proteins can induce immune responses, cytokine/chemokine secretion, and antigen processing and presentation by liver sinusoidal endothelial cells (SECs). Another molecule that has been shown to induce similar types of responses in Kupffer cells (KCs) is lipopolysaccharide (LPS). Because these materials induce similar responses, it was the purpose of this study to investigate the relationship between LPS and MAA-modified proteins in the development of proinflammatory responses by SECs and KCs., Methods: For these studies, SECs and KCs were isolated from chow-fed, pair-fed, and ethanol-fed rats. Cells were stimulated with media alone, bovine serum albumin (Alb), or MAA-modified Alb (MAA-Alb) in the presence or absence of LPS 1 ng/ml, and the supernatants were assayed by enzyme-linked immunosorbent assay for tumor necrosis factor alpha, macrophage chemotactic protein 1, and macrophage inhibitory protein., Results: All three cytokines/chemokines were 3 to 5 times higher when SECs or KCs were stimulated by MAA-Alb in the presence of LPS, in contrast to cells stimulated with Alb or media in the presence of LPS. Chronic ethanol consumption (6 weeks) had variable effects on the secretion of these cytokines/chemokines but in general did not alter the increased secretion in response to MAA-Alb in the presence of LPS., Conclusions: These studies strongly suggest that the sensitization of SECs and KCs by LPS plays a significant role in the development and/or progression of alcoholic liver disease, and the subsequent activation by MAA-modified proteins may be a mechanism by which proinflammatory processes are initiated.

While most of the investigations into the causative events in the development of alcoholic liver disease (ALD) have been focused on multiple factors, increasing interest has centered around the possible role of immune mechanisms in the pathogenesis and perpetuation of ALD. This is because many of the clinical features of ALD suggest that immune effector mechanisms may be contributing to liver tissue damage, as evidenced by the detection of circulating autoantibodies, and the presence of CD4+ and CD8+ lymphoid cells in the livers of patients with ALD. One mechanism that has been associated with the development of autoimmune responses is the modification (haptenation or adduction) of liver proteins with aldehydes or other products of oxidative stress. This is because it has been shown that these adducted proteins can induce specific immune responses, to the adduct, the adduct plus protein (conformational antigens), as well as the unmodified parts of the protein. More importantly, it is possible to demonstrate that adducted self-proteins can induce reactivity to the normal self-protein and thereby induce autoimmune responses. Therefore, it is the purpose of this manuscript to outline the mechanism(s) by which these modified self proteins can induce autoimmune reactivity, and thus play a role in the development and/or progression of ALD.

Background/aims: Transforming growth factor-beta (TGF-beta) is a cytokine produced in abundance during liver injury. Recognizing the prominent roles that hepatic stellate cells (HSCs) and TGF-beta play in portal hypertension and fibrogenesis, respectively, we sought to evaluate the effect of TGF-beta on the contractility of activated HSCs., Methods: Spontaneous immortalized cell lines of HSC origin were used in this study. Cells were grown in three-dimensional collagen gel lattice, transferred to 60 mm dishes and exposed to varying concentrations of TGF-beta1 in serum-free medium at 37 degrees C for up to 120 h. The area of the floating gels was measured using a Fluor S-MultiImager (Biorad), the cellular smooth muscle-alpha actin (SMA) content quantified and PKC activation studies conducted., Results: TGF-beta1 induced a time- and dose-dependent decrease in lattice area up to 40% of control (P<0.05) that reflects the contraction of activated HSCs. This induced contraction was associated with increases in SMA content (3-fold, P<0.05) and PKC activation (5-fold, P<0.05) in these cells. Furthermore, pre-incubating with a PKC--specific inhibitor completely abrogated the TGF-beta-induced contraction., Conclusions: TGF-beta induces contraction of activated HSCs via an increase in SMA content and a PKC--mediated pathway.

A potential in vitro model for studying the mechanisms of alcohol-induced hepatocyte injury is the WIF-B cell line. It has many hepatocyte-like features, including a differentiated, polarized phenotype resulting in formation of bile canaliculi. The aim of this study was to examine the effects of ethanol treatment on this cell line. WIF-B cells were cultured up to 96 h in the absence or presence of 25 mM ethanol and subsequently were analyzed for ethanol-induced physiological and morphological changes. Initial studies revealed WIF-B cells exhibited alcohol dehydrogenase (ADH) activity, expressed cytochrome p4502E1 (CYP2E1), and efficiently metabolized ethanol in culture. This cell line also produced the ethanol metabolite acetaldehyde and exhibited low K(m) aldehyde dehydrogenase (ALDH) activity, comparable to hepatocytes. Ethanol treatment of the WIF-B cells for 48 h led to significant increases in the lactate/pyruvate redox ratio and cellular triglyceride levels. Ethanol treatment also significantly altered WIF-B morphology, decreasing the number of bile canaliculi, increasing the number of cells exhibiting finger-like projections, and increasing cell diameter. The ethanol-induced changes occurring in this cell line were negated by addition of the ADH inhibitor, 4-methylpyrazole (4-MP), indicating the effects were due to ethanol metabolism. In summary, the WIF-B cell line metabolizes ethanol and exhibits many ethanol-induced changes similar to those found in hepatocytes. Because of these similarities, WIF-B cells appear to be a suitable model for studying ethanol-induced hepatocyte injury.

Malondialdehyde-acetaldehyde (MAA) haptenated proteins have been described in disease processes related to prolonged oxidative stress (via malondialdehyde production), such as alcohol liver disease (ALD), non-alcoholic non-steatohepatitis (NASH) and atherosclerosis. Experimentally, high titer IgG1 antibody responses are seen after immunization without adjuvant; however, T cell proliferative responses and the role of scavenger receptors in this immunogenicity has not previously been described. In this study, T cell proliferative responses to the carrier protein, but not the MAA hapten itself, were identified in vitro. Moreover, these T proliferative responses were inhibited when MAA-hen egg lysozyme (HEL) was co-immunized with excess scavenger receptor ligand polyG (poly-guanylic acid), implicating the role of (a) scavenger receptor(s) in initiating the T helper cell response. Activated B cells were unable to process and present MAA-HEL preferentially to T cells, while thioglycollate-elicited (but not Con A-elicited) macrophages and dendritic cells (DC) did so with approximately 32-fold less MAA-HEL than native antigen necessary to initiate equal proliferative responses. While this preferential processing and presentation may be related to several factors, preferential binding of MAA haptenated proteins mediated by scavenger receptors may be one mechanism. IL-4 was absent from the supernatants of T proliferative assays despite a strong IgG1 response in vivo, although the TH2 cytokines IL-6 and IL-10 were expressed. Since the modification of proteins by the MAA have previously been shown to occur after ethanol consumption in vivo, the ability of MAA haptens to experimentally enhance immune responses, specifically humoral and T cell responses, may represent mechanisms by which autoimmune phenomena found in ALD occur.

Alcoholic liver disease has been associated with abnormalities in receptor-mediated endocytosis (RME) which results in abnormal degradation of metabolically altered proteins. Model systems using formaldehyde-modified albumin (f-Alb) have shown an impairment in RME following chronic alcohol consumption utilizing both in situ perfused rat livers and isolated rat liver endothelial cells (LECs). The discovery that alcohol metabolite derived aldehydes can modify proteins prompted a study to determine if malondialdehyde-acetaldehyde-modified albumin (MAA-Alb) would be degraded similar to that reported for f-Alb, and whether ethanol-fed rats would demonstrate an impaired RME with respect to this ligand which occurs as a consequence of chronic ethanol consumption. MAA-Alb was degraded slightly more than f-Alb in both in situ perfused livers and at the single cell level. This degradation was completely inhibited with 100x unlabeled f-Alb, which suggests the use of a similar receptor. Following alcohol consumption there was a 50-60% decrease in MAA-Alb degradation in whole livers and isolated LECs. Utilizing isolated LECs it was determined that impairment in internalization was the most likely mechanism for the decrease in the amount of MAA-Alb that was degraded. These data show that chronic alcohol consumption by rats does in fact impair RME of alcohol metabolite-derived adducted proteins, and this impairment is due to a defect in the post-internalization step rather than the binding or degradation of the modified protein.

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.

Previous studies showed that chronic ethanol administration inhibits methionine synthase activity, resulting in impaired homocysteine remethylation to form methionine. This defect in homocysteine remethylation was shown to increase plasma homocysteine and to interfere with the production of hepatic S-adenosylmethionine (SAM) in ethanol-fed rats. These changes were shown to be reversed by the administration of betaine, an alternative methylating agent. This study was undertaken to determine additional effects of ethanol on methionine metabolism and their functional consequences. The influences of methionine loading and betaine supplementation were also evaluated. Adult Wistar rats were fed ethanol or a control Lieber-DeCarli liquid diet for 4 wk, and metabolites of the methionine cycle were measured in vitro in isolated hepatocytes under basal and methionine-supplemented conditions. S-Adenosylhomocysteine (SAH) concentrations were elevated in hepatocytes isolated from ethanol-fed rats compared with controls and in hepatocytes from both groups when supplemented with methionine. The addition of betaine to the methionine-supplemented incubation media reduced the elevated SAH levels. The decrease in the intracellular SAH:SAM ratio due to ethanol consumption inhibited the activity of the liver-specific SAM-dependent methyltransferase, phosphatidylethanolamine methyltransferase. Our data indicate that betaine, by remethylating homocysteine and removing SAH, overcomes the detrimental effects of ethanol consumption on methionine metabolism and may be effective in correcting methylation defects and treating liver diseases.

Previous studies using the Hep G2-based VA cells showed that ethanol metabolism resulted in both cytotoxicity and impaired DNA synthesis, causing reduced accumulation of cells in culture. To further characterize the ethanol oxidation-mediated impairment of DNA synthesis we analyzed the cell-cycle progression of VA cells. These studies showed approximately a 6-fold increase in the percentage of cells in the G2/M phase of the cell cycle after 4 days of ethanol exposure. The G2/M transition requires activity of the cyclin-dependent kinase, Cdc2. Cdc2 is positively regulated by association with cyclin B1, and negatively regulated by phosphorylation of amino acids Thr14 and Tyr15. Immunoblot analysis revealed that ethanol metabolism had little affect on total Cdc2 content in these cells, but resulted in the accumulation of up to 20 times the amount of cyclin B1, indicating that cyclin B1 was available for formation of Cdc2/cyclin B1 complexes. Co-immunoprecipitation revealed that 6 times more Cdc2/cyclin B1 complexes were present in the ethanol-treated cells compared with the controls. Investigation of the phosphorylation state of Cdc2 revealed that ethanol oxidation increased the amount of the phosphorylated inactive form of Cdc2 by approximately 3-fold. Thus, the impairment in cell-cycle progression could not be explained by a lack of cyclin B1, or the ability of Cdc2 and cyclin B1 to associate, but instead resulted, at least in part, from impaired Cdc2 activity. In conclusion, ethanol oxidation by VA cells results in a G2/M cell-cycle arrest, mediated by accumulation of the phosphorylated inactive form of Cdc2.

Apoptotic cell death is a well-defined process that is controlled by intrinsic cellular mechanisms followed by the generation of apoptotic bodies and their subsequent rapid elimination through the action of phagocytic cells. Within the liver, the asialoglycoprotein receptor (ASGP-R) has been shown to be involved in the phagocytosis of apoptotic hepatocytes, as well as altered cellular endocytic events after ethanol administration. The goal of the present study was to further clarify the capacity of ASGP-R to phagocytose apoptotic cells in relationship to the damaging events that occur with alcohol consumption. For these experiments, we used an in vitro suspension assay coupled with flow cytometry to measure apoptotic cell engulfment by rat hepatocytes after chronic ethanol administration. The results of this assay indicated that the phagocytosis of apoptotic cells was decreased significantly (30% to 42%, P <.05) in the presence of antibody specific for ASGP-R as well as the introduction of competing sugars in the media. In addition, uptake of apoptotic cells was impaired by 40% to 60% (P <.05) in cells obtained from ethanol-fed animals as compared with controls. In conclusion, the ASGP-R is involved in the recognition and uptake of apoptotic cells and this process is altered significantly by ethanol treatment. These findings may play a role in a better understanding of the clinical manifestations of alcohol-induced liver injury as altered uptake of apoptotic cells via ASGP-R may result in the release of proinflammatory mediators, the introduction of autoimmune responses, and inflammatory injury to the tissue.

We have created a number of recombinant Hep G2 cell lines, designated VA cells, that constitutively express alcohol dehydrogenase. Oxidation of ethanol by the VA cells results in the production and accumulation of acetaldehyde, and a dramatic increase in the nicotinamide adenine dinucleotide, reduced (NADH)/nicotinamide adenine dinucleotide (NAD(+)) ratio (redox-state). It is believed that production of acetaldehyde, and the increase in the redox-state of hepatocytes, are responsible for many of the dysfunctions associated with alcoholic liver disease. When the VA cells were cultured in the presence of ethanol, we observed a dramatic reduction in cell accumulation. This reduction was more pronounced in cells that metabolized ethanol more efficiently. Inhibition of alcohol dehydrogenase activity abolished this reduction, demonstrating that ethanol oxidation was required for this dysfunction. Subsequent investigations indicated that this ethanol oxidation-mediated reduction in cell accumulation was the result of both cytotoxicity and impaired DNA synthesis. To dissociate the increase in the cellular redox-state from acetaldehyde production, VA cells were cultured in the presence of isopropanol. The oxidation of isopropanol results in similar redox changes, but the metabolic by-product of isopropanol oxidation is acetone. The metabolism of isopropanol by VA cells resulted in very little reduction in cell number. Furthermore, treatment of ethanol-metabolizing VA cells with the aldehyde dehydrogenase inhibitor, cyanamide, increased the levels of acetaldehyde and resulted in an additional reduction in cell number. In conclusion, these studies indicated that exposure to acetaldehyde caused cytotoxicity, as well as the ethanol oxidation-mediated reduction in cell number.

System A, the Na(+)-dependent amino acid transport activity, is encoded by the ATA2 gene and up-regulated following partial hepatectomy (PH), and its competitive inhibition interferes with liver regeneration. Rabbit polyclonal antibody was raised against a portion of the ATA2 gene product followed by immunodetection of ATA2 in isolated liver plasma membrane and lysate. The level of ATA2 increased in the plasma membrane following PH, while the relatively high quantity of ATA2 found in liver lysate remained constant. We also have shown that Northern analysis of steady-state ATA2 mRNA revealed no significant change following PH. These data show that ATA2-mediated transport is not regulated by the steady-state level of ATA2 mRNA but is regulated by the amount of ATA2 and redistribution to the plasma membrane. We hypothesize that ATA2 activity is regulated by recruitment of ATA2 protein from an intracellular compartment. In addition, the pattern of expression of System A activity in oocytes, transport kinetics, and sensitivity to chemical modification indicate the presence of a second System A isoform in liver that differs substantially from ATA2.

Recent studies have demonstrated that circulating antibodies against malondialdehyde-acetaldehyde (MAA)-haptenated proteins are significantly increased in patients with alcohol-induced cirrhosis and hepatitis and correlate with the severity of liver damage. Additionally, when proteins are haptenated with MAA, they become highly immunogenic in vivo in the absence of adjuvants. However, the mechanism(s) of this immunogenicity are currently unknown. Initial in vitro studies on the effects of MAA-modified proteins on cells demonstrated an increase in cell death at concentrations that were cell type specific and time-dependent. Since immunogenicity due to cell death has been described, we investigated the mechanism(s) by which cell death was occurring. Assessment of cell death in splenocytes after 1 h found significant levels of apoptosis as compared to controls. After 5 h, a significant and dose-dependent necrosis occurred in which cells were exposed to >62.5 microg/ml (43.1 mM) MAA-haptenated protein. After 24 h, exposure to >31.3 microg/ml (21.6 mM) MAA-haptenated protein resulted in significant levels of necrosis, although DNA laddering studies found apoptosis was occurring as well. Morphological changes in the cells were observed by light microscopy that correlated with a "low" forward scatter population by flow cytometry. Since necrosis has been implicated in enhancing both primary and secondary immune responses, and necrosis was predominantly occurring in response to MAA-haptenated proteins, a possible mechanism by which the immunogenicity of MAA modification of proteins in vivo may occur is suggested. Specifically, MAA modification of self proteins may result in the death of various cell types, most likely those in the liver. These necrotic materials may induce anti-MAA antibodies and other auto antibodies, whose levels may then correlate with the severity of ALD.

The increased deposition of extracellular matrix by hepatic stellate cells following liver injury, in a process known as activation, is considered a key mechanism for increased collagen content of liver during the development of liver fibrosis. We report that N-(methylamino)isobutyric acid (MeAIB), a specific inhibitor of System A-mediated amino acid uptake, reduces the accumulation of collagen in CFSC-2G hepatic stellate cell cultures and in a rat model of liver injury and fibrosis. Rat CFSC-2G cells were cultured in 0-5mM MeAIB, and the accumulation and synthesis of collagen were measured by binding to Sirius red F3B and pulse-labeling with [3H]-proline, respectively. The effect of MeAIB on collagen accumulation in vivo was evaluated utilizing a rat model of hepatic fibrosis. MeAIB inhibited collagen accumulation in CFSC-2G cultures in a concentration-dependent manner with 5mM MeAIB reducing collagen 44.6+/-1.2% compared with the control. In CFSC-2G cultures, MeAIB selectively inhibited the incorporation of proline into cellular macromolecules by 43+/-4%, while the synthesis of proteins containing leucine was not affected. In vivo, oral administration of 160mg MeAIB/kg body weight per day to rats significantly reduced the hepatic collagen accumulation in response to 1 week of CCl(4)-induced liver injury. MeAIB reduces the accumulation of collagen in CFSC-2G hepatic stellate cell cultures and in a CCl(4)-induced rat model of liver injury and fibrosis.

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.

Previous studies from our laboratory have shown that malondialdehyde-acetaldehyde-protein adducts (MAA adducts) are formed in hepatocytes of ethanol-fed rats and directly influence the hepatic stellate cells (HSCs) to induce their secretion of chemokines and to up-regulate their expression of adhesion molecules. Since protein kinase C (PKC) is known to play a major role in many diverse intracellular signal transduction processes, we investigated whether MAA adducts influence the function of HSCs via a PKC-dependent pathway. HSCs in culture were exposed to MAA adducts, and PKC activity was determined. We observed a time- and concentration-dependent activation of PKC when cultures were exposed to BSA-MAA as compared with unmodified BSA. Using PKC isoform-specific inhibitors, we also showed that BSA-MAA induces the activation of a specific isoform of PKC, PKC-alpha, in HSCs. No activation of PKC was observed when HSCs were exposed to other aldehyde adducts such as BSA-acetaldehyde or BSA-malondialdehyde, indicating that the effects of MAA adducts on HSCs were somewhat specific. We further examined whether the observed increase in PKC activation induced by MAA adducts in HSCs, in turn, causes a functional effect. We observed that BSA-MAA induces the increased secretion of urokinase-type plasminogen activator, a key component of the plasmin-generating system, and that PKC activation is necessary for this enhanced urokinase-type plasminogen activator secretion. These results indicate that MAA adducts via a PKC-mediated pathway may regulate plasmin-mediated matrix degradation in the liver, thereby contributing to the progression of hepatic fibrosis.

Among the most important pathways for liver integrity in the body are the two that synthesize methionine and S-adenosylmethionine (SAM) through methylation of homocysteine. Results of studies in this laboratory have demonstrated ethanolic inhibition of one of these pathways catalyzed by methionine synthetase. It has been shown elsewhere that alcohol per se does not inhibit the enzyme, but that the metabolite of ethanol, acetaldehyde, is responsible through the formation of an inhibiting covalent adduct. Because hepatic SAM has been shown to be essential in the transport of fat from the liver, avoiding steatosis and further liver damage, it is entirely feasible that this repression of methionine synthase is an important site of the injurious action of alcohol metabolism in the liver. This loss of activity is particularly important in human beings who cannot produce methionine and SAM by means of the alternate pathway mediated by betaine:homocysteine:transmethylase, because of the lack of production of the betaine substrate for this enzyme.

Background: Circulating antibodies against malondialdehyde-acetaldehyde (MAA) haptenated proteins are increased significantly in patients with alcohol-induced cirrhosis and hepatitis and are associated with severity of liver damage. Additionally, MAA haptenated proteins are highly immunogenic without the use of adjuvant and have been suggested to induce autoreactive responses. The mechanism of this immunogenicity is currently unknown but may be mediated by cell death in a similar manner as other autoimmune diseases such as systemic lupus erythematosus, mixed connective tissue disease, myositis, and Sjögren's disease., Methods: Antigen-presenting cells, lymphocytes, and hepatocytes were exposed to different levels of MAA haptenated hen egg lysozyme and assessed by [3H]thymidine incorporation and (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) conversion for viability., Results: The cells investigated in this study were those that potentially may be involved in the development of autoimmune liver damage; they include antigen-presenting cells, lymphocytes, and cells of the liver itself. Each cell type was found to be sensitive to MAA haptenated protein-induced cell death at levels between 690 microM (10 microg/ml) and 6.9 mM (100 microg/ml), which may be found locally after chronic ethanol consumption. Antigen-presenting cells (macrophage and B cells) were found to be activated at concentrations just under those levels associated with cell death., Conclusions: A dose response to MAA haptenated protein-induced cell death is seen in antigen-presenting cells, lymphocytes, and hepatocytes in vitro. Recent reports have associated both apoptotic and necrotic cell death with the development of autoimmune disease; thus, it is possible that this may be one mechanism by which in vivo immunogenicity is mediated by aldehyde haptenated proteins.

Background: The alcohol metabolites malondialdehyde and acetaldehyde can combine to form stable adducts (MAA) which are found in the livers of humans and rats after significant alcohol ingestion. While adducted proteins induce antibody responses in the absence of adjuvants, the mechanisms by which these responses occur are unknown. Thus, it was the purpose of these studies to investigate how MAA modification stimulates antibody and T-cell responses in the absence of adjuvants., Methods: Hen egg lysozyme (HEL) was modified with increasing levels of MAA and was used as an immunogen, and antibody and T-cell responses were determined. The role of scavenger receptors in the immunogenicity of MAA-adducted proteins was also investigated., Results: Maximum antibody response was induced after immunization with 1.8 nM MAA/nM HEL, and was primarily an IgG1 response to HEL as determined by inhibition ELISAs. T-cell proliferative responses after immunization with HEL-MAA were solely to HEL. Immunization with a scavenger receptor ligand in conjunction with HEL-MAA increased the predominant IgG1 response and sharply decreased the IgG2a response by approximately 50%. Binding of HEL-MAA by splenocytes was determined by flow cytometry to be approximately 15% greater than HEL alone, showing a doubling of the geometric mean fluorescence. Also, most of the cells that bound HEL-MAA were class II positive, indicating that antigen-presenting cells can bind the MAA-adducted HEL, and potentially initiate immune responses., Conclusions: MAA modification of proteins induces antibody and T-cell proliferative responses in vivo. Initial studies suggest that these responses may be mediated by scavenger receptors that recognize MAA-adducted proteins. This suggests a mechanism by which proteins modified with oxidative products associated with chronic ethanol consumption may alter immune responses that may play an active role in the development and/or progression of alcoholic liver disease.

Background: For normal function and survival, hepatocytes require proper cell-extracellular matrix (ECM) contacts mediated by integrin receptors and focal adhesions. Previous studies have shown that chronic ethanol consumption selectively impairs perivenous (PV) hepatocyte attachment and spreading on various ECM substrates but increases expression of the beta1 integrin subunit, the common beta subunit for two major hepatocyte-ECM receptors, alpha1beta1 and alpha5beta1 integrins. This study examined the effects of ethanol treatment on the expression and cytoskeletal distribution of alpha1, alpha5, and beta1 integrin subunits, the epidermal growth factor receptor (EGF-R), and the cytoskeletal proteins focal adhesion kinase, paxillin, vinculin, and actin in periportal and PV hepatocytes., Methods: Periportal and PV hepatocytes were isolated from control and ethanol-fed rats. For expression analysis, lysates were examined by SDS-PAGE and immunoblotting procedures. For cytoskeletal distribution studies, Triton-soluble and -insoluble (cytoskeletal) fractions from hepatocytes cultured on collagen IV were analyzed by SDS-PAGE and immunoblotting., Results: Chronic ethanol administration caused PV-specific increases in expression and cytoskeletal association of the integrin subunits. Although ethanol treatment did not affect expression of the EGF-R in either cell type, it did increase the association of the EGF-R with the cytoskeleton selectively in PV hepatocytes. Ethanol treatment had no significant effect on either the expression or the cytoskeletal distribution of focal adhesion kinase, paxillin, vinculin, or actin in either cell type., Conclusions: The increases in integrin expression and cytoskeletal association observed after chronic ethanol administration suggest that a process downstream of integrin-ECM interactions is impaired selectively in PV hepatocytes, possibly involving altered focal adhesion assembly or turnover, processes essential for efficient cell-ECM adhesion. Alterations in these processes could contribute to the impaired hepatocyte function and structure observed after chronic ethanol administration.

Previous study results have demonstrated that cigarette smoke or acetaldehyde rapidly stimulates protein kinase C (PKC)-mediated release of interleukin-8 (IL-8) in bovine bronchial epithelial cells (BECs). Low concentrations of acetaldehyde combine synergistically with malondialdehyde to increase significantly maximal BEC PKC activity at 48 to 96 h stimulation. Because more than 95% of alcoholics are cigarette smokers, we hypothesized that malondialdehyde, an inflammation product of lipid peroxidation, and acetaldehyde, both a product of ethanol metabolism and a component of cigarette smoke, might stimulate PKC-mediated IL-8 release in BECs by malondialdehyde-acetaldehyde (MAA) adduct formation, rather than as free aldehydes. Protein kinase C activity is maximally elevated in BECs treated with 50 microg/ml of BSA-MAA from approximately 1 to 3 h. This activity subsequently begins to decrease by 4 to 6 h, with a return to baseline unstimulated kinase activity levels by 24 h. No activation of cyclic AMP-dependent protein kinase (PKA) or cyclic GMP-dependent protein kinase (PKG) was observed in BSA-MAA-treated BECs. The MAA adduct activation of PKC was followed by a fourfold to tenfold greater release of IL-8 over that observed for both BECs exposed to media only and BSA control-treated BECs. Protein kinase C activation and IL-8 release were blocked by pretreating BECs with 1 microM calphostin C or 100 nM of the PKC alpha-specific inhibitor, Go 6976. Isoform-specific inhibitors to PKC beta, PKC delta, and PKC zeta failed to inhibit completely MAA adduct-stimulated PKC or IL-8 release. Results of these studies indicate that metabolites derived from ethanol and cigarette smoke, such as acetaldehyde and malondialdehyde, form adducts that stimulate airway epithelial cell PKC alpha-mediated release of promigratory cytokines.

Findings obtained from our recent studies have demonstrated that malondialdehyde, a product of lipid peroxidation, and acetaldehyde can react together with proteins in a synergistic manner and form hybrid protein conjugates, which have been designated as malondialdehyde-acetaldehyde (MAA)-protein adducts. These adducts have been detected in livers of ethanol-fed rats and are immunogenic because significant increases in circulating antibody titers against MAA-adducted proteins have been observed in ethanol-fed rats and more recently in human alcoholics. Although immunological factors may tend to perpetuate liver injury, little is known about the direct functional consequences of MAA-adducted proteins on the different cellular populations of the liver. Hepatic stellate cells (HSCs) have been shown to be pivotal in the pathogenesis of fibrosis and in the amplification and self-perpetuation of the inflammatory process. The present study was conducted to determine the effects of MAA-adducted proteins on the function of HSCs. Rat HSCs were exposed to various amounts of MAA-protein adducts and their unmodified controls, and the secretion of two chemokines, monocyte chemoattractant protein (MCP)-1 and macrophage inflammatory protein (MIP)-2, that are involved in the chemotaxis of monocytes/macrophages and neutrophils, respectively, was determined. We observed that bovine serum albumin-MAA induced a dose- and time-dependent increase in the secretion of both of these chemokines. These findings indicate that MAA-adducted proteins may play a role in the modulation of the hepatic inflammatory response and could contribute to the pathogenesis of alcoholic liver disease.

Results of previous studies have shown that chronic ethanol administration impairs methionine synthetase activity and decreases S-adenosylmethionine levels in the liver, indicating interference with homocysteine remethylation. The purpose of the present study was to investigate the effects of chronic ethanol feeding on the accumulation of homocysteine (Hcy), a potentially toxic agent. The research was divided into two experiments. In Experiment A, hepatocytes were isolated from pair-fed control and ethanol-fed rats after 2 weeks of feeding, and the release of Hcy into the medium was determined. Hepatocytes obtained from ethanol-fed rats released twice as much Hcy into the medium as did those obtained from controls. When hepatocytes were challenged by a methionine load, a marked increase in Hcy generation was observed, and the increase was further enhanced in hepatocytes obtained from ethanol-fed rats. In Experiment B, hepatocytes were isolated from pair-fed control and ethanol-fed rats after 4 weeks of feeding (the feeding time required for significant formation of alcoholic fatty liver in rats). In this experiment, similar results were obtained with Hcy generation as in Experiment A. In Experiment B, supplementation of the incubation medium with betaine prevented the increase in generation of Hcy by methionine-treated control cells as well as the generation of Hcy by cells of ethanol-treated rats. These results indicate that betaine may have the potential as a therapeutic agent against toxic Hcy formation.

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 animals. Our previous studies have shown that MAA adducts are comprised of two distinct products. One adduct is composed of two molecules of malondialdehyde and one molecule of acetaldehyde and was identified as the 4-methyl-1,4-dihydropyridine-3,5-dicarbaldehyde derivative of an amino group (MDHDC adduct). The other adduct is a 1:1 adduct of malondialdehyde and acetaldehyde and was identified as the 2-formyl-3-(alkylamino)butanal derivative of an amino group (FAAB adduct). In this study, information on the mechanism of MAA adduct formation was obtained, focusing on whether the FAAB adduct serves as a precursor for the MDHDC adduct. Upon the basis of chemical analysis and NMR spectroscopy, two initial reaction steps appear to be a prerequisite for MDHDC formation. One step involves the reaction of one molecule of malondialdehyde and one of acetaldehyde with an amino group of a protein to form the FAAB product, while the other step involves the generation of a malondialdehyde-enamine. It appears that generation of the MDHDC adduct requires the FAAB moiety to be transferred to the nitrogen of the MDA-enamine. For efficient reaction of FAAB with the enamine to take place, additional experiments indicated that these two intermediates likely must be in positions on the protein of close proximity to each other. Further studies showed that the incubation of liver proteins from ethanol-fed rats with MDA resulted in a marked generation of MDHDC adducts, indicating the presence of a pool of FAAB adducts in the liver of ethanol-fed animals. Overall, these findings show that MDHDC-protein adduct formation occurs via the reaction of the FAAB moiety with a malondialdehyde-enamine, and further suggest that a similar mechanism may be operative in vivo in the liver during prolonged ethanol consumption.

This article represents the proceedings of a workshop at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Geoffrey M. Thiele and Simon Worrall. The presentations were (1) The chemistry of malondialdehyde-acetaldehyde (MAA) adducts, by Dean J. Tuma; (2) The formation and clearance of MAA adducts in ethanol-fed rats, by Simon Worrall; (3) Immune responses to MAA adducts may play a role in the development of alcoholic liver disease, by Lynell W. Klassen; (4) Unique biological responses to MAA-modified proteins that may play a role in the development and/or progression of alcoholic liver disease, by Geoffrey M. Thiele; (5) MAA-adducted bovine serum albumin activates protein kinase C and stimulates interleukin-8 release in bovine bronchial epithelial cells, by Todd A. Wyatt; and (6) An enzyme immune assay for serum antiacetaldehyde adduct antibody using low-density lipoprotein-adduct and its significance in alcoholic liver injury and ALDH2 heterozygotes, by Naruhiko Nagata.

Previously, we have found that acetaldehyde, a volatile component of cigarette smoke, stimulates the protein kinase C (PKC) pathway and inhibits ciliary motility. A "smokeless" cigarette (Eclipse) now exists in which most of the tobacco is not burned, reducing the pyrolyzed components in the extract. We hypothesized that acetaldehyde is a component of cigarette smoke that activates PKC in the airway epithelial cell, and therefore the Eclipse cigarette would not activate epithelial cell PKC. In this study, bovine bronchial epithelial cells (BBEC) were incubated with cigarette smoke extract (CSE) or Eclipse smoke extract (ESE). We found that PKC activity was significantly higher in cells exposed to 5% CSE than cells exposed to 5% ESE or media. When acetaldehyde levels of both extracts were measured by gas chromatography, CSE was found to have 15-20 times greater concentration (microM) of acetaldehyde than ESE. When BBEC were treated with 5% CSE, ciliary beating was further decreased from baseline levels. This decrease in ciliary beating was not observed in cells treated with ESE, suggesting that acetaldehyde contained in CSE slows cilia. These results suggest that volatile components such as acetaldehyde in cigarette smoke may inhibit ciliary motility via a PKC-dependent mechanism.

Ethanol administration leads to altered function and impaired receptor-mediated endocytosis of the hepatocyte asialoglycoprotein receptor (ASGP-R). The purpose of the present study was to examine the effects of ethanol on the phosphorylation of the ASGP-R to determine whether this post-translational modification could contribute mechanistically to the observed ethanol-induced alterations. The methodological approach of this work involved the measurement of the phosphorylation state of the receptor obtained from isolated rat hepatocytes, using a combination of experimental designs from the biosynthetic incorporation of phosphate to the determination of steady-state phosphotyrosine levels. We report here that both short-term (1- to 2-week) and chronic (5- to 7-week) periods of ethanol administration resulted in a significant increase in the steady-state phosphotyrosine protein in the ASGP-R. In addition, in vitro incorporation of [gamma-(32)P]ATP using a permeabilized cell assay system similarly showed an increase in tyrosine-phosphorylated receptors. Furthermore, metabolic radiolabeling of hepatocytes with [(32)P]orthophosphate demonstrated hyperphosphorylation of the ASGP-R in cells obtained from chronically ethanol-fed animals. Finally, our results revealed that dephosphorylation of the ASGP-R was unaffected by ethanol administration, indicating that kinase activity rather than impaired phosphatase action contributes to the increased phosphorylation state of the receptor. Overall, the results presented in this study demonstrated that the extent of tyrosine phosphorylation of the receptor is significantly higher in hepatocytes obtained from ethanol-fed animals. We conclude that hyperphosphorylation of the ASGP-R may be a contributing factor to the impaired function of the receptor elicited by ethanol administration.

Acetaldehyde and malonildialdehyde can form hybrid protein adducts, named MAA adducts that have strong immunogenic properties. The formation of MAA adducts in the liver of chronic alcohol-fed rats is associated with the development of circulating antibodies that specifically recognized these adducts. The aim of this study was to examine whether MAA adducts might participate in the immune response associated with human alcohol-induced liver disease. Circulating antibodies against MAA adducts were evaluated in 50 patients with alcohol-induced hepatitis or cirrhosis, in 40 patients with non-alcohol-induced liver disease, in 15 heavy drinkers without liver damage and in 40 healthy controls by enzyme-linked immunosorbent assays (ELISA). Immunoglobulin G (IgG) reacting with MAA-modified proteins were significantly increased in the patients with alcohol-induced cirrhosis or hepatitis. The individual levels of anti-MAA IgG in those patients were associated with the severity of liver damage. Anti-MAA antibodies were also positively correlated with the levels of IgG recognizing epitopes generated by acetaldehyde and malonildialdehyde. However, competitive inhibition experiments indicated that the anti-MAA antibodies were unrelated to those against acetaldehyde- or malonildialdehyde-derived antigens and mainly recognized a specific, cyclic MAA epitope. Some degree of immune reactivity towards MAA adducts was also observed in patients with non-alcohol-induced liver injury. However, competitive ELISA showed that the antigens recognized by these sera were not the cyclic MAA adducts. Altogether, these results showed the formation of MAA antigens during alcohol-induced liver disease and suggest their possible contribution to the development of immunologic reactions associated with alcohol-related liver damage.

Background: Hepatocytes require attachment and subsequent spreading on an extracellular matrix for their proper growth, function and survival. Our previous studies have shown that ethanol feeding selectively impairs perivenule hepatocyte attachment to various extracellular matrices. This study was undertaken to determine whether zonal differences in hepatocyte spreading in response to ethanol feeding occurs and to ascertain the influence of ethanol consumption on the zonal expression of the beta1 subunit of integrins, which are the major surface receptors responsible for matrix binding and subsequent interactions., Methods: Hepatocytes from the perivenous and periportal regions of the liver were isolated by digitonin/collagenase perfusion from rats that were pair-fed for 2 to 3 weeks with a liquid diet containing either ethanol or isocaloric carbohydrate. The ability of perivenous and periportal hepatocytes to spread on plates coated with either type IV collagen, laminin, fibronectin or polylysine was determined. In addition, the isolated cells were used for the analysis of total cellular and surface beta1 integrin expression., Results: With all of the matrix substrates tested, the spreading of perivenous hepatocytes isolated from the ethanol-fed animals was markedly impaired, while the spreading of periportal hepatocytes was essentially unaffected by ethanol feeding. Both the total cellular as well as the surface expression of the beta1 integrin subunit in perivenous cells from the ethanol-fed rats were significantly higher than from the perivenous control cells, whereas the total and surface expression of the beta1 integrin in periportal cells isolated from ethanol-fed and control rats were not significantly different., Conclusions: The results indicated that in addition to impairing hepatocyte attachment, ethanol feeding also impairs another critical step of the adhesion process, that of hepatocyte spreading on extracellular matrix substrates. This defect occurred preferentially in perivenous cells and not periportal cells and was associated with an increase in beta1 integrin expression, suggesting that a compensatory mechanism occurs as an attempt by the perivenous cells to overcome impaired cell-matrix interactions caused by ethanol. Overall, these alterations in extracellular matrix-hepatocyte interactions could lead to alterations of hepatocyte structure and function and potentially play a role in alcoholic liver injury.

Receptor-mediated endocytosis (RME) by a scavenger receptor on sinusoidal liver endothelial cells (LECs) for formaldehyde-treated bovine serum albumin (f-Alb) has previously been shown to be impaired following chronic ethanol consumption. These studies were initially performed by in situ perfusion, making it difficult to determine the point in the process at which RME is affected. Therefore, it was the purpose of this study to use isolated LECs to begin elucidating at what point in the process chronic ethanol consumption affects RME. Initial studies showed that degradation at the single-cell level were similarly decreased at levels that had been observed for in situ studies, suggesting that the ethanol effects can be repeated using isolated LECs, making them useful for in vitro studies. Binding studies with 125I-formaldehyde-treated bovine serum albumin (125I-f-Alb) demonstrated there was a slight, but significantly different, decrease in binding by LECs from ethanol-fed rats when compared with pair-fed or chow-fed rats. However, the affinity of these receptors was not different between these groups. In contrast, a defect in the initial stages of receptor-ligand internalization was indicated as less surface-bound ligand was internalized and subsequently degraded in cells from the ethanol-treated animals as compared with controls. Additionally, once the data were adjusted for the amount of ligand internalized, the degradation of the internalized ligand was only slightly impaired. These results indicate that chronic ethanol feeding impairs the process of RME by the liver; the major cause of this impairment appears to be caused by a decreased ability of these cells to internalize all of the surface-bound ligand, with a minimal defect in postinternalization events.

Many investigators have suggested that an immune reaction to acetaldehyde-protein adducts may be involved in the development and/or progression of alcohol liver disease. The most often reported acetaldehyde adduct is the reduced adduct prepared in vitro in the presence of strong reducing agents. However, the production of this adduct in vivo has been difficult to prove. Nevertheless, the detection of serum antibodies to this reduced adduct following alcohol exposure in animals and humans has been used to support the formation of this adduct in vivo. We have recently observed that when acetaldehyde-protein adducts prepared under nonreducing conditions are used to immunize animals, antibody to the reduced protein adduct is detected. Therefore, it was the purpose of this study to demonstrate that nonreduced (NR) adduct epitopes can be modified by intact cells to express reduced (R) adduct epitopes. This was accomplished using the monoclonal antibody RT1.1 that has been previously characterized by this laboratory and has been shown to recognize only R and not NR acetaldehyde adducts. In these studies, Balb/c mice were injected intraperitoneally (500 microg/animal) with either keyhole limpet hemocyanin (KLH)-NR or KLH-R adducted proteins. Immunization with KLH-NR produced significant amounts of antibodies that recognized both NR and R epitopes. In contrast, immunization with KLH-R produced antibodies to only R and not NR epitopes. Isolated peritoneal macrophages from nonimmunized mice were incubated in vitro with either KLH-NR, KLH-R, or unmodified KLH proteins, and the cell surface expression of the reduced epitope (RT1.1) and the activated macrophage marker (MAC-3) determined by double immunofluorescent staining. Activated macrophages incubated with KLH-NR expressed the R adduct on 11.5% of the cells, compared with 3.8% following incubation with unmodified KLH, and 19.4% following incubation with KLH-R. These data suggest that the NR adduct and/or the carrier protein are modified by peritoneal macrophages in vivo and present an epitope that is detected as a reduced adduct (RT1.1 positive). These observations may explain the presence of circulating antibodies to the reduced adduct that has been reported in human and animal studies.

It has been shown that malondialdehyde (MDA) and acetaldehyde react with proteins via the epsilon-amino group of a lysine residue to yield hybrid MDA-acetaldehyde (MAA)-protein adducts. The structure of one MAA adduct has been confirmed to be 4-methyl-1, 4-dihydropyridine-3,5-dicarbaldehyde (3). In this study, 13C NMR spectroscopy was used to identify the structure of a second MAA adduct as 2-formyl-3-(alkylamino)butanal (4). Isotopically labeled [1-13C]acetaldehyde was reacted with MDA and the protein, bovine serum albumin, under a variety of conditions, and the reactions were monitored at various time intervals by 13C NMR. In each experiment, new signals grew in at 50 and 22 ppm. By comparison to model compounds, the signals at 50 ppm correspond to a 2-formyl-3-(alkylamino)butanal adduct and the signals at 22 ppm correspond to the known 1,4-dihydropyridine-3,5-dicarbaldehyde adduct. Similar results were found when the BSA was replaced with polylysine. Overall, it appears that MAA-protein adducts are composed of two major products, 3 and 4.