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ABSTRACT: T cell activation and associated pro-inflammatory cytokine production is a pathological feature of inflammatory liver disease. It is also known that liver injury is associated with marked impairments in the function of many hepatic proteins including a hepatocyte-specific binding protein, the asialoglycoprotein receptor (ASGPR). Recently, it has been suggested that hepatic ASGPRs may play an important role in the physiological regulation of T lymphocytes, leading to our hypothesis that ASGPR defects correlate with inflammatory-mediated events in liver diseases. Therefore, in this study we investigated whether changes in hepatocellular ASGPR expression were related to the dysregulation of intrahepatic T lymphocytes and correlate with the development of T-cell mediated hepatitis. Mice lacking functional ASGPRs (receptor-deficient, RD), and wild-type (WT) controls were intravenously injected with T-cell mitogens, Concanavalin A (Con A) or anti-CD3 antibody. As a result of T cell mitogen treatment, RD mice lacking hepatic ASGPRs displayed enhancements in liver pathology, transaminase activities, proinflammatory cytokine expression, and caspase activation compared to that observed in normal WT mice. Furthermore, FACS analysis demonstrated that T-cell mitogen administration resulted in a significant rise in the percentage of CD8+ lymphocytes present in the livers of RD animals versus WT mice. Since these two mouse strains differ only in whether they express the hepatic ASGPR, it can be concluded that proper ASGPR function exerts a protective effect against T cell mediated hepatitis and that impairments to this hepatic receptor could be related to the accumulation of cytotoxic T cells that are observed in inflammatory liver diseases.
International immunopharmacology 03/2013; · 2.21 Impact Factor
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ABSTRACT: Acute and chronic ethanol administration increase autophagic vacuole (i.e., autophagosome; AV) content in liver cells. This enhancement depends on ethanol oxidation. Here, we used parental (nonmetabolizing) and recombinant (ethanol-metabolizing) Hep G2 cells to identify the ethanol metabolite that causes AV enhancement by quantifying AVs or their marker protein, microtubule-associated protein 1 light chain 3-II (LC3-II). The ethanol-elicited rise in LC3-II was dependent on ethanol dose, was seen only in cells that expressed alcohol dehydrogenase (ADH) and was augmented in cells that coexpressed cytochrome CYP2E1 (P450 2E1). Furthermore, the rise in LC3-II was inversely related to a decline in proteasome activity. AV flux measurements and colocalization of AVs with lysosomes or their marker protein Lysosomal-Associated Membrane Protein 1 (LAMP1) in ethanol-metabolizing VL-17A cells (ADH (+) /CYP2E1 (+) ) revealed that ethanol exposure not only enhanced LC3-II synthesis but also decreased its degradation. Ethanol-induced accumulation of LC3-II in these cells was similar to that induced by the microtubule inhibitor, nocodazole. After we treated cells with either 4-methylpyrazole to block ethanol oxidation or GSH-EE to scavenge reactive species, there was no enhancement of LC3-II by ethanol. Furthermore, regardless of their ethanol-metabolizing capacity, direct exposure of cells to acetaldehyde enhanced LC3-II content. We conclude that both ADH-generated acetaldehyde and CYP2E1-generated primary and secondary oxidants caused LC3-II accumulation, which rose not only from enhanced AV biogenesis, but also from decreased LC3 degradation by the proteasome and by lysosomes.
Autophagy 10/2012; 9(1). · 7.45 Impact Factor
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ABSTRACT: Although alcoholic liver disease is clinically well-described, the molecular basis for alcohol-induced hepatotoxicity is not well understood. Previously, we found that alcohol exposure led to increased microtubule acetylation and stability in polarized, hepatic WIF-B cells and in livers from ethanol-fed rats. Because microtubules are known to regulate transcription factor nuclear translocation, and that dynamic microtubules are required for translocation of at least a sub-set of these factors, we examined whether alcohol-induced microtubule acetylation and stability impairs nuclear translocation. We examined nuclear delivery of factors representing the two mechanisms by which microtubules regulate translocation. To represent factors that undergo directed delivery, we examined growth hormone-induced STAT5B translocation and interleukin-6-induced STAT3 translocation. To represent factors that are sequestered in the cytoplasm by microtubule attachment until ligand activation, we examined TGF-β-induced Smad2/3 translocation. We found that ethanol exposure selectively impaired the translocation of the STATs, but not Smad 2/3. STAT5B delivery was decreased to similar extents by addition of taxol (microtubule stabilizing drug) or trichostatin A (deacetylase inhibitor), agents that promote microtubule acetylation in the absence of alcohol. Thus, the alcohol-induced impairment of STAT nuclear translocation can be explained by increased microtubule acetylation and stability. Only ethanol-treatment impaired STAT5B activation, indicating that microtubules are not important for its activation by Jak2. Furthermore, nuclear exit was not changed in treated cells indicating this process is also independent of microtubule acetylation and stability. Together, these results raise the exciting possibility that deacetylase agonists may be effective therapeutics for the treatment of alcoholic liver disease.
AJP Gastrointestinal and Liver Physiology 10/2012; · 3.43 Impact Factor
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ABSTRACT: Steatosis, an early manifestation in alcoholic liver disease, is associated with the accumulation of hepatocellular lipid droplets (LDs). However, the role ethanol metabolism has in LD formation and turnover remains undefined. Here, we assessed LD dynamics following ethanol and oleic acid treatment to ethanol-metabolizing WIF-B cells (a hybrid of human fibroblasts (WI 38) and Fao rat hepatoma cells). An OA dose-dependent increase in triglyceride and stained lipids was identified which doubled (P < 0.05) in the presence of ethanol. This effect was blunted with the inclusion of an alcohol metabolism inhibitor. The ethanol/ OA combination also induced adipophilin, LD coat protein involved in the attenuation of lipolysis. Additionally, ethanol treatment resulted in a significant reduction in lipid efflux. These data demonstrate that the metabolism of ethanol in hepatic cells is related to LD accumulation, impaired fat efflux, and enhancements in LD-associated proteins. These alterations in LD dynamics may contribute to ethanol-mediated defects in hepatocellular LD regulation and the formation of steatosis.
International journal of hepatology. 01/2012; 2012:978136.
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ABSTRACT: 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.
International journal of hepatology. 01/2012; 2012:954157.
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ABSTRACT: 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.
International journal of hepatology. 01/2012; 2012:962183.
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ABSTRACT: The liver is the major site of ethanol metabolism and thus sustains the most injury from chronic alcohol consumption. Ethanol metabolism by the hepatocyte leads to the generation of reactive metabolites and oxygen radicals that can readily adduct DNA, lipids, and proteins. More recently, it has become apparent that ethanol consumption also leads to increased post-translational modifications of the natural repertoire, including lysine hyperacetylation. Previously, we determined that alcohol consumption selectively impairs clathrin-mediated internalization in polarized hepatocytes. However, neither the step at which the block occurs nor the mechanism responsible for the defect have been identified. To identify the specific step at which clathrin-mediated internalization is impaired, we examined the distributions, levels, and assembly of selected components of the clathrin machinery in control and ethanol-treated cells. To determine whether the impairment is caused by ethanol-induced lysine acetylation, we also examined the same coat components in cells treated with trichostatin A (TSA), a deacetylase inhibitor that leads to protein hyperacetylation in the absence of ethanol. Conclusion: We determined that both ethanol and TSA impair internalization at a late stage before vesicle fission. We further determined that this defect is likely the result of decreased dynamin recruitment to the necks of clathrin-coated invaginations resulting in impaired vesicle budding. These results also raise the exciting possibility that agents that promote lysine deacetylation may be effective therapeutics for the treatment of alcoholic liver disease.
Hepatology 11/2011; 55(4):1260-70. · 11.66 Impact Factor
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ABSTRACT: In addition to cigarette smoking, alcohol exposure is also associated with increased lung infections and decreased mucociliary clearance. However, little research has been conducted on the combination effects of alcohol and cigarette smoke on lungs. Previously, we have demonstrated in a mouse model that the combination of cigarette smoke and alcohol exposure results in the formation of a very stable hybrid malondialdehyde-acetaldehyde (MAA)-adducted protein in the lung. In in vitro studies, MAA-adducted protein stimulates bronchial epithelial cell interleukin-8 (IL-8) via the activation of protein kinase C epsilon (PKCɛ). We hypothesized that direct MAA-adducted protein exposure in the lungs would mimic such a combination of smoke and alcohol exposure leading to airway inflammation. To test this hypothesis, C57BL/6J female mice were intranasally instilled with either saline, 30μL of 50μg/mL bovine serum albumin (BSA)-MAA, or unadducted BSA for up to 3 weeks. Likewise, human lung surfactant proteins A and D (SPA and SPD) were purified from human pulmonary proteinosis lung lavage fluid and successfully MAA-adducted in vitro. Similar to BSA-MAA, SPD-MAA was instilled into mouse lungs. Lungs were necropsied and assayed for histopathology, PKCɛ activation, and lung lavage chemokines. In control mice instilled with saline, normal lungs had few inflammatory cells. No significant effects were observed in unadducted BSA- or SPD-instilled mice. However, when mice were instilled with BSA-MAA or SPD-MAA for 3 weeks, a significant peribronchiolar localization of inflammatory cells was observed. Both BSA-MAA and SPD-MAA stimulated increased lung lavage neutrophils and caused a significant elevation in the chemokine, keratinocyte chemokine, which is a functional homologue to human IL-8. Likewise, MAA-adducted protein stimulated the activation of airway and lung slice PKCɛ. These data support that the MAA-adducted protein induces a proinflammatory response in the lungs and that the lung surfactant protein is a biologically relevant target for malondialdehyde and acetaldehyde adduction. These data further implicate MAA-adduct formation as a potential mechanism for smoke- and alcohol-induced lung injury.
Alcohol (Fayetteville, N.Y.) 09/2011; 46(1):51-9. · 2.41 Impact Factor
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ABSTRACT: Most alcohol abusers smoke cigarettes and approximately half of all cigarette smokers consume alcohol. However, no animal models of cigarette and alcohol co-exposure exist to examine reactive aldehydes in the lungs. Cigarette smoking results in elevated lung acetaldehyde (AA) and malondialdehyde (MDA) levels. Likewise, alcohol metabolism produces AA via the action of alcohol dehydrogenase and MDA via lipid peroxidation. A high concentration of AA and MDA form stable hybrid protein adducts known as malondialdehyde-acetaldehyde (MAA) adducts. We hypothesized that chronic cigarette smoke and alcohol exposure in an in vivo mouse model would result in the in vivo formation of MAA adducts.
We fed C57BL/6 mice ad libitum ethanol (20%) in drinking water and exposed them to whole-body cigarette smoke 2 h/d, 5 d/wk for 6 weeks. Bronchoalveolar lavage fluid and lung homogenates were assayed for AA, MDA, and MAA adduct concentrations. MAA-adducted proteins were identified by Western blot and ELISA.
Smoke and alcohol exposure alone elevated both AA and MDA, but only the combination of smoke+alcohol generated protein-adducting concentrations of AA and MDA. MAA-adducted protein (~500 ng/ml) was significantly elevated in the smoke+alcohol-exposed mice. Of the 5 MAA-adducted proteins identified by Western blot, 1 protein band immunoprecipitated with antibodies to surfactant protein D. Similar to in vitro PKC stimulation by purified MAA-adducted protein, protein kinase C (PKC) epsilon was activated only in tracheal epithelial extracts from smoke- and alcohol-exposed mice.
These data demonstrate that only the combination of cigarette smoke exposure and alcohol feeding in mice results in the generation of significant AA and MDA concentrations, the formation of MAA-adducted protein, and the activation of airway epithelial PKC epsilon in the lung.
Alcoholism Clinical and Experimental Research 03/2011; 35(6):1106-13. · 3.34 Impact Factor
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ABSTRACT: Introduction: Nonalcoholic steatohepatitis (NASH) is an important cause of cirrhosis and over the past decade has accounted for an increasing proportion of liver transplants in the United States. Unfortunately there is no treatment for NASH except for risk factor modification. The aims of our study were to assess the impact of betaine on liver function tests, homocysteine levels and hepatic fibrosis in a prospective cohort of NASH patients Materials and Methodology: Between July 2003 and June 2006, consecutive patients with NASH were screened to deter-mine treatment eligibility. Eligibility criteria included elevated aminotransferases and a liver biopsy within twelve months of study entry satisfying the Brunt criteria for NASH. Patients were treated with betaine anhydrous 10 grams twice a day for one year. Liver function tests, homocysteine levels and liver biopsy were performed prior to and at the end of treat-ment. Outcomes were calculated using intention to treat analysis. Results: 35 patients were eligible. 23 patients completed treatment, seven were intolerant and five dropped out and were lost to follow up. Improvement or normalization in aminotransferases occurred in 62.9% of patients (p<0.05) and in ho-mocysteine in 45.7% (p> 0.05). Resolution or improvement in steatosis occurred in 57.1% (p<0.05), improvement or sta-bilization of inflammation in 60% (p<0.05) and fibrosis in 62.9% (p<0.05). Conclusion: Betaine appears to improve hepatic function tests, homocysteine levels and histology in this cohort of NASH patients. Large randomized studies with long-term follow up are required to assess the effect of betaine for this growing epidemic.
01/2011; 3.
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ABSTRACT: 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 nonsyngeneic 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.
This study sought to test whether MAA-modified syngeneic liver cytosolic proteins administered daily in the absence of adjuvant into C57BL/6 mice abrogates tolerance to initiate a MAA-induced autoimmune-like hepatitis.
In mice immunized with MAA-modified cytosols, there was an increase in liver damage as assessed by aspartate aminotransferase/alanine aminotransferase levels that correlated with liver pathology scores and the presence of the pro-fibrotic factors, smooth muscle actin, TGF-β, and collagen. IgG antibodies and T-cell proliferative responses specific for cytosolic proteins were also detected. Pro-inflammatory cytokines were produced in the livers of animals exposed to MAA-modified cytosols. Finally, transfer of immunized T cells to naïve animals caused biochemical and histological evidence of liver damage.
These data demonstrate that a disease with an autoimmune-like pathophysiology can be generated in this animal model using soluble MAA-modified syngeneic liver cytosols as the immunogen. These studies provide insight into potential mechanism(s) that the metabolites of alcohol may play in contributing to the onset of an autoimmune-like disease in patients with alcoholic liver disease.
Alcoholism Clinical and Experimental Research 12/2010; 34(12):2126-36. · 3.34 Impact Factor
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ABSTRACT: Antibodies to malondialdehyde (MDA)-modified macromolecules (adducts) have been detected in the serum of patients with atherosclerosis and correlate with the progression of this disease. However, the epitope and its formation have not been characterized. Studies have shown that excess MDA can be degraded to acetaldehyde, which combines with proteins to from a stable dihydropyridine adduct. To investigate, mice were immunized with MDA adducts in the absence of adjuvant and showed an increase in antibodies to MDA adducts and the carrier protein as the concentration of MDA was increased. In fact, a number of the commercially available antibodies to MDA-modified proteins were able to be inhibited by a chemical analogue, hexyl-MAA. Also, MDA-MAA adducts were detected in the serum and aortic tissue of JCR diabetic/atherosclerotic rats. These studies determined that commercially available antibodies to MDA predominantly react with the MAA adduct and are present in the JCR model of atherosclerosis in both the serum and the aortic tissue. Therefore, the immune response to MDA-modified proteins is most probably to the dihydropyridine structure (predominant epitope in MAA), which suggests that MAA adducts may play a role in the development and/or progression of atherosclerosis.
Free radical biology & medicine 11/2010; 49(10):1480-6. · 5.42 Impact Factor
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ABSTRACT: Ethanol metabolism in the liver induces oxidative stress and altered cytokine production preceding myofibroblast activation and fibrogenic responses. The purpose of this study was to determine how ethanol affects the fibrogenic response in precision-cut liver slices (PCLS). PCLS were obtained from chow-fed male Wistar rats (200-300 g) and were cultured up to 96 h in medium, 25 mM ethanol, or 25 mM ethanol and 0.5 mM 4-methylpyrazole (4-MP), an inhibitor of ethanol metabolism. Slices from every time point (24, 48, 72, and 96 h) were examined for glutathione (GSH) levels, lipid peroxidation [thiobarbituric acid-reactive substance (TBARS) assay], cytokine production (ELISA and RT-PCR), and myofibroblast activation [immunoblotting and immunohistochemistry for smooth muscle actin (SMA) and collagen]. Treatment of PCLS with 25 mM ethanol induced significant oxidative stress within 24 h, including depletion of cellular GSH and increased lipid peroxidation compared with controls (P < 0.05). Ethanol treatment also elicited a significant and sustained increase in interleukin-6 (IL-6) production (P < 0.05). Importantly, ethanol treatment accelerates a fibrogenic response after 48 h, represented by significant increases in SMA and collagen 1alpha(I) production (P < 0.05). These ethanol-induced effects were prevented by the addition of 4-MP. Ethanol metabolism induces oxidative stress (GSH depletion and increased lipid peroxidation) and sustained IL-6 expression in rat PCLS. These phenomena precede and coincide with myofibroblast activation, which occurs within 48 h of treatment. These results indicate the PCLS can be used as in vitro model for studying multicellular interactions during the early stages of ethanol-induced liver injury and fibrogenesis.
AJP Gastrointestinal and Liver Physiology 09/2010; 299(3):G661-8. · 3.43 Impact Factor
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ABSTRACT: Although the clinical manifestations of alcoholic liver disease are well described, little is known about the molecular basis for liver injury. Recent studies have indicated that chronic alcohol consumption leads to the lysine-hyperacetylation of several hepatic proteins, and this list is growing quickly.
To identify other hyperacetylated proteins in ethanol-fed livers, we chose a proteomics approach. Cytosolic and membrane proteins (excluding nuclei) were separated on 2D gels, transferred to PVDF and immunoblotted with antibodies specific for acetylated lysine residues. Hyperacetylated proteins were selected for trypsin digestion and mass spectrometric analysis.
In all, 40 proteins were identified, 11 of which are known acetylated proteins. Remarkably, the vast majority of hyperacetylated membrane proteins were mitochondrial residents. Hyperacetylated cytosolic proteins ranged in function from metabolism to cytoskeletal support. Notably, 3 key anti-oxidant proteins were identified whose activities are impaired in ethanol-treated cells. We confirmed that the anti-oxidant enzyme, glutathione peroxidase 1, actin and cortactin are hyperacetylated in ethanol-treated livers.
Alcohol-induced hyperacetylation of multiple proteins may contribute to the development of liver injury. The abundance of acetylated mitochondrial proteins further suggests that this modification is important in regulating liver metabolism and when perturbed, may contribute to the progression of a variety of metabolic diseases.
Alcoholism Clinical and Experimental Research 11/2009; 34(2):280-91. · 3.34 Impact Factor
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ABSTRACT: To investigate the role of reactive oxygen species (ROS) in ethanol-mediated cell death of polarized hepatic (WIF-B) cells.
In this work, WIF-B cultures were treated with pyrazole (inducer of cytochrome P4502E1, CYP2E1) and/or L-buthionine sulfoximine (BSO), a known inhibitor of hepatic glutathione (GSH), followed by evaluation of ROS production, antioxidant levels, and measures of cell injury (apoptosis and necrosis).
The results revealed that ethanol treatment alone caused a significant two-fold increase in the activation of caspase-3 as well as a similar doubling in ROS. When the activity of the CYP2E1 was increased by pyrazole pretreatment, an additional two-fold elevation in ROS was detected. However, the CYP2E1-related ROS elevation was not accompanied with a correlative increase in apoptotic cell injury, but rather was found to be associated with an increase in necrotic cell death. Interestingly, when the thiol status of the cells was manipulated using BSO, the ethanol-induced activation of caspase-3 was abrogated. Additionally, ethanol-treated cells displayed enhanced susceptibility to Fas-mediated apoptosis that was blocked by GSH depletion as a result of diminished caspase-8 activity.
Apoptotic cell death induced as a consequence of ethanol metabolism is not completely dependent upon ROS status but is dependent on sustained GSH levels.
World Journal of Gastroenterology 07/2009; 15(21):2609-16. · 2.47 Impact Factor
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ABSTRACT: Although alcoholic liver disease is clinically well-described, the molecular basis for alcohol-induced hepatotoxicity is not well understood. Previously, we determined that the clathrin-mediated internalization of asialoglycoprotein receptor was impaired in ethanol-treated WIF-B cells whereas the internalization of a glycophosphatidylinositol-anchored protein thought to be endocytosed via a caveolae/raft-mediated pathway was not changed suggesting that clathrin-mediated endocytosis is selectively impaired by ethanol. To test this possibility, we examined the internalization of a panel of proteins and compounds internalized by different mechanisms in control and ethanol-treated WIF-B cells. We determined that the internalization of markers known to be internalized via clathrin-mediated mechanisms was impaired. In contrast, the internalization of markers for caveolae/raft-mediated endocytosis, fluid phase internalization or non-vesicle-mediated uptake was not impaired in ethanol-treated cells. We further determined that clathrin heavy chain accumulated at the basolateral surface in small puncta in ethanol-treated cells while there was decreased dynamin-2 membrane association. Interestingly, the internalization of resident apical proteins that lack any known internalization signals was also disrupted by ethanol suggesting that these proteins are internalized via clathrin-mediated mechanisms. This conclusion is consistent with our findings that dominant negative dynamin-2 overexpression impaired internalization of known clathrin markers and single spanning apical residents, but not of markers of fluid phase or raft-mediated internalization. Together these results indicate that ethanol exposure selectively impairs hepatic clathrin-mediated internalization by preventing vesicle fission from the plasma membrane.
Biochemical pharmacology 06/2009; 78(6):648-55. · 4.25 Impact Factor
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ABSTRACT: The effect of relaxin administration before (prevention) or after (treatment) the establishment of hepatic fibrosis in a mouse model was examined. In the prevention study, relaxin reduced collagen and smooth muscle actin content and significantly reduced serum levels of the liver enzymes alanine aminotransferase and aspartate aminotransferase. In the treatment study, administration of relaxin for 1 week reduced collagen and smooth muscle actin but not liver enzyme levels. Relaxin administered for 2 weeks had no significant effect. In conclusion, the data suggest that relaxin treatment before fibrosis can reduce collagen and improve liver function but that there is little effect of short-term relaxin treatment after fibrosis is established.
Annals of the New York Academy of Sciences 05/2009; 1160:348-9. · 3.15 Impact Factor
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ABSTRACT: 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 10(6) 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 microg/ml and 1092 microg/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.
International Immunopharmacology 09/2008; 8(8):1112-8. · 2.38 Impact Factor
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ABSTRACT: Alcohol abuse results in liver injury, but investigations into the mechanism(s) for this injury have been hampered by the lack of appropriate in vitro culture models in which to conduct in depth and specific studies. In order to overcome these shortcomings, we have developed the use of precision-cut liver slices (PCLS) as an in vitro culture model in which to investigate how ethanol causes alcohol-induced liver injury. In these studies, it was shown that the PCLS retained excellent viability as determined by lactate dehydrogenase and adenosine triphosphate (ATP) levels over a 96-h period of incubation. More importantly, the major enzymes of ethanol detoxification; alcohol dehydrogenase, aldehyde dehydrogenase, and cytochrome P4502E1, remained active and PCLS readily metabolized ethanol and produced acetaldehyde. Within 24 h and continuing up to 96h the PCLS developed fatty livers and demonstrated an increase in the redox state. These PCLS secreted albumin, and albumin secretion was decreased by ethanol treatment. All of these impairments were reversed following the addition of 4-methylpyrazole, which is an inhibitor of ethanol metabolism. Therefore, this model system appears to mimic the ethanol-induced changes in the liver that have been previously reported in human and animal studies, and may be a useful model for the study of alcoholic liver disease.
Biochemical pharmacology 09/2008; 76(3):426-36. · 4.25 Impact Factor
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ABSTRACT: We have been using polarized, hepatic WIF-B cells to examine ethanol-induced liver injury. These cells polarize in culture and maintain numerous liver-specific activities including the ability to metabolize alcohol. Previously, we found that microtubules were more highly acetylated and more stable in ethanol-treated WIF-B cells and that increased microtubule acetylation required ethanol metabolism and was likely mediated by acetaldehyde. This study was aimed at identifying the mechanism responsible for increased microtubule acetylation. We examined the expression of two known microtubule deacetylases, histone deacetylase 6 (HDAC6) and Sirtuin T2 (SirT2), in WIF-B cells. Immunoblotting, immunofluorescence microscopy, and assays using the SirT2 inhibitor nicotinamide revealed that WIF-B cells do not express SirT2. In contrast, HDAC6 was highly expressed in WIF-B cells. Addition of trichostatin A (TSA), an HDAC6 inhibitor, induced microtubule acetylation to the same extent as in ethanol-treated cells (approximately threefold). Although immunofluorescence labeling revealed that HDAC6 distribution did not change in ethanol-treated cells, immunoblotting showed HDAC6 protein levels slightly decreased. HDAC6 solubility was increased in nocodazole-treated cells, suggesting impaired microtubule binding. Direct microtubule binding assays confirmed this hypothesis. The decreased microtubule binding was partially prevented by 4-methyl pyrazole, indicating the effect was in part mediated by acetaldehyde. Interestingly, HDAC6 from ethanol-treated cells was able to bind and deacetylate exogenous tubulin to the same extent as control, suggesting that ethanol-induced tubulin modifications prevented HDAC6 binding to endogenous microtubules. CONCLUSION: We propose that lower HDAC6 levels combined with decreased microtubule binding lead to increased tubulin acetylation in ethanol-treated cells.
Hepatology 07/2008; 48(5):1671-9. · 11.66 Impact Factor
