Ethanol exposure modulates hepatic S-adenosylmethionine and S-adenosylhomocysteine levels in the isolated perfused rat liver through changes in the redox state of the NADH/NAD(+) system.

Methionine metabolism is disrupted in patients with alcoholic liver disease, resulting in altered hepatic concentrations of S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), and other metabolites. The present study tested the hypothesis that reductive stress mediates the effects of ethanol on liver methionine metabolism. Isolated rat livers were perfused with ethanol or propanol to induce a reductive stress by increasing the NADH/NAD(+) ratio, and the concentrations of SAM and SAH in the liver tissue were determined by high-performance liquid chromatography. The increase in the NADH/NAD(+) ratio induced by ethanol or propanol was associated with a marked decrease in SAM and an increase in SAH liver content. 4-Methylpyrazole, an inhibitor the NAD(+)-dependent enzyme alcohol dehydrogenase, blocked the increase in the NADH/NAD(+) ratio and prevented the alterations in SAM and SAH. Similarly, co-infusion of pyruvate, which is metabolized by the NADH-dependent enzyme lactate dehydrogenase, restored the NADH/NAD(+) ratio and normalized SAM and SAH levels. The data establish an initial link between the effects of ethanol on the NADH/NAD(+) redox couple and the effects of ethanol on methionine metabolism in the liver.

Integrated hepatic transcriptome and proteome analysis of mice with high-fat diet-induced nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is the most common form of liver disease in the US and refers to a wide spectrum of liver damage, including simple steatosis, steatohepatitis, fibrosis and cirrhosis. The goal of the present study was to achieve a more detailed understanding of the molecular changes in response to high fat-induced liver steatosis through the identification of a differentially expressed liver transcriptome and proteome. Male C57/BL6 mice fed a high-fat lard diet for 8 weeks developed visceral obesity and hepatic steatosis characterized by significantly increased liver and plasma free fatty acid and triglyceride levels and plasma alanine aminotransferase activities. Transcriptome analysis demonstrated that, compared to the control diet (CD), high-fat diet changed the expression of 309 genes (132 up- and 177 down-regulated; by a twofold change and more, P<.05). Multiple genes encoding proteins involved in lipogenesis were down-regulated, whereas genes involved in fatty acid oxidation were up-regulated. Proteomic analysis revealed 12 proteins which were differentially expressed. Of these, glutathione S-transferases mu1 and pi1 and selenium-binding protein 2 were decreased at both the gene and protein levels. This is the first study to perform a parallel transcriptomic and proteomic analysis of diet-induced hepatic steatosis. Several key pathways involving xenobiotic and lipid metabolism, the inflammatory response and cell-cycle control were identified. These pathways provide targets for future mechanistic and therapeutic studies as related to the development and prevention of NAFLD.

Inhibition of adiponectin production by homocysteine: a potential mechanism for alcoholic liver disease.

UNLABELLED: Although recent evidence suggests that down-regulation of production of the adipocyte hormone adiponectin has pathophysiological consequences for the development of alcoholic liver disease (ALD), the underlying mechanisms are elusive. Abnormal hepatic methionine-homocysteine metabolism induced by prolonged alcohol exposure has been reported both in clinical and experimental studies of ALD. Here, we conducted both in vivo and in vitro experiments to examine the effects of prolonged alcohol exposure on homocysteine levels in adipose tissue, its potential involvement in regulating adiponectin production, and the consequences for ALD. Chronic alcohol exposure decreased the circulating adiponectin concentration and adiponectin messenger RNA (mRNA) and protein levels in epididymal fat pads. Alcohol feeding induced modest hyperhomocysteinemia and increased homocysteine levels in the epididymal fat pad, which was associated with decreased mRNA levels of cystationine beta-synthase. Betaine supplementation (1.5%, wt/vol) in the alcohol-fed mice reduced homocysteine accumulation in adipose tissue and improved adiponectin levels. Moreover, exogenous homocysteine administration reduced gene expression, protein levels, and secretion of adiponectin in primary adipocytes. Furthermore, rats fed a high-methionine diet (2%, wt/wt) were hyperhomocysteinemic and had decreased adiponectin levels in both plasma and adipose tissue, which was associated with suppressed AMP-activated protein kinase activation in the liver. Mechanistic studies revealed that both inactivation of the extracellular signal regulated kinase 1/2 pathway and induction of endoplasmic reticulum stress response, specifically C/EBP homologous protein expression, may contribute to the inhibitory effect exerted by homocysteine. CONCLUSION: Chronic alcohol feeding caused abnormal accumulation of homocysteine in adipocytes, which contributes to decreased adiponectin production in ALD.

Genome-wide transcriptome expression in the liver of a mouse model of high carbohydrate diet-induced liver steatosis and its significance for the disease.

PURPOSE: To perform a large-scale gene profiling of the liver in a mouse model of fatty liver induced by high carbohydrate (sucrose) diet (HCD) to gain a deeper insight into potential mechanisms of diet-induced hepatic steatosis. METHODS: C57BL/6 male mice were fed either a purified, control diet or a HCD for 16 weeks. HCD feeding led to marked liver steatosis without inflammation or necrosis. The expression of 42,500 genes/sequences was assessed. RESULTS: A number of genes (471) underwent significant expression changes in HCD- as compared to standard diet-fed mice (n = 5/group; P < 0.01). Of these genes, 211 were down- and 260 up-regulated. The latter group includes 20 genes encoding enzymes involved in carbohydrate conversion to fat. The genes that underwent expression changes perform a large variety of molecular functions, and the vast majority of these have never been tested before in non-alcoholic fatty liver of nutritional origin. They reveal novel aspects of the disease and allow identification of candidate genes that may underlie the initiation of hepatic steatosis and progression to non-alcoholic steatohepatitis. CONCLUSIONS: HCD-fed laboratory animals provide a model of early non-alcoholic fatty liver disease resembling the disease in humans. The genome wide gene profiling of the liver reveals the complexity of the disease, unravels novel aspects of HCD-induced hepatic steatosis, and helps elucidate its nature and mechanisms.

Silymarin prevents palmitate-induced lipotoxicity in HepG2 cells: involvement of maintenance of Akt kinase activation.

Whereas adipocytes have a unique capacity to store excess free fatty acids in the form of triglyceride in lipid droplets, non-adipose tissues, such as liver, have a limited capacity for storage of lipids. Saturated long-chain fatty acids, such as palmitate, are the major contributors to lipotoxicity. Silymarin is a mixture of flavonolignans, extracted from the milk thistle (Silibum marianum). Its hepatoprotective properties have been studied both in vitro and in vivo; however, its effect on palmitate-induced lipotoxicity has not been investigated. The objective of this study was to investigate (i) whether silymarin could protect HepG2 cells from palmitate-induced cell death in an in vitro model, and (ii) possible mechanisms involved in this hepatoprotective role of silymarin. HepG2 cells were treated with palmitate in the absence or presence of silymarin and supernatants or cell lysates were collected at varying time-points. Cell death was assayed by measuring DNA fragmentation, caspase-3 activity and lactate dehydrogenase release. Lipid peroxidation was assessed by measuring malondialdehyde and 4-hydroxyalkenals. Akt kinase activity was also measured. Incubation with palmitate caused significant death in HepG2 cells. Palmitate incubation did not cause significant changes in reactive oxygen species production or intracellular glutathione content, but markedly inhibited Akt kinase activity. Pre-treatment of HepG2 cells with silymarin prevented palmitate-induced inhibition of Akt kinase activity and attenuated cell death. Our results suggest that silymarin may be an effective agent in protecting hepatocytes from saturated fatty acids-induced cell death. These data also provide a further rationale for exploration of the use of silymarin in the treatment of non-alcoholic steatohepatitis.

Involvement of AMP-activated protein kinase in beneficial effects of betaine on high-sucrose diet-induced hepatic steatosis.

Although simple steatosis was originally thought to be a pathologically inert histological change, fat accumulation in the liver may play a critical role not only in disease initiation, but also in the progression to nonalcoholic steatohepatitis and cirrhosis. Therefore, prevention of fat accumulation in the liver may be an effective therapy for multiple stages of nonalcoholic fatty liver disease (NAFLD). Promising beneficial effects of betaine supplementation on human NAFLD have been reported in some pilot clinical studies; however, data related to betaine therapy in NAFLD are limited. In this study, we examined the effects of betaine on fat accumulation in the liver induced by high-sucrose diet and evaluated mechanisms by which betaine could attenuate or prevent hepatic steatosis in this model. Male C57BL/6 mice weighing 20 +/- 0.5 g (means +/- SE) were divided into four groups (8 mice per group) and started on one of four treatments: standard diet (SD), SD+betaine, high-sucrose diet (HS), and HS + betaine. Betaine was supplemented in the drinking water at a concentration of 1% (wt/vol) (anhydrous). Long-term feeding of high-sucrose diet to mice caused significant hepatic steatosis accompanied by markedly increased lipogenic activity. Betaine significantly attenuated hepatic steatosis in this animal model, and this change was associated with increased activation of hepatic AMP-activated protein kinase (AMPK) and attenuated lipogenic capability (enzyme activities and gene expression) in the liver. Our findings are the first to suggest that betaine might serve as a therapeutic tool to attenuate hepatic steatosis by targeting the hepatic AMPK system.

Alcohol-induced S-adenosylhomocysteine accumulation in the liver sensitizes to TNF hepatotoxicity: possible involvement of mitochondrial S-adenosylmethionine transport.

Hepatocytes are resistant to tumor necrosis factor-alpha- (TNF) induced killing/apoptosis under normal circumstances, but primary hepatocytes from rats chronically fed alcohol have increased TNF cytotoxicity. Therefore, there must be mechanism(s) by which alcohol exposure "sensitizes" to TNF hepatotoxicity. Abnormal metabolism of methionine and S-adenosylmethionine (SAM) are well-documented acquired metabolic abnormalities in ALD. S-adenosylhomocysteine (SAH) is the product of SAM in hepatic transmethylation reactions, and SAH hydrolase (SAHH) is the only enzyme to metabolize SAH to homocysteine and adenosine. Our previous studies demonstrated that chronic intracellular accumulation of SAH sensitized hepatocytes to TNF cytotoxicity in vitro. In the current study, we extended our previous observations by further characterizing the effects of chronic alcohol intake on mitochondrial SAM levels in liver and examining its possible involvement in SAH sensitization to TNF hepatotoxicity. Chronic alcohol consumption in mice not only increased cytosolic SAH levels, but also decreased mitochondrial SAM concentration, leading to decreased mitochondrial SAM to SAH ratio. Moreover, accumulation of hepatic SAH induced by administration of 3-deaza-adenosine (DZA-a potent inhibitor of SAHH) enhanced lipopolysaccharide (LPS)/TNF hepatotoxicity in mice in vivo. Inhibition of SAHH by DZA resulted not only in accumulation of cytoplasmic SAH, but also in depletion of the mitochondrial SAM pool. Further studies using mitochondrial SAM transporter inhibitors showed that inhibition of SAM transport into mitochondria sensitized HepG2 cells to TNF cytotoxicity. In conclusion, our results demonstrate that depletion of the mitochondrial SAM pool by SAH, which is elevated during chronic alcohol consumption, plays a critical role in SAH induced sensitization to TNF hepatotoxicity.

4-Hydroxynonenal-induced apoptosis in rat hepatic stellate cells: mechanistic approach.

BACKGROUND AND AIM: Although lipid peroxidation products act as apoptotic signals for several cell types, including hepatic stellate cells, the underlying mechanisms are not well understood. In this study we determined if: (i) 4-hydroxy-2,3-nonenal (HNE) induces apoptosis in two rat stellate cell lines, HSC-T6 and CFSC-2G; and (ii) if apoptosis is regulated at the transcriptional and/or translational level. METHODS: HSC-T6 and CFSC-2G cells were treated with HNE and total RNA and protein extracted. mRNA and protein expression levels of pro- and antiapoptotic factors were determined. The effects of HNE on activation, morphology and cell death by apoptosis were also studied. RESULTS: HNE caused dose-dependent apoptosis in both HSC-T6 and CFSC-2G cell lines. Apoptosis in HSC-T6 cells was associated with increased mRNA expression of the pro-apoptotic adaptors/regulators FasR, FasL, Bax, and caspases-2 and -3. In contrast, CFSC-2G cells showed no changes in FasR, Bax and caspase-3 mRNA levels. Caspase-3 activity was elevated in T6 but not in 2G cells. Changes in protein expression generally paralleled the mRNA findings. CONCLUSIONS: HNE-induced apoptosis of both CFSC-2G and HSC-T6 rat hepatic stellate cells is associated with changes in mRNA and protein expression of several apoptotic adaptors/regulators. The underlying mechanism for HNE-induced apoptosis may involve both transcriptional and translational regulatory steps.

Nonalcoholic fatty liver disease: predisposing factors and the role of nutrition.

More than 20% of Americans have nonalcoholic fatty liver disease (NAFLD), and this is, by far, the leading cause of abnormal liver enzymes in the United States. Nonalcoholic steatohepatitis (NASH), a more serious form of NAFLD, can proceed to cirrhosis and even hepatocellular carcinoma. These liver diseases represent the hepatic component of the metabolic syndrome, and this spectrum of liver disease represents a major health problem both in the United States and worldwide. Hepatic steatosis is closely linked to nutrition, including obesity, possibly high-fructose corn syrup consumption and consumption of certain types of fats. There are a variety of second insults or "hits" that appear to transform simple steatosis into NASH, with some of these second hits including certain proinflammatory cytokines, oxidative stress and possibly industrial toxins. In certain underdeveloped countries, it appears likely that industrial toxins play a role in NASH, and there is increasing interest in the potential interaction of industrial toxins and nutrients. Moreover, optimal therapy for NAFLD appears to include lifestyle modification with exercise, diet and weight loss. Certain nutrients may also be of benefit. Important areas for future research are the effect(s) of nutritional supplements on NAFLD/NASH and the effects of industrial toxins.

Interactions of cytokines, S-Adenosylmethionine, and S-Adenosylhomocysteine in alcohol-induced liver disease and immune suppression.

Alcoholic liver disease (ALD) remains a leading cause of death in the USA. Defining mechanisms for liver cell death in ALD in order to develop potential new agents for therapeutic intervention is a major focus of the authors' work. Abnormal cytokine metabolism is a major feature of ALD, and a thorough understanding of both mechanisms and interactions of cytokine overproduction and sensitization are critical to developing a possible treatment for ALD. S-Adenosylmethionine has been used in a variety of animal studies and clinical trials and has been reported to improve biochemical parameters of liver function. Last, immunosuppression associated with chronic alcohol abuse is an important predisposing factor to opportunistic infections and cancer. It is the authors' working hypothesis that alcohol consumption leads to chronic activation of the immune system.

Lipopolysaccharide-induced liver apoptosis is increased in interleukin-10 knockout mice.

Although IL-10 down-regulates pro-inflammatory cytokine secretion by hepatic Kupffer cells, the mechanisms underlying its hepatoprotective effects are not fully clear. This study tested the hypothesis that IL-10 protects the liver against pro-inflammatory cytokines by counteracting their pro-apoptotic effects. Wild type and IL-10 knockout mice were treated with bacterial lipopolysaccharide and sacrificed 1, 4, 8, and 12 h later. Plasma ALT activity was measured as a marker of liver injury. Liver pathology and TUNEL response were assessed by histology. Plasma levels and whole liver mRNA levels were measured for TNF-alpha, IL-1 beta, TGF-beta1, IL-10, and their respective receptors. Hepatic mRNA levels were measured for several pro-apoptotic adaptors/regulators, including FasL, Fas receptor, FADD, TRADD, Bad, Bak, Bax, and Bcl-X(S), and anti-apoptotic regulators, including Bcl-w, Bcl-X(L), Bcl-2, and Bfl-1. Caspase-3 activity in the liver was determined as well as immunohistochemistry for IL-1RII, TGF-betaRII and Fas receptor. At all time points the livers from IL-10 knockout mice displayed a significantly increased number of apoptotic nuclei compared to wild type mice. Changes in plasma cytokine levels and their liver mRNA levels were consistent with suppression by IL-10 of pro-inflammatory cytokine secretion. In addition, pro-inflammatory cytokine receptor mRNA levels (TNF-alpha, TGF-beta, and IL-1 beta) were markedly up-regulated by LPS at all time points in IL-10 knockout mice as compared to wild type mice. Expression of the pro-inflammatory cytokine receptor IL-1RII was similarly increased as shown by immunostaining. The mRNA levels of a typical pro-apoptotic cytokine, TRAIL, were increased and LPS also up-regulated the mRNA expression of other apoptotic factors to a larger extent in IL-10 knockout mice than in their wild type counterparts, suggestive of an IL-10 anti-apoptotic effect. In the livers of knockout mice, markedly increased caspase-3 activity was already evident at the 1-h time point following LPS administration, while in the wild type animals this increase was delayed. Immunostaining also indicated that LPS increased hepatic expression of the pro-apoptotic receptors Fas and TGF-betaRII in IL-10 knockout mice. The data presented in this study show that: (i) IL-10 modulates not only the secretion of pro-inflammatory cytokines, but also the receptors of these cytokines, and ii) IL-10 protects the liver against LPS-induced injury at least in part by counteracting pro-inflammatory cytokine-induced liver apoptosis.

Silymarin protects against acute ethanol-induced hepatotoxicity in mice.

BACKGROUND: Accumulated evidence has demonstrated that both oxidative stress and abnormal cytokine production, especially tumor necrosis factor-alpha (TNF), play important etiological roles in the pathogenesis of alcoholic liver disease (ALD). Agents that have both antioxidant and anti-inflammation properties, particularly anti-TNF production, represent promising therapeutic interventions for ALD. We investigated the effects and the possible mechanism(s) of silymarin on liver injury induced by acute ethanol (EtOH) administration. METHODS: Nine-week-old mice were divided into 4 groups, control, silymarin treatment, EtOH treatment, and silymarin/EtOH treatment, with 6 mice in each group. Because control and silymarin values were virtually identical, only control treatment is shown for ease of viewing. Ethanol-treated mice received EtOH [5 g/kg body weight (BW)] by gavage every 12 hours for a total of 3 doses. Control mice received an isocalorical maltose solution. In the silymarin/EtOH group, silymarin was dissolved in the EtOH and gavaged simultaneously with EtOH at a dose of 200 mg/kg BW. At 4 hours after the last dosing, the mice were anesthetized and subsequent serum alanine aminotransferase (ALT) level, hepatic lipid peroxidation, enzymatic activity of hepatic cytochrome P450 2E1, hepatic TNF-alpha, and glutathione (GSH) levels were measured. Histopathological change was assessed by hematoxylin and eosin staining. RESULTS: Acute EtOH administration caused prominent hepatic microvesicular steatosis with mild necrosis and an elevation of serum ALT activity, induced a significant decrease in hepatic GSH in conjunction with enhanced lipid peroxidation, and increased hepatic TNF production. Supplementation with a standardized silymarin attenuated these adverse changes induced by acute EtOH administration. CONCLUSIONS: Silymarin protects against the liver injury caused by acute EtOH administration. In view of its nontoxic nature, it may be developed as an effective therapeutic agent for alcohol-induced liver disease by its antioxidative stress and anti-inflammatory features.

Dysregulated cytokine metabolism, altered hepatic methionine metabolism and proteasome dysfunction in alcoholic liver disease.

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.

Clinical implications of oxidative stress and antioxidant therapy.

Oxidative stress occurs when there is an imbalance between generation of reactive oxygen species and inadequate antioxidant defense systems. Oxidative stress can cause cell damage either directly or through altering signaling pathways. Oxidative stress is a unifying mechanism of injury in many types of disease processes, including gastrointestinal diseases. For example, in alcoholic liver disease, reactive oxygen species have been detected through direct spin-trapping techniques and through indirect markers, such as products of lipid peroxidation. A host of antioxidants have protected against liver injury in animal models of alcoholic liver disease. Similarly, in inflammatory bowel disease, oxidative stress has been postulated to play a role in disease initiation and progression, and antioxidant therapy, such as green tea polyphenols and gene therapy with superoxide dismutase, has a markedly attenuated disease. Downregulation of specific detoxification genes may play a role in the pathogenesis of inflammatory bowel disease, especially in ulcerative colitis. Oxidative stress is postulated to play a sustaining role in acute and chronic pancreatitis. Antioxidant supplementation has been used with some success in the treatment of chronic pancreatitis. This review covers recent findings related to oxidative stress in liver disease, inflammatory bowel disease, and pancreatitis.

Lessons from large-scale gene profiling of the liver in alcoholic liver disease.

This review examines the studies pertaining to large-scale gene profiling of liver cells and the whole liver as performed with the aid of macro- or microarray gene detection technology under the conditions of alcohol-induced liver injury. The review emphasizes the variability of the data as a function of strain, species, and model of alcohol-induced liver injury employed in different studies. Further, the review highlights the importance of determining if changes in transcriptome expression are parallelled by changes in proteome and metabolome of the liver. On the basis of such data, models can be constructed to unravel new mechanistic aspects of alcohol-induced liver injury and to design novel therapies for alcoholic liver disease.

Modulation of endotoxin stimulated interleukin-6 production in monocytes and Kupffer cells by S-adenosylmethionine (SAMe).

Interleukin-6 (IL-6) is a multifunctional cytokine having primarily anti-apoptotic and anti-inflammatory effects. Recent reports have documented that IL-6 plays a key role in liver regeneration. Intracellular deficiency of S-adenosylmethionine (SAMe) is a hallmark of toxin-induced liver injury. Although the administration of exogenous SAMe attenuates liver injury, its mechanisms of action are not fully understood. Here we investigated the effects of exogenous SAMe on IL-6 production in monocytes and Kupffer cells. RAW 264.7 cells, a murine monocyte cell line, and isolated rat Kupffer cells were stimulated with lipopolysaccharide (LPS) in the absence or presence of exogenous SAMe. IL-6 production was assayed by ELISA and intracellular SAMe concentrations were measured by HPLC. We have found that exogenous SAMe administration enhanced both IL-6 protein production and gene expression in LPS-stimulated monocytes and Kupffer cells. Cycloleucine (CL), an inhibitor for extrahepatic methionine adenosyltransferases (MAT), inhibited LPS-stimulated IL-6 production. The enhancement of LPS-stimulated IL-6 production by SAMe was inhibited by ZM241385, a specific antagonist of adenosine (A2) receptor. Our results demonstrate that SAMe administration may exert its anti-inflammatory and hepatoprotective effects, at least in part, by enhancing LPS-stimulated IL-6 production.

Gene expression in the liver of rats fed alcohol by means of intragastric infusion.

It has become increasingly evident that one of the most fruitful approaches to understanding cellular processes and their relation to disease consists of large-scale gene profiling of cells, tissues, and organs. This also constitutes a first step in exploring the molecular biologic basis of various diseases. In the current study, we used cDNA microarray technology to assess possible changes in the expression of a large number of genes in the liver of rats fed alcohol (ethanol) chronically (4 weeks) by means of intragastric infusion. This animal model resembles closely the alcoholic liver disease in human beings. Of a total of 8,740 probe sets arrayed on the microchip, 2,069 were expressed by the liver. After a correction for false discovery rate at 10%, 72 genes were found to be significantly up-regulated (40) or down-regulated (32). Forty-two genes were suppressed, and four genes were induced, by alcohol. These genes are involved in a wide spectrum of cellular functions. Also, the genes that underwent significant changes were categorized into two groups: genes that have been implicated in alcoholic liver disease and genes that have not been tested for possible changes in expression. Large-scale gene profiling of the liver reveals changes in the expression of a number of genes that have never been implicated in alcohol-induced injury. Further investigation of such genes may cast light on mechanisms underlying alcohol-induced liver injury and help in the design of new therapeutic approaches.

Recent advances in alcoholic liver disease. IV. Dysregulated cytokine metabolism in alcoholic liver disease.

Alcoholic liver disease (ALD) remains a leading cause of death from liver disease in the United States for which there is no FDA-approved therapy. Abnormal cytokine metabolism is a major feature of ALD. Elevated serum concentration levels of TNF-alpha and TNF-alpha-inducible cytokines/chemokines, such as IL-6, -8, and -18, have been reported in patients with alcoholic hepatitis and/or cirrhosis, and levels correlated with markers of the acute phase response, liver function, and clinical outcome. Studies in animal models support an etiologic role for cytokines in the liver injury of ALD. Cytokines, such as transforming growth factor-beta, play a critical role in the fibrosis of ALD. Multiple new strategies are under investigation to modulate cytokine metabolism as a form of therapy for ALD.

Microarray gene analysis of the liver in a rat model of chronic, voluntary alcohol intake.

The mechanisms underlying alcoholic liver disease are not fully understood. It has been established that alcohol interferes with transcriptional and translational regulatory steps of cell function. To understand such an effect, assessment of alcohol-induced changes in the simultaneous expression of a large number of genes may prove very useful. The purpose of the current study was to test a large number of genes ( approximately 8700) for possible changes in expression induced by alcohol alone or in addition to treatment with lipopolysaccharide (LPS), a putative mediator of alcohol effects on the liver. Male rats were fed an alcohol-containing liquid diet (Lieber-DeCarli) for 14-15 weeks, injected with Escherichia coli LPS (0.8 mg x kg(-1)), and killed 24 h later. Blood samples were taken for determination of plasma liver enzyme activity, and liver samples were obtained for histologic evaluation and total RNA extraction. Total RNA was analyzed for gene expression (Rat Toxicology U34 Array; Affymetrix, Santa Clara, CA). Of 8740 genes on the microchip, 2259 were expressed in the liver. Seven hundred ninety-eight genes underwent significant changes induced by either alcohol or LPS, but listed in this article are only those that significantly increased or decreased expression twofold or more. The genes were assigned to functional groups and reviewed. Gene changes were discussed from two viewpoints: relevance to established hypotheses of alcohol and LPS mechanisms of action and revealing of novel mechanisms of alcohol-induced liver injury. Application of DNA microarray technology to the study of alcohol-induced liver injury generated novel theoretical and experimental approaches to alcohol-induced liver injury.