This paper is based upon the “Charles Lieber Satellite Symposia” organized by Manuela G. Neuman at the Research Society on Alcoholism (RSA) Annual Meetings, 2013 and 2014. The present review includes pre-clinical, translational and clinical research that characterize alcoholic liver disease (ALD) and non-alcoholic steatohepatitis (NASH). In addition, a literature search in the discussed area was performed. Strong clinical and experimental evidence lead to recognition of the key toxic role of alcohol in the pathogenesis of ALD. The liver biopsy can confirm the etiology of NASH or alcoholic steatohepatitis (ASH) and assess structural alterations of cells, their organelles, as well as inflammatory activity. Three histological stages of ALD are simple steatosis, ASH, and chronic hepatitis with hepatic fibrosis or cirrhosis. These latter stages may also be associated with a number of cellular and histological changes, including the presence of Mallory's hyaline, megamitochondria, or perivenular and perisinusoidal fibrosis. Genetic polymorphisms of ethanol metabolizing enzymes such as cytochrome p450 (CYP) 2E1 activation may change the severity of ASH and NASH. Alcohol mediated hepatocarcinogenesis, immune response to alcohol in ASH, as well as the role of other risk factors such as its comorbidities with chronic viral hepatitis in the presence or absence of human deficiency virus are discussed. Dysregulation of hepatic methylation, as result of ethanol exposure, in hepatocytes transfected with hepatitis C virus (HCV), illustrates an impaired interferon signaling. The hepatotoxic effects of ethanol undermine the contribution of malnutrition to the liver injury. Dietary interventions such as micro and macronutrients, as well as changes to the microbiota are suggested. The clinical aspects of NASH, as part of metabolic syndrome in the aging population, are offered. The integrative symposia investigate different aspects of alcohol-induced liver damage and possible repair. We aim to (1) determine the immuno-pathology of alcohol-induced liver damage, (2) examine the role of genetics in the development of ASH, (3) propose diagnostic markers of ASH and NASH, (4) examine age differences, (5) develop common research tools to study alcohol-induced effects in clinical and pre-clinical studies, and (6) focus on factors that aggravate severity of organ-damage. The intention of these symposia is to advance the international profile of the biological research on alcoholism. We also wish to further our mission of leading the forum to progress the science and practice of translational research in alcoholism.

Maternal diet can affect fetal gene expression through epigenetic mechanisms. Wilson disease (WD), which is caused by autosomal recessive mutations in ATP7B encoding a biliary copper transporter, is characterized by excessive hepatic copper accumulation, but variability in disease severity. We tested the hypothesis that gestational supply of dietary methyl groups modifies fetal DNA methylation and expression of genes involved in methionine and lipid metabolism that are impaired prior to hepatic steatosis in the toxic milk (tx-j) mouse model of WD. Female C3H control and tx-j mice were fed control (choline 8 mmol/Kg of diet) or choline-supplemented (choline 36 mmol/Kg of diet) diets for 2 weeks throughout mating and pregnancy to gestation day 17. A second group of C3H females, half of which were used to cross foster tx-j pups, received the same diet treatments that extended during lactation to 21 d postpartum. Compared with C3H, fetal tx-j livers had significantly lower copper concentrations and significantly lower transcript levels of Cyclin D1 and genes related to methionine and lipid metabolism. Maternal choline supplementation prevented the transcriptional deficits in fetal tx-j liver for multiple genes related to cell growth and metabolism. Global DNA methylation was increased by 17% in tx-j fetal livers after maternal choline treatment (P<0.05). Maternal dietary choline rescued the lower body weight of 21 d tx-j mice. Our results suggest that WD pathogenesis is modified by maternal in utero factors, including dietary choline.

We previously reported the mechanisms involved in the formation of Mallory-Denk bodies (MDBs) in mice fed DDC. To further provide clinical evidence as to how ubiquitin-like protein (Ubls) modification, gene transcript expression in Ufmylation and FATylation were investigated in human archived formalin-fixed, paraffin-embedded (FFPE) liver biopsies and frozen liver sections from DDC re-fed mice were used. Real-time PCR analysis showed that all Ufmylation molecules (Ufm1, Uba5, Ufc1, Ufl1 and UfSPs) were significantly down regulated, both in DDC re-fed mice livers and patients’ livers where MDBs had formed, indicating that gene transcript changes were limited to MDB-forming livers where the protein quality control system was down regulated. FAT10 and subunits of the immunoproteasome (LMP2 and LMP7) were both up regulated as previously shown. An approximate 176- and 5-fold up regulation (respectively) of FAT10 were observed in the DDC re-fed mice liver and in the livers of human alcoholic hepatitis with MDBs present, implying that there was an important role played by this gene. The FAT10-specific E1 and E2 enzymes Uba6 and USE1, however, were found to be down regulated both in patients’ livers and in the liver of DDC re-fed mice. Interestedly, the down regulation of mRNA levels was proportionate to MDB abundance in the liver tissues. Our results show the first systematic demonstration of transcript regulation of Ufmylation and FATylation in the liver of patients who form MDBs, where protein quality control is down regulated. This was also shown in livers of DDC re-fed mice where MDBs had formed.

There is a need for a nontoxic antioxidant agent to be identified which will prevent alcoholic liver disease (ALD) in alcoholic patients. We tested 4 candidate agents: quercetin, EGCG, catechin and betaine, all of which occur naturally in food. HepG2 cells over expressing CYP2E1 were subjected to arachidonic acid, iron and 100 mM ethanol with or without the antioxidant agent. All the agents prevented oxidative stress and MDA/4HNE formation induced by ethanol, except for EGCG. Catechin prevented CYP2E1 induction by ethanol. All the agents tended to down regulate the ethanol-induced increased expression of glutathionine peroxidase 4 (GPX4). All the agents, except catechin, tended to reduce the expression of SOD2 induced by ethanol. Heat shock protein 70 was up regulated by ethanol alone and betaine tended to prevent this. All 4 agents down regulated the expression of Gadd45b in the presence of ethanol, which could explain the mechanism of DNA demethylation associated with the up regulation of the gene expression observed in experimental ALD. In conclusion, the in vitro model of oxidative stress induced by ethanol provided evidence that all 4 agents tested prevented some aspect of liver cell injury caused by ethanol.

Activation of Toll-like receptor (TLR) signaling which stimulates inflammatory and proliferative pathways is the key element in the pathogenesis of Mallory-Denk bodies (MDBs) in mice fed DDC. However, little is known as to how TLR signaling is regulated in MDB formation during chronic liver disease development. The first systematic study of TLR signaling pathway transcript regulation in human archived formalin-fixed, paraffin-embedded (FFPE) liver biopsies with MDB formation is presented here. When compared to the activation of Toll-like signaling in alcoholic hepatitis (AH) and Non alcoholic steatohepatitis (NASH) patients, striking similarities and obvious differences were observed. Similar TLRs (TLR3 and TLR4, etc.), TLR downstream adaptors (MyD88 and TRIF, etc.) and transcript factors (NFκB and IRF7, etc.) were all up regulated in the patients’ livers. MyD88, TLR3 and TLR4 were significantly induced in the livers of AH and NASH compared to normal subjects, while TRIF and IRF7 mRNA were only slightly up regulated in AH patients. This is a different pathway from the induction of the TLR4-MyD88-independent pathway in the AH and NASH patients with MDBs present. Importantly, chemokine receptor 4 and 7 (CXCR4/7) mRNAs were found to be induced in the patients livers in FAT10 positive hepatocytes. The CXCR7 pathway was significantly up regulated in patients with AH and the CXCR4 was markedly up regulated in patients with NASH, indicating that CXCR4/7 is crucial in liver MDB formation. This data constitutes the first demonstration of the up regulation of the MyD88-dependent TLR4/NFκB pathway in AH and NASH where MDBs formed, via the NFκB-CXCR4/7 pathway, and provides further insight into the mechanism of MDB formation in human liver diseases.

Promoter CpG island hypermethylation is an important mechanism for inactivating key cellular enzymes that mediate epigenetic processes in hepatitis-related hepatocellular carcinoma (HCC). The ubiquitin-fold modifiers 1 (Ufm1) conjugation pathway (Ufmylation) plays an essential role in protein degradation, protein quality control and signal transduction. Previous studies showed that the Ufmylation pathway was down regulated in alcoholic hepatitis (AH), non alcoholic steatohepatitis (NASH) and in mice fed DDC, resulting in the formation of Mallory-Denk bodies (MDBs). In this study, we further discovered that betaine, a methyl donor, fed together with DDC significantly prevents the increased expression of Ufmylation in drug-primed mice fed DDC. Betaine significantly prevented transcript silencing of Ufm1, Uba5 and UfSP1 where MDBs developed and also prevented the increased expression of FAT10 and LMP7 caused by DDC re-fed mice. Similar down regulation of Ufmylation was observed in multiple AH and NASH biopsies which had formed MDBs. The DNA methylation levels of Ufm1, Ufc1 and UfSP1 in the promoter CpG region were significantly increased both in AH and NASH patients compared to normal subjects. DNA (cytosine-5-)-methyltransferase 1 (DNMT1) and DNA (cytosine-5-)-methyltransferase 3 beta (DNMT3B) mRNA levels were markedly up regulated in AH and NASH patients, implying that the maintenance of Ufmylation methylation might be mediated by DNMT1 and DNMT3B together. These data show that MDB formation results from Ufmylation expression epigenetically in AH and NASH patients. Promoter CpG methylation may be a major mechanism silencing Ufmylation expression.

Chemokines and their receptors are involved in oncogenesis and in tumor progression, invasion, and metastasis. Various chemokines also promote cell proliferation and resistance to apoptosis of stressed cells. The chemokine CXCL8, also known as interleukin-8 (IL-8), is a proinflammatory molecule that has functions within the tumor microenvironment. Deregulation of IL-8 signaling is shown to play pivotal roles in tumorigenesis and progression. Mallory-Denk Bodies (MDBs) are prevalent in various liver diseases including alcoholic hepatitis (AH) and are formed in mice livers by feeding DDC. By comparing AH livers where MDBs had formed with normal livers, there were significant changes of IL-8 signaling by RNA sequencing (RNA-Seq) analyses. Real-time PCR analysis of CXCR2 further shows a 6-fold up regulation in AH livers and a 26-fold up regulation in the livers of DDC re-fed mice. IL-8 mRNA was also significantly up regulated in AH livers and DDC re-fed mice livers. This indicates that CXCR2 and IL-8 may be crucial for liver MDB formation. MDB containing balloon hepatocytes in AH livers had increased intensity of staining of the cytoplasm for both CXCR2 and IL-8. Over expression of IL-8 leads to an increase of the mitogen activated protein kinase (MAPK) cascade and exacerbates the inflammatory cycle. These observations constitute a demonstration of the altered regulation of IL-8 signaling in the livers of AH and mice fed DDC where MDBs formed, providing further insight into the mechanism of MDB formation mediated by IL-8 signaling in AH.

Mallory-Denk bodies (MDBs) are aggresomes composed of undigested ubiqutinated short lived proteins which have accumulated because of a decrease in the rate of their degradation by the 26s proteasome. The decrease in the activity of the proteasome is due to a shift in the activity of the 26s proteasome to the immunoproteasome triggered by an increase in expression of the catalytic subunits of the immunoproteasome which replaces the catalytic subunits of the 26s proteasome. This switch in the type of proteasome in liver cells is triggered by the binding of IFNγ to the IFNγ sequence response element (ISRE) located on the FAT10 promoter. To determine if either FAT10 or IFNγ are essential for the formation of MDBs we fed both IFNγ and FAT10 knock out (KO) mice DDC added to the control diet for 10 weeks in order to induce MDBs. Mice fed the control diet and Wild type mice fed the DDC or control diet were compared. MDBs were located by immunofluorescent double stains using antibodies to ubiquitin to stain MDBs and FAT10 to localize the increased expression of FAT10 in MDB forming hepatocytes. We found that MDB formation occurred in the IFNγ KO mice but not in the FAT10 KO mice. Western blots showed an increase in the ubiquitin smears and decreases β 5 (chymotrypsin-like 26S proteasome subunit) in the Wild type mice fed DDC but not in the FAT10 KO mice fed DDC. To conclude, we have demonstrated that FAT10 is essential to the induction of MDB formation in the DDC fed mice.