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This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Hidekazu Tsukamoto and Yoshiyuki Takei. The presentations were (1) Tribute to Professor Rajendar K. Chawla, by Craig J. McClain; (2) Dysregulated TNF signaling in alcoholic liver disease, by Craig J. McClain, S. Joshi-Barve, D. Hill, J Schmidt, I. Deaciuc, and S. Barve; (3) The role of mitochondria in ethanol-mediated sensitization of the liver, by Anna Colell, Carmen Garcia-Ruiz, Neil Kaplowitz, and Jose C. Fernandez-Checa; (4) A peroxisome proliferator (bezafibrate) can prevent superoxide anion release into hepatic sinusoid after acute ethanol administration, by Hirokazu Yokoyama, Yukishige Okamura, Yuji Nakamura, and Hiromasa Ishii; (5) S-adenosylmethionine affects tumor necrosis factor-α gene expression in macrophages, by Rajendar K. Chawla, S. Barve, S. Joshi-Barve, W. Watson, W. Nelson, and C. McClain; (6) Iron, retinoic acid and hepatic macrophage TNFα gene expression in ALD, by Hidekazu Tsukamoto, Min Lin, Mitsuru Ohata, and Kenta Motomura; and (7) Role of Kupffer cells and gut-derived endotoxin in alcoholic liver injury, by N. Enomoto, K. Ikejima, T. Kitamura, H. Oide, Y. Takei, M. Hirose, B. U. Bradford, C. A. Rivera, H. Kono, S. Peter, S. Yamashina, A. Konno, M. Ishikawa, H. Shimizu, N. Sato, and R. Thurman. [ABSTRACT FROM AUTHOR]

Background: Recently, we have demonstrated that acute alcohol exposure due to binge drinking leads to hepatic steatosis with the deregulation of hepatic histone deacetylase (HDAC) expression. Various class I, II, and IV HDACs were down-regulated, whereas expression of HDAC3 was solely up-regulated. Hence, in the present work, we specifically examined the mechanistic role of HDAC3 in the development of hepatic steatosis occurring in response to binge alcohol administration., Methods: C57BL/6 mice were gavaged 3 times with ethanol (EtOH) at a dose of 4.5 g/kg. HDAC inhibitor, Trichostatin A (TSA) was simultaneously injected intraperitoneally at a dose of 1 mg/kg. Hepatic steatosis, injury, expression of HDAC3 and carnitine palmitoyltransferase 1α (CPT1α) were evaluated. HDAC3 and histone H3 acetylation levels at the Cpt1α promoter were analyzed by chromatin immunoprecipitation (ChIP)., Results: The binge EtOH-mediated increase in HDAC3 was prevented by simultaneous administration of HDAC inhibitor, TSA, which markedly attenuated hepatic steatosis and injury. Importantly, HDAC3 inhibition was able to normalize the down-regulation of Cpt1α expression. Causal role of HDAC3 in the transcriptional repression of Cpt1α was demonstrated by increased HDAC3 binding at the thyroid receptor element site in the Cpt1α distal promoter region. Further, a resultant decrease in the transcriptionally permissive histone H3 lysine 9 acetylation in the proximal promoter region near the transcriptional start site was observed. Notably, TSA treatment reduced HDAC3 binding and increased H3K9 acetylation at Cpt1α promoter leading to increased Cpt1α expression. These molecular events resulted in attenuation of binge alcohol-induced hepatic steatosis., Conclusions: These findings provide insights into potential epigenetic mechanisms underlying transcriptional regulation of Cpt1α in the hepatic steatosis occurring in response to binge EtOH administration., (Copyright © 2013 by the Research Society on Alcoholism.)

Data from several laboratories have shown that ethanol (EtOH) feeding impairs many essential methylation reactions that contribute to alcoholic liver disease (ALD). EtOH is also a comorbid factor in the severity of hepatitis C virus-induced liver injury. The presence of viral proteins further exacerbates the methylation defects to disrupt multiple pathways that promote the pathogenesis of liver disease. This review is a compilation of presentations that linked the methylation reaction defects with proteasome inhibition, decreased antigen presentation, and impaired interferon (IFN) signaling in the hepatocytes and dysregulated TNFα expression in macrophages. Two therapeutic modalities, betaine and S-adenosylmethionine, can correct methylation defects to attenuate many EtOH-induced liver changes, as well as improve IFN signaling pathways, thereby overcoming viral treatment resistance., (Copyright © 2012 by the Research Society on Alcoholism.)

Background: Binge, as well as chronic, alcohol consumption affects global histone acetylation leading to changes in gene expression. It is becoming increasingly evident that these histone-associated epigenetic modifications play an important role in the development of alcohol-mediated hepatic injury., Methods: C57BL/6 mice were gavaged 3 times (12-hour intervals) with ethanol (EtOH; 4.5 g/kg). Hepatic histone deacetylase (Hdac) mRNAs were assessed by qRT-PCR. Total HDAC activity was estimated by a colorimetric HDAC activity/inhibition assay. Histone acetylation levels were evaluated by Western blot. Liver steatosis and injury were evaluated by histopathology, plasma aminotransferase (ALT) activity, and liver triglyceride accumulation. Expression of fatty acid synthase (Fas) and carnitine palmitoyl transferase 1a (Cpt1a) was also examined. HDAC 9 association with Fas promoter was analyzed., Results: Binge alcohol exposure resulted in alterations of hepatic Hdac mRNA levels. Down-regulation of HDAC Class I (Hdac 1), Class II (Hdac 7, 9, 10), and Class IV (Hdac 11) and up-regulation of HDAC Class I (Hdac 3) gene expression were observed. Correspondent to the decrease in HDAC activity, an increase in hepatic histone acetylation was observed. These molecular events were associated with microvesicular hepatic steatosis and injury characterized by increased hepatic triglycerides (48.02 ± 3.83 vs. 19.90 ± 3.48 mg/g liver, p < 0.05) and elevated plasma ALT activity (51.98 ± 6.91 vs. 20.8 ± 0.62 U/l, p < 0.05). Hepatic steatosis was associated with an increase in FAS and a decrease in CPT1a mRNA and protein expression. Fas promoter analysis revealed that binge EtOH treatment decreased HDAC 9 occupancy at the Fas promoter resulting in its transcriptional activation., Conclusions: Deregulation of hepatic Hdac expression likely plays a major role in the binge alcohol-induced hepatic steatosis and liver injury by affecting lipogenesis and fatty acid β-oxidation., (Copyright © 2012 by the Research Society on Alcoholism.)

Background: Alcohol abuse has long-term deleterious effects on the immune system, and results in a depletion and loss of function of CD4(+) T lymphocytes, which regulate both innate and adaptive immunity. T-lymphocyte activation via T-cell receptor (TCR) involves the lipid raft colocalization and aggregation of proteins into the immunological signalosome, which triggers a signaling cascade resulting in the production of interleukin-2 (IL-2). IL-2 regulates the proliferation and clonal expansion of activated T cells and is essential for an effective immune response. The present work examines the mechanisms underlying ethanol-induced dysfunction of CD4(+) T lymphocytes based on the hypothesis that ethanol downregulates lipid raft-mediated TCR signal transduction and resultant IL-2 production., Methods: Primary or cultured human T lymphocytes were exposed to ethanol for 24 hours prior to stimulation with anti-CD3/anti-CD28 antibodies or phytohemagglutinin. Effects of ethanol exposure on TCR-signaling (including activation of Lck, ZAP70, LAT, and PLCγ1) and IL-2 gene expression were examined., Results: Exposure of both primary and cultured human CD4(+) T lymphocytes to physiologically relevant concentrations of ethanol leads to down-regulation of IL-2 mRNA and protein via inhibition of DNA-binding activity of NFAT, the essential transcription factor for IL-2. Ethanol decreases tyrosine phosphorylation and activation of upstream signaling proteins PLCγ1, LAT, ZAP70, and Lck. These effects are prevented by inhibition of metabolism of ethanol. Sucrose density gradient fractionation and confocal microscopy revealed that ethanol inhibited essential upstream lipid raft-mediated TCR-dependent signaling events, namely colocalization of Lck, ZAP70, LAT, and PLCγ1 with plasma membrane lipid rafts., Conclusions: Overall, our data demonstrate that ethanol inhibits lipid raft-mediated TCR-signaling in CD4(+) T lymphocytes, resulting in suppression of IL-2 production. These findings may represent a novel mechanism underlying alcohol abuse-associated immune suppression and may be particularly relevant in diseases such as HIV/AIDS and hepatitis C virus infection where alcohol abuse is a known comorbidity., (Copyright © 2011 by the Research Society on Alcoholism.)

The ubiquitin-proteasome system has come to be known as a vital constituent of mammalian cells. The proteasome is a large nonlysosomal enzyme that acts in concert with an 8.5 kDa polypeptide called ubiquitin and a series of conjugating enzymes, known as E1, E2 and E3, that covalently bind multiple ubiquitin moieties in a polyubiquitin chain to protein substrates in a process called ubiquitylation. The latter process targets protein substrates for unfolding and degradation by the 26S proteasome. This enzyme system specifically recognizes and degrades polyubiquitylated proteins, many of which are key proteins involved in cell cycle regulation, apoptosis, signal transduction, and antigen presentation. The 26S proteasome contains a cylinder-shaped 20S catalytic core that, itself, degrades proteins in an ATP- and ubiquitin-independent manner. The 20S form is actually the predominant enzyme form in mammalian cells. Proteolysis by the constitutive 20S proteasome is vital in removing oxidized, misfolded and otherwise modified proteins. Such degradation is critical as a means of cellular detoxification, as intracellular accumulation of damaged and misfolded proteins is potentially lethal. Studies have shown that inhibition of proteasome activity can lead to cell death. Ethanol and its metabolism cause partial inhibition of the proteasome. This leads to a number of pleiotropic effects that can affect a variety of cellular processes. This critical review describes important aspects of ethanol metabolism and its influence on the proteasome. The review will summarize recent findings on: (1) the interactions between the proteasome and the ethanol metabolizing enzyme, CYP2E1; (2) the dynamics of proteasome inhibition by ethanol in animal models and cultured cells; (3) ethanol-elicited suppression of proteasome activity and its effect on signal transduction; (4) The role of proteasome inhibition in cytokine production by liver cells; and (5) ethanol elicited suppression of peptide hydrolysis and the potential effects on antigen presentation. While the principal focus is on alcohol-induced liver injury, the authors foresee that the findings presented in this review will prompt further research on the role of this proteolytic system in other tissues injured by excessive alcohol consumption.

Alcoholic liver disease (ALD) remains an important complication and cause of morbidity and mortality from alcohol abuse. Major developments in our understanding of the mechanisms of ALD over the past decade are now being translated into new forms of therapy for this disease process which currently has no FDA approved treatment. Cytokines are low molecular weight mediators of cellular communication, and the pro-inflammatory cytokine tumor necrosis factor (TNF) has been shown to play a pivotal role in the development of experimental ALD. Similarly, TNF levels are elevated in the serum of alcoholic hepatitis patients. Abnormal methionine metabolism is well documented in patients with ALD, with patients having elevated serum methionine levels, but low S-adenosylmethionine levels in the liver. On the other hand, S-adenosylhomocysteine and homocysteine levels are elevated in ALD. Recent studies have documented potential interactions between homocysteine and S-adenosylhomocysteine with TNF in the development of ALD. Altered proteasome function also is now well documented in ALD, and decreased proteasome function can cause hepatocyte apoptosis. Recently it has been shown that decreased proteasome function can also act synergistically to enhance TNF hepatotoxicity. Hepatocytes dying of proteasome dysfunction release pro-inflammatory cytokines such as Interleukin-8 to cause sustained inflammation. This article reviews the interactions of cytokines, altered methionine metabolism, and proteasome dysfunction in the development of ALD.

Background: Clinical and experimental studies have shown that an important deleterious consequence of excessive alcohol consumption is immunosuppression, specifically, a depletion in the mature CD4+ T-cell population. A predominant mechanism involved in T-cell depletion is activation-induced cell death (AICD). Although it is well documented that ethanol intake can cause depletion of CD4+ T cells, the mechanism of how alcohol mediates its effects is unclear., Methods: The results were based on data from three separate experiments presented as mean +/- standard deviation (SD). Jurkat CD4+ T cells and peripheral blood lymphocytes were treated with 25 mM of ethanol (12-18 hr), followed by stimulation with mitogens Conconavalin A (5 microg/ml) and Phytohemmaglutinin (1 microg/ml) or T-cell receptor ligation (anti-CD3 antibody (5 microg/ml)) for 6 hr, and then harvested for measurement. The apoptotic cell death markers measured include cell viability, Caspase-3-like activity, and DNA fragmentation., Results: We demonstrate that alcohol pretreatment enhances AICD of Jurkat CD4+ T cells and peripheral blood lymphocytes upon activation by CD3-crosslinking or stimulation with Conconavalin A and Phytohemmaglutinin. Furthermore, we find that the ethanol-mediated enhancement of T cells to apoptosis involves increased activation of Caspase-3 and can be abrogated by treatment with a specific inhibitor of Caspase-3., Conclusions: Our data indicate that ethanol can sensitize CD4+ T cells to enhanced stimulation-induced Caspase-3 activation and to subsequent AICD. This is, perhaps, an important mechanism in alcohol-induced immunosuppression.

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Terrence M. Donohue, Jr, and Dahn L. Clemens. The presentations were (1) Characterization of single and double recombinant hepatoma cells that express ethanol-metabolizing enzymes, by Terrence M. Donohue, Jr; (2) Inhibition of cell growth by ethanol metabolism, by Dahn L. Clemens; (3) Use of transfected HeLa cells to study the genesis of alcoholic fatty liver, by Andrea Galli and David Crabb; (4) CYP2E1-mediated oxidative stress induces COL1A2 mRNA in hepatic stellate cells and in a coculture system of HepG2 and stellate cells, by Natalia Nieto; (5) Transforming growth factor-alpha secreted from ethanol-exposed hepatocytes contributes to development of alcoholic hepatic fibrosis, by Junji Kato; and (6) Effect of ethanol on Fas-dependent caspase-3 activation and apoptosis in CD4+ T cells, by Shirish S. Barve.

Increased levels of hepatic and serum tumor necrosis factor (TNF) have been documented in animal models of alcoholic liver disease and in human alcoholic liver disease. This dysregulated TNF metabolism has been postulated to play a role in many of the metabolic complications and the liver injury of alcoholic liver disease. One potential therapy for alcoholic liver disease may be agents that downregulate TNF production or block TNF activity. Indeed, agents such as prostaglandins and glucocorticoids (both inhibit TNF production) have been used in both human liver disease and experimental models of liver injury, and anti-TNF antibody has recently been shown to attenuate the hepatotoxicity in an animal model of alcoholic-related liver disease. In this study, we demonstrate that a simple ex vivo system can be used to initially assess potential efficacy of anticytokine agents when administered to humans. Both prednisone and a prostaglandin analog were effective in downregulating TNF and interleukin-8 production. The liver is normally resistant to TNF cytotoxicity. Sensitivity to TNF cytotoxicity is thought to occur when there is inadequate production of hepatic protective factors. In this study, we showed that, when patients with acute alcoholic hepatitis were matched with trauma patients for serum levels of interleukin-6, they had similar depressions in the negative acute phase protein, albumin, but markedly different increases in the major acute phase protein, C reactive protein. Patients with alcoholic hepatitis had a very blunted response. We also showed that inhibiting activation of the redox sensitive transcription factor NFkappaB sensitizes to TNF-induced hepatocyte death in vitro. This transcription factor is important for the production of both cytokines and many acute phase protective factors. Several hepatic protective factors are induced by TNF. One possible mechanism for liver injury in alcoholic hepatitis may be inadequate generation of hepatic protective factors. Our future understanding of mechanisms of alcoholic liver disease will involve understanding the balance between noxious and protective factors in the liver, and this should lead to rational therapy for this disease process.

There is increased activity of the proinflammatory cytokine, tumor necrosis factor (TNF) in alcoholic liver disease (ALD). Hepatic neutrophil infiltration is a principal injurious manifestation of ALD. TNF can induce cellular oxidative injury directly, and indirectly by inducing neutrophil chemotactic factor (IL-8) production by hepatocytes. IL-8 activates and chemotactically attracts neutrophils to the liver where they release oxidizing substances. Patients with ALD also have decreased protective factors for cellular oxidative injury. Manganous superoxide dismutase (MnSOD) is an antioxidant protective factor. The objectives of these studies were to investigate mechanisms for induction of an injurious factor (IL-8) and a protective factor (MnSOD) in the HepG2 human hepatoma cell line. In the first set of experiments, IL-8 gene reporter constructs were used to transiently transfect a derivative (MVh2E1-9) of the HepG2 cell line which expresses P-4502E1 and metabolizes ethanol. Inactivation of the NF-kappaB and 3'NF-IL-6 DNA binding sites decreased IL-8 gene transcriptional activation in response to TNF while inactivation of the 5'NF-IL-6 binding site increased IL-8 gene transcriptional activity in response to TNF. This system may be useful to assess the effects of ethanol on TNF-induced hepatocyte IL-8 production. In the second set of experiments, HepG2 cells were cultured in 25 to 100 mmol concentrations of ethanol. Both TNF and ethanol increased HepG2 cell MnSOD activity in short-term (72 hr) cultures with ethanol. However, after long-term (10 weeks) culture with ethanol, there was no induction of MnSOD by ethanol and there was a diminished induction of MnSOD in response to TNF. Further studies are needed to assess the effect of this diminished induction of MnSOD with chronic ethanol culture on HepG2 cell susceptibility to TNF cytotoxicity. We conclude that transfected liver cell lines can be used to evaluate mechanisms for increased injurious factors and decreased protective factors in alcoholic liver injury.