Commentary with regard to the role of alcohol and cofactors in the development of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a primary cancer of the liver. It is clear that chronic alcohol abuse is associated with the development of HCC, but the mechanistic role of alcohol in HCC is not well studied. The research that is presented in the Brandon-Warner and colleagues' (2012) article approaches an important outcome of chronic alcohol abuse. The most important study results that are presented in their article show convincing data that chronic alcohol consumption by male mice resulted in a robust response in the liver to a carcinogen. It was shown that female mice responded significantly less to alcohol and the carcinogen than the male animals. The overall research data covered a number of aspects of the liver disease associated with chronic alcohol consumption and the presence of a carcinogen that will support a great deal of further research to develop mechanistic data.

Association of chronic alcohol consumption and increased susceptibility to and pathogenic effects of pulmonary infection with respiratory syncytial virus in mice.

Chronic alcohol abuse by human beings has been shown to be associated with increased susceptibility to pulmonary infections and severity of inflammatory responses associated with pulmonary infection. On the basis of the higher likelihood of exposure to respiratory viruses, people who abuse alcohol would logically be susceptible to respiratory viral infections. To test this hypothesis, mice were provided alcohol in drinking water for 13-16 weeks with the Meadows-Cook protocol and infected intranasally with respiratory syncytial virus. At various times after infection, severity of infection was determined by evaluation of cellular and cytokine composition of bronchoalveolar lavage fluid (BALF) and histologic evaluation of inflammation. Infection was associated with neutrophil infiltration in both groups, but the proportion and number of neutrophils in BALF were significantly greater in the alcohol consumption group than in the control group. Concentrations of tumor necrosis factor-alpha and monocyte chemoattractant protein-1 in BALF in the alcohol consumption group were increased. Interferon (IFN)-gamma concentrations were lower in the alcohol consumption group at later times of infection. Pulmonary inflammation was cleared by 3-5 days after infection in the control group. In contrast, pulmonary inflammation was evident in the alcohol consumption group after 7 days of infection, and some mice showed severe inflammation with hemorrhage and edema. IFN-alpha/beta was evident in BALF at low concentrations in the alcohol consumption group for several days after infection, and increased mRNA for IFN-alpha/beta was also evident in the alcohol consumption group. This was accompanied by the presence of virus in this group at these times of infection. Chronic alcohol consumption increased severity of pulmonary infection with a virus that naturally infects hosts by an aerosol route. Infection of mice that had consumed alcohol chronically was more severe in terms of increased proinflammatory cytokine production, inflammation, and a failure to clear the virus from the lungs.

Thymocytes, pre-B cells, and organ changes in a mouse model of chronic ethanol ingestion--absence of subset-specific glucocorticoid-induced immune cell loss.

BACKGROUND: The well-known immune deficiency of the chronic alcoholic dictates the need for a long-term rodent ethanol administration model to evaluate the baseline immunologic effects of chronic ethanol abuse, and investigate the genetic determinants of those effects. Much published work with rodents has shown clearly that acute ethanol administration and short-term ethanol-containing liquid diets both cause elevated corticosterone and can cause significant thymocyte, pre-B cell and peripheral lymphocyte losses. Such losses may mask more subtle alterations in immune homeostasis, and in any case are generally short-lived compared with the span of chronic ethanol abuse. Thus, it is important to have a model in which long-term immune alterations can be studied free of corticosteroid-induced cell losses. METHODS: We have utilized chronic 20% (w/v) ethanol in water administration to several mouse strains for prolonged periods of time and evaluated serum corticosterone, immunologic stress parameters, and other organ changes by standard methods. RESULTS: We now confirm earlier reports that chronic ethanol in water administration to mice does not produce net elevations of corticosterone, although diurnal variation is altered. Importantly, there is neither selective loss of immune cell populations known to be corticosteroid sensitive, CD4+CD8+ thymocytes and pre-B cells, nor are changes observed in the histologic appearance of the thymus. Nonetheless, there are significant chronic ethanol effects in other tissues, including reduced heart weight, mild hepatic steatosis, alterations of gut flora, increased serum peptidoglycan, and as published elsewhere, immune system abnormalities. CONCLUSIONS: This model of ethanol administration is convenient, sustainable for up to 1 year, demonstrably feasible in several mouse strains, permits good weight gains in most strains, and results in significant changes in a number of organs. The administration method also will permit modeling of long-term steady abuse punctuated by major binges, and is suitable for supplementation studies using water soluble additives. Overall, the method is useful for a wide range of studies requiring a chronic low-stress method of ethanol administration.

Alcoholic pancreatitis: mechanisms of viral infections as cofactors in the development of acute and chronic pancreatitis and fibrosis.

Acute and chronic pancreatitis is associated with alcohol abuse, but symptomatic pancreatitis develops in only a small proportion of persons (10-20%) who abuse alcohol. This apparent paradox has led to the notion that additional cofactors are involved in the development of alcoholic pancreatitis. Potential cofactors, such as diet and smoking, have been suggested, but there are no compelling epidemiologic data to support this idea. A number of viruses and some bacteria have been shown to infect the pancreas and produce pancreatitis. One important mediator of pancreatitis in persons with a compromised immune system is a viral infection. The increased susceptibility of immunocompromised persons to viral pancreatitis led to the hypothesis, described in this paper, that the well-known immunosuppression associated with alcohol abuse would result in a more severe viral pancreatitis in mice, which are provided ethanol, than in control animals. To test this hypothesis, C57BL/6 mice were infected with a virulent strain of coxsackievirus B3, which preferentially induces pancreatitis, or with a strain that is naturally avirulent. The study findings presented in this paper show that ethanol consumption alone does not produce pancreas damage but results in a more severe and prolonged pancreatitis after infection with a virulent virus and interestingly, after infection with the avirulent strain of virus. This was associated with an increased number of viruses in the pancreas and spleen, which correlated with decreased humoral immune responses to the virus.

Inhibition of protein kinase C epsilon causes ciliated bovine bronchial cell detachment.

This study defines the in vitro phenomenon of ciliated bovine bronchial epithelial cell (BBEC) detachment from the basal epithelium and regulation of cilia motility mediated through protein kinase C epsilon (PKCepsilon). The authors determined the time course of activation and downregulation of PKCepsilon by the known PKC activator phorbol 12-myristate 13-acetate (PMA) and demonstrate that chemical inhibition of PKC by calphostin C or the novel PKC isoform inhibitor Ro 31-8220 induced striking detachment of ciliated BBECs from the basal cell monolayer within 1 hour, independent of apoptosis or necrotic cell death. The results of this study support a possible novel PKCepsilon-mediated signaling pathway through which ciliated cell attachment is maintained.

Acute and chronic alcohol abuse modulate immunity.

This article represents the proceedings of the Alcohol and Immunology Research Interest Group (AIRIG) meeting, a satellite workshop held at the 37th Annual Meeting of the Society for Leukocyte Biology. The meeting was sponsored by the AIRIG and the National Institute on Alcohol Abuse and Alcoholism. The presentations were as follows: (1) Effects of Ethanol on Immune Response to Hepatitis C Virus by Jack R. Wands, (2) Alcohol and Alveolar Macrophage Dysfunction: The Role of Chronic Oxidant Stress by Lou Ann S. Brown, (3) T Cell Responses to Listeria monocytogenes in Mice on a Chronic Ethanol Exposure Protocol by Robert T. Cook, (4) Mechanisms of Acute and Chronic Alcohol Consumption on Severity of Viral Infections by the Liver and Pancreas by Thomas R. Jerrells, (5) Acute and Chronic Effects on Macrophage Ectodomain Shedding: Implications for Lung Host Defenses by Jay K. Kolls, (6) Increased Susceptibility to Pseudomonas Infection of Burn-Injured Mice Given Alcohol Before Injury by Elizabeth J. Kovacs, (7) Regulation of Tumor Necrosis Factor alpha Expression in Macrophages by Chronic Ethanol by Laura E. Nagy, and (8) Hepatitis C Virus Infection and Alcohol Use by Gyongyi Szabo. Meeting coorganizers were Elizabeth J. Kovacs, Lou Ann S. Brown, Thomas R. Jerrells, and Robert T. Cook.

Metabolic basis of ethanol-induced cytotoxicity in recombinant HepG2 cells: role of nonoxidative metabolism.

Chronic alcohol abuse, a major health problem, causes liver and pancreatic diseases and is known to impair hepatic alcohol dehydrogenase (ADH). Hepatic ADH-catalyzed oxidation of ethanol is a major pathway for the ethanol disposition in the body. Hepatic microsomal cytochrome P450 (CYP2E1), induced in chronic alcohol abuse, is also reported to oxidize ethanol. However, impaired hepatic ADH activity in a rat model is known to facilitate a nonoxidative metabolism resulting in formation of nonoxidative metabolites of ethanol such as fatty acid ethyl esters (FAEEs) via a nonoxidative pathway catalyzed by FAEE synthase. Therefore, the metabolic basis of ethanol-induced cytotoxicity was determined in HepG2 cells and recombinant HepG2 cells transfected with ADH (VA-13), CYP2E1 (E47) or ADH + CYP2E1 (VL-17A). Western blot analysis shows ADH deficiency in HepG2 and E47 cells, compared to ADH-overexpressed VA-13 and VL-17A cells. Attached HepG2 cells and the recombinant cells were incubated with ethanol, and nonoxidative metabolism of ethanol was determined by measuring the formation of FAEEs. Significantly higher levels of FAEEs were synthesized in HepG2 and E47 cells than in VA-13 and VL-17A cells at all concentrations of ethanol (100-800 mg%) incubated for 6 h (optimal time for the synthesis of FAEEs) in cell culture. These results suggest that ADH-catalyzed oxidative metabolism of ethanol is the major mechanism of its disposition, regardless of CYP2E1 overexpression. On the other hand, diminished ADH activity facilitates nonoxidative metabolism of ethanol to FAEEs as found in E47 cells, regardless of CYP2E1 overexpression. Therefore, CYP2E1-mediated oxidation of ethanol could be a minor mechanism of ethanol disposition. Further studies conducted only in HepG2 and VA-13 cells showed lower ethanol disposition and ATP concentration and higher accumulation of neutral lipids and cytotoxicity (apoptosis) in HepG2 cells than in VA-13 cells. The apoptosis observed in HepG2 vs. VA-13 cells incubated with ethanol appears to be mediated by release of mitochondrial cytochrome c via activation of caspase-9 and caspase-3. These results strongly support our hypothesis that diminished hepatic ADH activity facilitates nonoxidative metabolism of ethanol and the products of ethanol nonoxidative metabolism cause apoptosis in HepG2 cells via intrinsic pathway.

Rescue of in vivo FAS-induced apoptosis of hepatocytes by corticosteroids either associated with alcohol consumption by mice or provided exogenously.

The pathogenic effects of many hepatic viral infections are mediated, at least in part, by the immune response to the infected hepatocyte. The immune response in the infected liver involves the interaction of cytotoxic T cells (CTL) with the hepatocytes through the interaction of FAS-ligand on the CTL and FAS on the hepatocyte. The initial hypothesis for this study was that alcohol consumption by mice would sensitize the liver to apoptosis induced by ligation of FAS. C57Bl/6 mice fed ethanol in a liquid diet did show an increased percentage of apoptotic cells 2 h after injection with anti-FAS as compared with the percentage in the control mice. However, 4 and 6 h after anti-FAS injection, control mice showed high percentages of apoptotic cells (20% to 41%) compared with 5% and 4% apoptotic cells in the ethanol-fed mice. The decreased apoptosis of ethanol-fed mice correlated closely with corticosterone levels in the sera. This was confirmed by the finding that adrenalectomized (ADX) mice provided a high level of corticosterone in drinking water were protected against FAS-induced hepatocyte apoptosis. Ethanol-fed mice showed a significant elevation of serum alanine aminotransferase (ALT) levels indicating the development of hepatitis in spite of the relatively low proportion of apoptotic cells in the liver. In conclusion, high levels of corticosterone protect hepatocytes from FAS-mediated apoptosis, but do not prevent the ultimate development of liver damage. In experiments where mice were provided ethanol chronically in drinking water, where stress is minimal, higher levels of ALT were noted in animals in the ethanol group as compared with animals in the control group. These data support the suggestion that ethanol increases hepatocyte sensitivity to FAS-mediated damage.

Alcohol and immunology: introduction to and summary of the 2003 Alcohol and Immunology Research Interest Group (AIRIG) meeting.

The 8th Meeting of the Alcohol and Immunology Research Interest Group (AIRIG) was held at Loyola University Medical Center, Maywood, Illinois, USA, on November 21, 2003. Reports from multiple laboratories reveal that the functional integrity of the immune system is of paramount importance to the survival of the individual after infection or injury. Evidence supports the idea that exposure to alcohol causes dysregulation of both the innate and the adaptive arms of the immune system. Gaining a better understanding of how alcohol interferes with normal inflammatory and immunoregulatory processes will aid researchers in the design of therapeutic interventions that can be used to improve these responses to better fight infection and maintain the health of the individual. At this meeting, nine speakers presented a summary of their recent work on the combined effects of ethanol and injury, infection, or inflammatory challenge. Topics were (1) T-cell activation after chronic ethanol ingestion in mice, (2) effect of ethanol consumption on the severity of acute viral-mediated pancreatitis, (3) ethanol and alveolar macrophage dysfunction, (4) impaired intestinal immunity and barrier function: a cause for enhanced bacterial translocation in alcohol intoxication and burn injury, (5) immune consequences of the combined insult of acute ethanol exposure and burn injury, (6) consequences of alcohol-induced dysregulation of immediate hemodynamic and inflammatory responses to trauma/hemorrhage, (7) regulation of tumor necrosis factor-alpha production by Kupffer cells after chronic exposure to ethanol, (8) acute exposure to ethanol and suppression of cytokine responses induced through Toll-like receptors, and (9) inhibition of antigen-presenting cell functions by alcohol: implications for hepatitis C virus infection. We anticipate that the work presented at the 8th Meeting of AIRIG, summarized in this article, and presented in the nine articles to follow in this Special Issue of Alcohol will stimulate ideas that will develop into research projects in these topical areas.

Ethanol consumption potentiates viral pancreatitis and may inhibit pancreas regeneration: preliminary findings.

Alcohol abuse is often associated with acute pancreatitis. The pathogenesis of alcoholic pancreatitis remains poorly understood, in part because of the lack of a suitable animal model to study the mechanism or mechanisms of this disease. It has been proposed that ethanol predisposes or sensitizes the pancreas to the effects of co-factors, and the combination of the effects of ethanol on the pancreas and the actions of these co-factors results in alcoholic pancreatitis. A number of viruses are known to infect the pancreas, and we have suggested that one co-factor that could be involved in the development of alcoholic pancreatitis is a viral infection. One of the most-studied groups of viruses that infect the pancreas and cause pancreatitis in human beings is the coxsackieviruses. We have shown that short-term (5-14 days) and subchronic (>28 days) administration of ethanol to mice increases the severity of coxsackie B3-induced pancreas damage. We hypothesize that consumption of ethanol would result in an impairment of pancreas regeneration after injury, similar to the effect of ethanol on liver regeneration. With the use of the murine model of coxsackie B3-mediated alcoholic pancreatitis we have obtained preliminary data to support the hypothesis. Specifically, consumption of ethanol by mice is associated with changes in the replication of acinar cells and their organization into acini after viral-mediated injury. We believe that this model will be a valuable tool to study the biochemical and molecular mechanisms involved in alcoholic pancreatitis.

Animal model of alcoholic pancreatitis: role of viral infections.

Pancreatitis is clearly associated with alcohol abuse, but only a relatively small percentage of people who abuse alcohol develops obvious pancreatitis. These observations have led to the concept that the development of alcoholic pancreatitis requires cofactors. Although diet and smoking have been studied, a clear cofactor has not been identified. The study results presented in this paper were obtained to determine whether viral infection of the pancreas would be a cofactor for alcoholic pancreatitis similar to the role of hepatitis virus infections in the development of alcoholic liver disease. To test this hypothesis, mice were fed ethanol with a liquid diet protocol and infected with coxsackievirus B3 (CVB3). It was found that consumption of alcohol alone did not result in pancreatitis as determined by serum levels of amylase or histologic changes in the pancreas. Two strains of CVB3 that are tropic for the pancreas were used; a virulent and an avirulent strain. Infection of alcohol-fed animals with the virulent CVB3 strain 28 resulted in a more severe pancreatitis than the pancreatitis noted in control animals. Alcohol-fed mice infected with the avirulent strain (GA) showed severe pancreatitis, whereas the infection of control mice did not result in obvious pathologic effects in the pancreas. This model allows mechanistic studies to define the role of viral infection as a cofactor for alcoholic pancreatitis.

Modulation of immunity and viral-host interactions by alcohol.

This manuscript represents the proceedings of a symposium at the 2001 RSA Meeting in Montreal, Canada. The organizers/chairs were Gyongyi Szabo and Geoffrey M. Thiele. The presentations were (1) Introduction, by Gyongyi Szabo; (2) Chemokine dysregulation after acute ethanol exposure, by Elizabeth J. Kovacs; (3) Chemokine production and innate immunity in the livers of simian immunodeficiency virus-infected Macaca mulatta following chronic alcohol administration, by Abraham P. Bautista; (4) Influence of ethanol consumption on the severity and progression of hepatitis associated with cytomegalovirus infection, by Laura Sosa and Thomas R. Jerrells; (5) Scavenger receptor involvement in the immune response to the metabolites of chronic ethanol ingestion, by Geoffrey M. Thiele; and (6) Mechanisms of impaired accessory cell functions due to alcohol exposure and hepatitis C infection, by Gyongyi Szabo.

Influence of ethanol consumption on experimental viral hepatitis.

BACKGROUND: One important contributor to pathologic effects on the liver associated with alcohol abuse is viral hepatitis, especially hepatitis C virus (HCV) infection. Alcohol consumption has been shown to be associated with more severe HCV infection and hepatitis. The mechanisms of the more severe viral infection of the liver are unclear, and studies have been hampered by the lack of an animal model of hepatotropic viral infections. METHODS: We have established a murine model system of viral hepatitis in which C57BL/6 mice are infected with murine cytomegalovirus, a herpesvirus that produces self-limiting hepatitis in immunocompetent mice. Mice were fed a liquid diet containing 36% ethanol-derived calories with a pair-feeding protocol. After infection with a sublethal dose of murine cytomegalovirus, the severity of liver infection was determined by measuring serum levels of alanine aminotransferase and by histological evaluation. Other parameters determined included the serum levels of cytokines, cytokine RNA in liver samples, and viral concentration in liver samples. RESULTS: Ethanol-fed mice showed more severe hepatitis in the later stages of the infection as compared with the hepatitis noted in control mice. The ethanol-fed mice did not control the virus replication in the liver, which was associated with a greater mononuclear cell inflammatory response, composed predominantly of cells with morphological characteristics of lymphocytes and macrophages. The early production of interferon gamma, as well as production throughout the infection, was significantly lower in the ethanol-fed mice. The early production of interleukin 12 was also less in ethanol-fed mice. CONCLUSIONS: The more severe hepatitis seen in the ethanol-fed mice is likely to be the result of an inability to control the growth of the virus, which is associated with a continued inflammatory response. The inability to control the virus in the liver may be related to the decreased production of interferon gamma and interleukin 12.

Association of alcohol consumption and exaggerated immunopathologic effects in the liver induced by infectious organism.

The cause of alcoholic liver disease (ALD) is multifactorial and poorly understood. It is clear that alcohol alone is not responsible for most of the changes associated with ALD and that cofactors are involved in initiation and production of ALD. One cofactor that has received a great deal of attention recently is the concomitant infection with hepatitis C virus (HCV) and alcohol abuse. The interactive effects of HCV and alcohol abuse are still unclear, but apparently they are the result of an inability of the immune system to control the viral infection and exaggerated hepatocyte damage mediated by either the cells of the inflammatory response or factors produced by the inflammatory cells. A major effort in my laboratory has been focused on defining the effects of alcohol consumption on immunity to various infectious agents. Efforts have also been directed to elucidating the pathologic effects in the liver of inflammatory and immune responses to microorganisms that either specifically or ultimately infect the liver from an initial site of infection other than the liver. This review will focus on one aspect of the possible pathogenic effects associated with alcohol abuse and hepatic infections: the possible role of the immune system, notably the cytotoxic T lymphocyte (CTL) response. It is clear that the development of a CTL response is critical for the control of HCV and other infections, and it is also likely that this response is involved in liver damage. In this review, the evidence that shows the importance of the CD8+ CTL in bacterial and viral clearance and the role for pathogenesis will be presented. Findings obtained from animal studies that support the suggestion that activated CD8+ CTLs can induce liver damage will be presented, as will results of recent studies from my laboratory that provide evidence for an effect of alcohol to enhance the liver damage mediated by activated CD8+ T cells.

Role of activated CD8+ T cells in the initiation and continuation of hepatic damage.

The cause of alcoholic liver disease (ALD) is multifactorial and poorly understood. It is clear that alcohol alone is not responsible for most of the changes associated with ALD and that cofactors are involved in initiation and production of ALD. One cofactor that has received a great deal of attention recently is the concomitant infection with hepatitis C virus (HCV) and alcohol abuse. The interactive effects of HCV and alcohol abuse are still unclear, but apparently they are the result of an inability of the immune system to control the viral infection and exaggerated hepatocyte damage mediated by either the cells of the inflammatory response or factors produced by the inflammatory cells. This review will focus on one aspect of the possible pathogenic effects associated with alcohol abuse and HCV infection: the possible role of the immune system, notably the cytotoxic T lymphocyte (CTL) response. It is clear that the development of a CTL response is critical for the control of HCV infections, and it is also likely that this response is involved in liver damage. In this review, the evidence that shows the importance of the CD8(+) CTL in viral clearance and the role for pathogenesis will be presented. Findings obtained from animal studies that support the suggestion that activated CD8(+) CTLs can induce liver damage will be presented, as will results of recent studies from my laboratory that provide evidence for an effect of alcohol to enhance the liver damage mediated by activated CD8(+) T cells.

Relationship between acetaldehyde levels and cell survival in ethanol-metabolizing hepatoma cells.

We have created a number of recombinant Hep G2 cell lines, designated VA cells, that constitutively express alcohol dehydrogenase. Oxidation of ethanol by the VA cells results in the production and accumulation of acetaldehyde, and a dramatic increase in the nicotinamide adenine dinucleotide, reduced (NADH)/nicotinamide adenine dinucleotide (NAD(+)) ratio (redox-state). It is believed that production of acetaldehyde, and the increase in the redox-state of hepatocytes, are responsible for many of the dysfunctions associated with alcoholic liver disease. When the VA cells were cultured in the presence of ethanol, we observed a dramatic reduction in cell accumulation. This reduction was more pronounced in cells that metabolized ethanol more efficiently. Inhibition of alcohol dehydrogenase activity abolished this reduction, demonstrating that ethanol oxidation was required for this dysfunction. Subsequent investigations indicated that this ethanol oxidation-mediated reduction in cell accumulation was the result of both cytotoxicity and impaired DNA synthesis. To dissociate the increase in the cellular redox-state from acetaldehyde production, VA cells were cultured in the presence of isopropanol. The oxidation of isopropanol results in similar redox changes, but the metabolic by-product of isopropanol oxidation is acetone. The metabolism of isopropanol by VA cells resulted in very little reduction in cell number. Furthermore, treatment of ethanol-metabolizing VA cells with the aldehyde dehydrogenase inhibitor, cyanamide, increased the levels of acetaldehyde and resulted in an additional reduction in cell number. In conclusion, these studies indicated that exposure to acetaldehyde caused cytotoxicity, as well as the ethanol oxidation-mediated reduction in cell number.