Oxidative mechanisms in the pathogenesis of alcoholic liver disease

Although the capacity of ethanol to induce oxidative stress in the liver is well established, the mechanisms by which oxidative damage contributes to the pathogenesis of alcoholic liver disease (ALD) is still incompletely understood. Recent reports have implicated oxidative mechanisms in the onset of alcoholic steatosis and in the formation of Mallory's bodies. Moreover, by inducing mitochondrial alterations, oxidative stress promotes hepatocyte necrosis and contributes to alcohol-induced sensitization of hepatocyte to the pro-apoptotic action of TNF-alpha. Oxidative mechanisms play also a role in the progression of liver fibrosis by triggering the release of pro-fibrotic cytokines and activating collagen gene expression in hepatic stellate cells. Finally, immune responses towards antigens originating from the reactions of lipid peroxidation products with hepatic proteins might represent one of the mechanisms that contribute to perpetuate chronic hepatic inflammation in ALD. Altogether these observations give a rationale to the possible clinical application of antioxidants in the therapy of ALD.     

Recent advances in alcoholic liver disease III. Role of the innate immune response in alcoholic hepatitis

Ethanol consumption is known to cause significant acute liver damage resulting in hepatic fibrosis and eventual cirrhosis when consumed chronically. The mechanism(s) by which ethanol exerts its damaging effects on the liver are not well understood; however, recent scientific investigation has begun to delineate the earliest events in alcoholic liver disease. From these studies, it is apparent that components of the innate immune system and, in particular, Kupffer cells, play a significant role in this process. It is also becoming clear that other parts of the immune system including T cells may also be responsible for mediating the devastating effects of chronic alcohol consumption on the liver. This review will highlight recent experiments demonstrating a role for the innate immune response in the initiation and progression of alcohol-induced liver hepatitis and subsequent organ damage.     

Identification of oxidized mitochondrial proteins in alcohol-exposed human hepatoma cells and mouse liver

Heavy alcohol consumption can damage various cells and organs partly through production of reactive oxygen species (ROS) and mitochondrial dysfunction. Treatment with antioxidants can significantly reduce the degree of damage. Despite well established roles of ROS in alcohol-induced cell injury, the proteins that are selectively oxidized by ROS are poorly characterized. We hypothesized that certain cysteinyl residues of target proteins are oxidized by ROS upon alcohol exposure, and these modified proteins may play roles in mitochondrial dysfunction. A targeted proteomics approach utilizing biotin-N-maleimide (biotin-NM) as a specific probe to label oxidized cysteinyl residues was employed to investigate which mitochondrial proteins are modified during and after alcohol exposure. Human hepatoma HepG2 cells with transduced CYP2E1 (E47 cells) were used as a model to generate ROS through CYP2E1-mediated ethanol metabolism. Following exposure to 100 mM ethanol for 4 and 8 h, the biotin-NM-labeled oxidized proteins were purified with agarose coupled to either streptavidin or monoclonal antibody against biotin. The purified proteins were resolved by two-dimensional gel electrophoresis and protein spots that displayed differential abundances were excised from the gel, in-gel digested with trypsin and analyzed for identity utilizing either matrix-assisted laser desorption-time of flight mass spectrometry or microcapillary reversed-phase liquid chromatography-tandem mass spectrometry. The results demonstrate that heat shock protein 60, protein disulfide isomerase, mitochondrial aldehyde dehydrogenases, prohibitin, and other proteins were oxidized after alcohol exposure. The identity of some of the proteins purified with streptavidin-agarose was also confirmed by immunoblot analyses using the specific antibody to each target protein. This method was also used to identify oxidized mitochondrial proteins in the alcohol-fed mouse liver. These results suggest that exposure to ethanol causes oxidation of various mitochondrial proteins that may negatively affect their function and contribute to alcohol-induced mitochondrial dysfunction and cellular injury.     

Protein targeting to destinations of the secretory pathway in the malaria parasite Plasmodium falciparum

The secretory pathway in the malaria parasite Plasmodium falciparum has many unique aspects in terms of protein destinations and trafficking mechanisms. Recently, several exciting insights into protein trafficking within this intracellular parasite have been unveiled: these include signals that are required for targeting of proteins to the red blood cell and the relict plastid (known as the apicoplast); and the elucidation of the pathways of the haemoglobin proteases targeted to the food vacuole. Protein-targeting to the apical organelles in P. falciparum, however, is still not very well understood, but available research offers a tantalising glimpse of the system.     

Recent Advances in Alcoholic Liver Disease I. Role of intestinal permeability and endotoxemia in alcoholic liver disease

A significant body of evidence indicates that endotoxemia and endotoxin-mediated hepatocellular damage play a crucial role in the pathogenesis of alcoholic liver disease. A close correlation between endotoxemia and the severity of alcohol-induced liver injury is supported by a number of clinical and experimental studies. Elevated intestinal permeability appears to be the major factor involved in the mechanism of alcoholic endotoxemia and the pathogenesis of alcoholic liver disease. Ethanol and its metabolic derivatives, acetaldehyde in particular, alter intracellular signal-transduction pathways leading to the disruption of epithelial tight junctions and an increase in paracellular permeability to macromolecules. Studies addressing the mechanisms of such epithelial disruption and the protective factors that prevent ethanol and acetaldehyde-mediated disruption of epithelial tight junctions are critically important in the investigations toward the search of preventive and therapeutic strategies for alcoholic liver disease.     

A dynamic plasma membrane proteome analysis of alcohol-induced liver cirrhosis

ABSTRACT: Alcohol-induced injury has become one of the major causes for liver cirrhosis. However, the molecular mechanisms of ethanol-induced injury are not fully understood. To this end, we performed a dynamic plasma membrane proteomic research on rat model. A rat model from hepatitis to liver cirrhosis was developed. Plasma membrane from liver tissue with liver fibrosis stage of 2 and 4 (S2 and S4) was purified by sucrose density gradient centrifugation. Its purification was verified by western blotting. Proteins from plasma membrane were separated by two-dimensional electrophoresis (2DE) and differentially expressed proteins were identified by tandem mass spectrometry. 16 consistent differentially expressed proteins from S2 to S4 were identified by mass spectrometry. The expression of differentially expressed proteins annexin A6 and annexin A3 were verified by western blotting, and annexin A3 was futher verified by immunohistochemistry. Our research suggests a possible mechanism by which ethanol alters protein expression to enhance the liver fibrosis progression. These differentially expressed proteins might be new drug targets for treating alcoholic liver cirrhosis.     

Differential protein expression profiles in the hippocampus of human alcoholics

Mild to severe cognitive impairments are frequently observed symptoms in chronic alcoholics. Decline of cognitive function significantly affects patients' recovery process and prognosis. The hippocampal region is sensitive to the effects of alcohol and it has been suggested that alcohol-induced hippocampal damage and/or changes in neuronal circuitry play an important role in generating these symptoms. Although various hypotheses have been proposed, molecular mechanisms underlying these alterations in the hippocampus are largely unknown. In the present study, we employed a 2DE-based proteomics approach to compare the protein expression profiles of the hippocampus in human alcoholic and healthy control brains. In the alcoholic hippocampus, 20 protein spots were found to be differentially regulated, 2 increased and 18 decreased. Seventeen proteins were identified using mass spectroscopy and were subcategorized into three energy metabolic proteins, six protein metabolic proteins, four signalling proteins, two oxidative stress-related proteins, one vesicle trafficking protein and one cytoskeletal protein. Some of these proteins have been previously implicated in alcohol-induced brain pathology. Based upon the results, several hypotheses were generated to explain the mechanisms underlying possible functional and/or structural alterations induced by chronic alcohol use in this brain region.     

Interactions between an inflammatory response to infection and protein trafficking pathways favor correction of defective protein trafficking in Cystic Fibrosis

One unresolved issue in Cystic Fibrosis research is how functional loss of CFTR, a protein involved in chloride transport, results in chronic lung inflammation. Large scale experiments investigating protein or gene expression changes due to altered trafficking of the most common disease causing CFTR mutation (ΔF508) have produced long lists of changes with no apparent connection to inflammation. Likewise, experiments documenting the effects of inflammation in bronchial epithelial cell lines have yielded no insights into CFTR trafficking. We used MetaMiner CF to combine and analyze results of several CFTR trafficking and epithelial response to infection studies which were on different platforms using different methodologies and had different objectives. The program searches a manually curated database for published experiments linking proteins or genes and displays the interactions in a more easily understood graphic format. Numerous connections were established between genes documented to correct ΔF508 trafficking and a list of genes differentially expressed in bronchial epithelial cells after exposure to bacteria or virus. Of 34 genes documented to correct ΔF508 trafficking, 9 were directly linked by positive expression activation mechanisms to the immune inflammatory response. Looking at interactions among the results as a whole and in detail, it is apparent that an inflammatory response produces numerous changes which favor correct trafficking of ΔF508. One can take a view of the inflammatory process as potentially a corrective mechanism for dysfunctional ΔF508 trafficking. This opens up a new research direction and provides new targets in the search for disease treatments.     

Increased Sensitivity to Binge Alcohol-Induced Gut Leakiness and Inflammatory Liver Disease in HIV Transgenic Rats

The mechanisms of alcohol-mediated advanced liver injury in HIV-infected individuals are poorly understood. Thus, this study was aimed to investigate the effect of binge alcohol on the inflammatory liver disease in HIV transgenic rats as a model for simulating human conditions. Female wild-type (WT) or HIV transgenic rats were treated with three consecutive doses of binge ethanol (EtOH) (3.5 g/kg/dose oral gavages at 12-h intervals) or dextrose (Control). Blood and liver tissues were collected at 1 or 6-h following the last dose of ethanol or dextrose for the measurements of serum endotoxin and liver pathology, respectively. Compared to the WT, the HIV rats showed increased sensitivity to alcohol-mediated gut leakiness, hepatic steatosis and inflammation, as evidenced with the significantly elevated levels of serum endotoxin, hepatic triglycerides, histological fat accumulation and F4/80 staining. Real-time PCR analysis revealed that hepatic levels of toll-like receptor-4 (TLR4), leptin and the downstream target monocyte chemoattractant protein-1 (MCP-1) were significantly up-regulated in the HIV-EtOH rats, compared to all other groups. Subsequent experiments with primary cultured cells showed that both hepatocytes and hepatic Kupffer cells were the sources of the elevated MCP-1 in HIV-EtOH rats. Further, TLR4 and MCP-1 were found to be upregulated by leptin. Collectively, these results show that HIV rats, similar to HIV-infected people being treated with the highly active anti-retroviral therapy (HAART), are more susceptible to binge alcohol-induced gut leakiness and inflammatory liver disease than the corresponding WT, possibly due to additive or synergistic interaction between binge alcohol exposure and HIV infection. Based on these results, HIV transgenic rats can be used as a surrogate model to study the molecular mechanisms of many disease states caused by heavy alcohol intake in HIV-infected people on HAART.     

Metallothionein protection against alcoholic liver injury through inhibition of oxidative stress

Antioxidants are likely potential pharmaceutical agents for the treatment of alcoholic liver disease. Metallothionein (MT) is a cysteine-rich protein and functions as an antioxidant. This study was designed to determine whether MT confers resistance to acute alcohol-induced hepatotoxicity and to explore the mechanistic link between oxidative stress and alcoholic liver injury. MT-overexpressing transgenic and wild-type mice were administrated three gastric doses of alcohol at 5 g/kg. Liver injury, oxidative stress, and ethanol metabolism-associated changes were determined. Acute ethanol administration in the wild-type mice caused prominent microvesicular steatosis, along with necrosis and elevation of serum alanine aminotransferase. Ultrastructural changes of the hepatocytes include glycogen and fat accumulation, organelle abnormality, and focal cytoplasmic degeneration. This acute alcohol hepatotoxicity was significantly inhibited in the MT-transgenic mice. Furthermore, ethanol treatment decreased hepatic-reduced glutathione, but increased oxidized glutathione along with lipid peroxidation, protein oxidation, and superoxide generation in the wild-type mice. This hepatic oxidative stress was significantly suppressed in the MT-transgenic mice. However, MT did not affect the ethanol metabolism-associated decrease in NAD(+)/NADH ratio or increase in cytochrome P450 2E1. In conclusion, MT is an effective agent in cytoprotection against alcohol-induced liver injury, and hepatic protection by MT is likely through inhibition of alcohol-induced oxidative stress.     

Protein trafficking mechanisms associated with neurite outgrowth and polarized sorting in neurons.

Neuronal differentiation in vitro and in vivo involves coordinated changes in the cellular cytoskeleton and protein trafficking processes. I review here recent progress in our understanding of the membrane trafficking aspects of neurite outgrowth of neurons in culture and selective microtubule-based polarized sorting in fully polarized neurons, focusing on the involvement of some key molecules. Early neurite outgrowth appears to involve the protein trafficking machineries that are responsible for constitutive trans-Golgi network (TGN) to plasma membrane exocytosis, utilizing transport carrier generation mechanisms, SNARE proteins, Rab proteins and tethering mechanisms that are also found in non-neuronal cells. This vectorial TGN-plasma membrane traffic is directed towards several neurites, but can be switch to concentrate on the growth of a single axon. In a mature neuron, polarized targeting to the specific axonal and dendritic domains appears to involve selective microtubule-based mechanisms, utilizing motor proteins capable of distinguishing microtubule tracks to different destinations. The apparent gaps in our knowledge of these related protein transport processes will be highlighted.     

Proteome map of the normal murine ventricular myocardium

Cardiovascular disease is the leading cause of morbidity and mortality in developed countries. The underlying mechanisms involved in cardiac dysfunction and heart failure are poorly understood. In this study, 2-DE was utilised to map, for the first time, proteins of normal, nonfailing mouse ventricular tissues to form a basis for future comparative analysis of mouse models with cardiovascular disorders. Proteins were obtained from ventricles of C57BL6 mice, aged 18 wk, and separated by 2-DE. A total of 150 protein spots, corresponding to 77 distinct proteins, were identified by MALDI-TOF MS. The proteins identified in mouse ventricles covered a wide range of biological processes (e.g. cell cycle, muscle contraction and signal transduction), with the majority of proteins contributing to cardiomyocyte energetics and cell structure. This 2-D gel map of mouse myocardial proteins will be an invaluable tool in proteomic research for the detection of protein changes and identifying cardiac biomarkers of cardiovascular disease.     

MAP kinase signaling in diverse effects of ethanol

Chronic ethanol abuse is associated with liver injury, neurotoxicity, hypertension, cardiomyopathy, modulation of immune responses and increased risk for cancer, whereas moderate alcohol consumption exerts protective effect on coronary heart disease. However, the signal transduction mechanisms underlying these processes are not well understood. Emerging evidences highlight a central role for mitogen activated protein kinase (MAPK) family in several of these effects of ethanol. MAPK signaling cascade plays an essential role in the initiation of cellular processes such as proliferation, differentiation, development, apoptosis, stress and inflammatory responses. Modulation of MAPK signaling pathway by ethanol is distinctive, depending on the cell type; acute or chronic; normal or transformed cell phenotype and on the type of agonist stimulating the MAPK. Acute exposure to ethanol results in modest activation of p42/44 MAPK in hepatocytes, astrocytes, and vascular smooth muscle cells. Acute ethanol exposure also results in potentiation or prolonged activation of p42/44MAPK in an agonist selective manner. Acute ethanol treatment also inhibits serum stimulated p42/44 MAPK activation and DNA synthesis in vascular smooth muscle cells. Chronic ethanol treatment causes decreased activation of p42/44 MAPK and inhibition of growth factor stimulated p42/44 MAPK activation and these effects of ethanol are correlated to suppression of DNA synthesis, impaired synaptic plasticity and neurotoxicity. In contrast, chronic ethanol treatment causes potentiation of endotoxin stimulated p42/44 MAPK and p38 MAPK signaling in Kupffer cells leading to increased synthesis of tumor necrosis factor. Acute exposure to ethanol activates pro-apoptotic JNK pathway and anti-apoptotic p42/44 MAPK pathway. Apoptosis caused by chronic ethanol treatment may be due to ethanol potentiation of TNF induced activation of p38 MAPK. Ethanol induced activation of MAPK signaling is also involved in collagen expression in stellate cells. Ethanol did not potentiate serum stimulated or Gi-protein dependent activation of p42/44 MAPK in normal hepatocytes but did so in embryonic liver cells and transformed hepatocytes leading to enhanced DNA synthesis. Ethanol has a 'triangular effect' on MAPK that involve direct effects of ethanol, its metabolically derived mediators and oxidative stress. Acetaldehyde, phosphatidylethanol, fatty acid ethyl ester and oxidative stress, mediate some of the effects seen after ethanol alone whereas ethanol modulation of agonist stimulated MAPK signaling appears to be mediated by phosphatidylethanol. Nuclear MAPKs are also affected by ethanol. Ethanol modulation of nuclear p42/44 MAPK occurs by both nuclear translocation of p42/44 MAPK and its activation in the nucleus. Of interest is the observation that ethanol caused selective acetylation of Lys 9 of histone 3 in the hepatocyte nucleus. It is plausible that ethanol modulation of cross talk between phosphorylation and acetylations of histone may regulate chromatin remodeling. Taken together, these recent developments place MAPK in a pivotal position in relation to cellular actions of ethanol. Furthermore, they offer promising insights into the specificity of ethanol effects and pharmacological modulation of MAPK signaling. Such molecular signaling approaches have the potential to provide mechanism-based therapy for the management of deleterious effects of ethanol or for exploiting its beneficial effects.     

Zinc prevention and treatment of alcoholic liver disease

Alcoholic liver disease (ALD) is associated with decreases in zinc (Zn) and its major binding protein, metallothionein (MT), in the liver. Studies using animal models have shown that Zn supplementation prevents alcohol-induced liver injury under both acute and chronic alcohol exposure conditions. There are hepatic and extrahepatic actions of Zn in the prevention of alcoholic liver injury. Zn supplementation attenuates ethanol-induced hepatic Zn depletion and suppresses ethanol-elevated cytochrome P450 2E1 (CYP2E1) activity, but increases the activity of alcohol dehydrogenase in the liver; an action that is likely responsible for Zn suppression of alcohol-induced oxidative stress. Zn also enhances glutathione-related antioxidant capacity in the liver. At the cellular level, Zn inhibits alcohol-induced hepatic apoptosis partially through suppression of the Fas/FasL-mediated pathway. Zn supplementation preserves intestinal integrity and prevents endotoxemia, leading to inhibition of endotoxin-induced tumor necrosis factor-alpha (TNF-alpha) production in the liver. Zn also directly inhibits the signaling pathway involved in endotoxin-induced TNF-alpha production. These hepatic and extrahepatic effects of Zn are independent of MT. However, low levels of MT in the liver sensitize the organ to alcohol-induced injury, and elevation of MT enhances the endogenous Zn reservoir and makes Zn available when oxidative stress is imposed. Zn has a high potential to be developed as an effective agent in the prevention and treatment of ALD.     

Ethanol induced teratogenesis: characterization, mechanisms and diagnostic approaches

Extensive research has been aimed at characterising FAS and FAE. Whereas the symptomology for FAS has been established, that of FAE remains to be fully characterized. Various mechanisms of ethanol induced teratogenesis have been proposed however it remains to be defined how these mechanisms combine to produce the entire constellation of teratogenic characteristics observed. At present, impaired placental transport, abnormal muscle organogenesis and fetal hypoxia have limitations in explaining the entire spectrum of defects in FAS. The role of prostaglandins and hormones requires further research. Also, other as yet unidentified mechanisms may exist. Even if the composite mechanism can be established, ethanol effects in utero will likely not be preventable by any approach other than consumption modification. In light of this notion, future research into identifying high-risk pregnant drinkers for clinical intervention is emphasized. Intervention leading to abstinence or, if this is not possible, removing the infant as early as possible from an intrauterine environment that is causing growth retardation and fetal distress, are the only solutions available at present.     

Ethanol-induced iron mobilization: role of acetaldehyde-aldehyde oxidase generated superoxide

Superoxide radicals, a species known to mobilize ferritin iron, and their interaction with catalytic iron have been implicated in the pathogenesis of alcohol-induced liver injury. The mechanism(s) by which ethanol metabolism generates free radicals and mobilizes catalytic iron, however, is not fully defined. In this investigation the role of hepatic aldehyde oxidase in the mobilization of catalytic iron from ferritin was studied in vitro. Iron mobilization due to the metabolism of ethanol to acetaldehyde by alcohol dehydrogenase was increased 100% by the addition of aldehyde oxidase. Iron release was favored by low pH and low oxygen concentration. Mobilization of iron due to acetaldehyde metabolism by aldehyde oxidase was completely inhibited by superoxide dismutase but not by catalase suggesting that superoxide radicals mediate mobilization. Acetaldehyde-aldehyde oxidase mediated reduction of ferritin iron was facilitated by incubation with menadione, an electron acceptor for aldehyde oxidase. Mobilization of ferritin iron due to the metabolism of acetaldehyde by aldehyde oxidase may be a fundamental mechanism of alcohol-induced liver injury.     

Protein solubility and folding enhancement by interaction with RNA

While basic mechanisms of several major molecular chaperones are well understood, this machinery has been known to be involved in folding of only limited number of proteins inside the cells. Here, we report a chaperone type of protein folding facilitated by interaction with RNA. When an RNA-binding module is placed at the N-terminus of aggregation-prone target proteins, this module, upon binding with RNA, further promotes the solubility of passenger proteins, potentially leading to enhancement of proper protein folding. Studies on in vitro refolding in the presence of RNA, coexpression of RNA molecules in vivo and the mutants with impaired RNA binding ability suggests that RNA can exert chaperoning effect on their bound proteins. The results suggest that RNA binding could affect the overall kinetic network of protein folding pathway in favor of productive folding over off-pathway aggregation. In addition, the RNA binding-mediated solubility enhancement is extremely robust for increasing soluble yield of passenger proteins and could be usefully implemented for high-throughput protein expression for functional and structural genomic research initiatives. The RNA-mediated chaperone type presented here would give new insights into de novo folding in vivo.