Cyanamide Potentiates the Ethanol-Induced Impairment of Receptor-Mediated Endocytosis in a Recombinant Hepatic Cell Line Expressing Alcohol Dehydrogenase Activity

Ethanol administration has been shown to alter receptor-mediated endocytosis in the liver. We have developed a recombinant hepatic cell line stably transfected with murine alcohol dehydrogenase cDNA to serve as an in vitro model to investigate these ethanol-induced impairments. In the present study, transfected cells were maintained in the absence or presence of 25 mM ethanol for 7 days, and alterations in endocytosis by the asialoglycoprotein receptor were determined. The role of acetaldehyde in this dysfunction was also examined by inclusion of the aldehyde dehydrogenase inhibitor, cyanamide. Our results showed that ethanol metabolism impaired internalization of asialoorosomucoid, a ligand for the asialoglycoprotein receptor. The addition of cyanamide potentiated the ethanol-induced defect in internalization and also impaired degradation of the ligand in the presence of ethanol. These results indicate that the ethanol-induced impairment in endocytosis is exacerbated by the inhibition of aldehyde dehydrogenase, suggesting the involvement of acetaldehyde in this dysfunction

Effect of Malondialdehyde-Acetaldehyde-Protein Adducts on the Protein Kinase C-Dependent Secretion of Urokinase-Type Plasminogen Activator in Hepatic Stellate Cells

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-a, 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

Halothane potentiates the alcohol-adduct induced TNF-alpha release in heart endothelial cells

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-α (TNF-α) 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-α were measured by ELISA. RESULTS: Halothane pre-treated rat HECs released significantly greater TNF-α 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-α 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-α release measured following both MAA-Alb and LPS stimulation

Betaine Treatment Attenuates Chronic Ethanol-Induced Hepatic Steatosis and Alterations to the Mitochondrial Respiratory Chain Proteome

Introduction. Mitochondrial damage and disruption in oxidative phosphorylation contributes to the pathogenesis of alcoholic liver injury. Herein, we tested the hypothesis that the hepatoprotective actions of betaine against alcoholic liver injury occur at the level of the mitochondrial proteome. Methods. Male Wister rats were pair-fed control or ethanol-containing liquid diets supplemented with or without betaine (10 mg/mL) for 4-5 wks. Liver was examined for triglyceride accumulation, levels of methionine cycle metabolites, and alterations in mitochondrial proteins. Results. Chronic ethanol ingestion resulted in triglyceride accumulation which was attenuated in the ethanol plus betaine group. Blue native gel electrophoresis (BN-PAGE) revealed significant decreases in the content of the intact oxidative phosphorylation complexes in mitochondria from ethanol-fed animals. The alcohol-dependent loss in many of the low molecular weight oxidative phosphorylation proteins was prevented by betaine supplementation. This protection by betaine was associated with normalization of SAM : S-adenosylhomocysteine (SAH) ratios and the attenuation of the ethanol-induced increase in inducible nitric oxide synthase and nitric oxide generation in the liver. Discussion/Conclusion. In summary, betaine attenuates alcoholic steatosis and alterations to the oxidative phosphorylation system. Therefore, preservation of mitochondrial function may be another key molecular mechanism responsible for betaine hepatoprotection

Role of apoptotic hepatocytes in HCV dissemination: regulation by acetaldehyde

Alcohol consumption exacerbates hepatitis C virus (HCV) pathogenesis and promotes disease progression, although the mechanisms are not quite clear. We have previously observed that acetaldehyde (Ach) continuously produced by the acetaldehyde-generating system (AGS), temporarily enhanced HCV RNA levels, followed by a decrease to normal or lower levels, which corresponded to apoptosis induction. Here, we studied whether Ach-induced apoptosis caused depletion of HCV-infected cells and what role apoptotic bodies (AB) play in HCV-alcohol crosstalk. In liver cells exposed to AGS, we observed the induction of miR-122 and miR-34a. As miR-34a has been associated with apoptotic signaling and miR-122 with HCV replication, these findings may suggest that cells with intensive viral replication undergo apoptosis. Furthermore, when AGS-induced apoptosis was blocked by a pan-caspase inhibitor, the expression of HCV RNA was not changed. AB from HCV-infected cells contained HCV core protein and the assembled HCV particle that infect intact hepatocytes, thereby promoting the spread of infection. In addition, AB are captured by macrophages to switch their cytokine profile to the proinflammatory one. Macrophages exposed to HCV+ AB expressed more IL-1β, IL-18, IL-6, and IL-10 mRNAs compared with those exposed to HCV-AB. The generation of AB from AGS-treated HCV-infected cells even enhanced the induction of aforementioned cytokines. We conclude that HCV and alcohol metabolites trigger the formation of AB containing HCV particles. The consequent spread of HCV to neighboring hepatocytes via infected AB, as well as the induction of liver inflammation by AB-mediated macrophage activation potentially exacerbate the HCV infection course by alcohol and worsen disease progression

Increased immunogenicity to P815 cells modified with malondialdehyde and acetaldehyde

Aldehyde modified proteins have been associated with the development and/or progression of alcoholic liver disease (ALD). These protein adducts are capable of initiating many immunological responses that are harmful to the normal homeostasis of organism function. Previous studies have shown that malondialdehyde (MDA) and acetaldehyde (AA) synergistically form a unique adduct (MAA) with soluble proteins, which are capable of inducing cytokine release, T-cell proliferation, and antibody production. The purpose of this study was to determine whether MAA-adduction can elicit similar responses to cells using a well-defined tumor model. The mouse mastocytoma P815 tumor cell line was modified with MAA (P815-MAA) or left unmodified (P815) and 106 irradiated cells were injected into DBA/2 mice once a week for 5 weeks. Serum was collected and tested for antibody responses to P815 cells and the MAA epitope. Immunization of MAA-adducted P815 cells into syngeneic DBA/2 mice induced a strong antibody response to the MAA epitope as determined by ELISA on Alb and MAA-Alb (508 µg/ml and 1092 µg/ml, respectively). In addition, antibody to unmodified P815 cells was detected by fluorescent technique. Mice immunized with P815 cells or PBS showed little or no reactivity to the MAA epitope or P815 cells. Studies to assess IL-12 stimulation showed that peritoneal macrophages from P815 and PBS immunized animals produced modest amounts of IL-12 (20 and 35 pg/ml) when stimulated with Alb or MAA-Alb. However, macrophage from P815-MAA immunized mice responded to soluble MAA-adduct (142 pg/ml). Finally, in tumor survival studies the mean survival was 14.25 days in PBS treated mice; 15.75 days with P815 immunized mice and 18.25 days with P815-MAA immunized mice. Therefore, these data strongly suggest that antibody responses are induced by P815 cells modified with MAA-adducts. This may be a possible tool to begin looking at how alcohol metabolites potentially modify cells and/or cellular components making them recognizable to the immune system as foreign. It is thought that these studies define a model system that will be useful in assessing antibody and potentially T cell responses to cells that are modified by MAA

Autoimmune Hepatitis Induced by Syngeneic Liver Cytosolic Proteins Biotransformed by Alcohol Metabolites

Aldehydes that are produced following the breakdown of ethanol (acetaldehyde) and lipid peroxidation of membranes (malondialdehyde) have been shown to bind (adduct) proteins. Additionally, these two aldehydes can combine (MAA) on non-syngeneic and syngeneic proteins to initiate numerous immune responses to the unmodified part of the protein in the absence of an adjuvant. Therefore, these studies provide a potential mechanism for the development of antigen-specific immune responses resulting in liver damage should syngeneic liver proteins be adducted with MAA