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Lee YS, Seki E. In Vivo and In Vitro Models to Study Liver Fibrosis: Mechanisms and Limitations. Cell Mol Gastroenterol Hepatol 2023; 16:355-367. [PMID: 37270060 PMCID: PMC10444957 DOI: 10.1016/j.jcmgh.2023.05.010] [Citation(s) in RCA: 16] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/04/2023] [Revised: 05/26/2023] [Accepted: 05/26/2023] [Indexed: 06/05/2023]
Abstract
Liver fibrosis is a common result of liver injury owing to various kinds of chronic liver diseases. A deeper understanding of the pathophysiology of liver fibrosis and identifying potential therapeutic targets of liver fibrosis is important because liver fibrosis may progress to advanced liver diseases, such as cirrhosis and hepatocellular carcinoma. Despite numerous studies, the underlying mechanisms of liver fibrosis remain unclear. Mechanisms of the development and progression of liver fibrosis differ according to etiologies. Therefore, appropriate liver fibrosis models should be selected according to the purpose of the study and the type of underlying disease. Many in vivo animal and in vitro models have been developed to study liver fibrosis. However, there are no perfect preclinical models for liver fibrosis. In this review, we summarize the current in vivo and in vitro models for studying liver fibrosis and highlight emerging in vitro models, including organoids and liver-on-a-chip models. In addition, we discuss the mechanisms and limitations of each model.
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Affiliation(s)
- Young-Sun Lee
- Karsh Division of Gastroenterology and Hepatology, Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California; Department of Internal Medicine, Korea University College of Medicine, Seoul, South Korea
| | - Ekihiro Seki
- Karsh Division of Gastroenterology and Hepatology, Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California.
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Buyco DG, Martin J, Jeon S, Hooks R, Lin C, Carr R. Experimental models of metabolic and alcoholic fatty liver disease. World J Gastroenterol 2021; 27:1-18. [PMID: 33505147 PMCID: PMC7789066 DOI: 10.3748/wjg.v27.i1.1] [Citation(s) in RCA: 24] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/06/2020] [Revised: 11/01/2020] [Accepted: 12/06/2020] [Indexed: 02/06/2023] Open
Abstract
Non-alcoholic fatty liver disease (NAFLD) is a multi-systemic disease that is considered the hepatic manifestation of metabolic syndrome (MetS). Because alcohol consumption in NAFLD patients is common, there is a significant overlap in the pathogenesis of NAFLD and alcoholic liver disease (ALD). Indeed, MetS also significantly contributes to liver injury in ALD patients. This “syndrome of metabolic and alcoholic steatohepatitis” (SMASH) is thus expected to be a more prevalent presentation in liver patients, as the obesity epidemic continues. Several pre-clinical experimental models that couple alcohol consumption with NAFLD-inducing diet or genetic obesity have been developed to better understand the pathogenic mechanisms of SMASH. These models indicate that concomitant MetS and alcohol contribute to lipid dysregulation, oxidative stress, and the induction of innate immune response. There are significant limitations in the applicability of these models to human disease, such as the ability to induce advanced liver injury or replicate patterns in human food/alcohol consumption. Thus, there remains a need to develop models that accurately replicate patterns of obesogenic diet and alcohol consumption in SMASH patients.
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Affiliation(s)
- Delfin Gerard Buyco
- Division of Gastroenterology, University of Pennsylvania, Philadelphia, PA 19104, United States
| | - Jasmin Martin
- Division of Gastroenterology, University of Pennsylvania, Philadelphia, PA 19104, United States
| | - Sookyoung Jeon
- Division of Gastroenterology, University of Pennsylvania, Philadelphia, PA 19104, United States
| | - Royce Hooks
- Division of Gastroenterology, University of Pennsylvania, Philadelphia, PA 19104, United States
| | - Chelsea Lin
- Division of Gastroenterology, University of Pennsylvania, Philadelphia, PA 19104, United States
| | - Rotonya Carr
- Division of Gastroenterology, University of Pennsylvania, Philadelphia, PA 19104, United States
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Abstract
Apart from the classic knowledge that ethanol mediates its hepatotoxicity through its metabolism to acetaldehyde, a well-known hepatotoxic molecule, recent research has elucidated several key mechanisms that potentiate ethanol's damage to the liver parenchyma, such as generation of free radicals, activation of Kupffer cells, and alterations to the human bacterial and fungal microbiome. Genetic studies have suggested the role of PNPLA3 and TM6SF2 gene mutations in the progression of alcoholic liver disease.
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Affiliation(s)
- Themistoklis Kourkoumpetis
- Department of Gastroenterology, Baylor College of Medicine, 6620 Main Street, Suite 1450, Houston, TX 77030, USA
| | - Gagan Sood
- Department of Surgery, Division of Abdominal Transplantation, Baylor College of Medicine, 6620 Main Street, Suite 1450, Houston, TX 77030, USA.
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Gao B, Zakhari S. Epidemiology and Pathogenesis of Alcoholic Liver Disease. ZAKIM AND BOYER'S HEPATOLOGY 2018:334-344.e3. [DOI: 10.1016/b978-0-323-37591-7.00022-7] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 09/01/2023]
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Hashimoto JG, Wiren KM, Wilhelm CJ. A neurotoxic alcohol exposure paradigm does not induce hepatic encephalopathy. Neurotoxicol Teratol 2016; 56:35-40. [PMID: 27268733 DOI: 10.1016/j.ntt.2016.06.001] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2016] [Revised: 06/02/2016] [Accepted: 06/03/2016] [Indexed: 02/07/2023]
Abstract
Alcohol abuse is associated with neurological dysfunction, brain morphological deficits and frank neurotoxicity. Although these disruptions may be a secondary effect due to hepatic encephalopathy, no clear evidence of causality is available. This study examined whether a 72h period of alcohol intoxication known to induce physical dependence, followed by a single withdrawal, was sufficient to induce signs of hepatic encephalopathy in male and female mice. Animals were continuously intoxicated via alcohol vapor inhalation, a procedure previously shown to induce significant neurotoxicity in female mice. At peak synchronized withdrawal (8h following the end of alcohol exposure), blood samples were taken and levels of several liver-regulated markers and brain swelling were characterized. Glutathione levels were also determined in the medial frontal cortex (mFC) and hippocampus. Results revealed elevated levels of cholesterol, albumin, alkaline phosphatase (ALP), alanine aminotransferase (ALT) and decreased levels of blood urea nitrogen and total bilirubin in alcohol-exposed male and female groups compared to controls. Brain water weight was not affected by alcohol exposure, though males tended to have slightly more water weight overall. Alcohol exposure led to reductions in tissue levels of glutathione in both the hippocampus and mFC which may indicate increased oxidative stress. Combined, these results suggest that hepatic encephalopathy does not appear to play a significant role in the neurotoxicity observed following alcohol exposure in this model.
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Affiliation(s)
- Joel G Hashimoto
- Research & Development Service, VA Portland Health Care System, Portland, OR, USA; Department of Behavioral Neuroscience, Oregon Health & Science University, Portland, OR, USA
| | - Kristine M Wiren
- Research & Development Service, VA Portland Health Care System, Portland, OR, USA; Department of Behavioral Neuroscience, Oregon Health & Science University, Portland, OR, USA
| | - Clare J Wilhelm
- Research & Development Service, VA Portland Health Care System, Portland, OR, USA; Department of Psychiatry, Oregon Health & Science University, Portland, OR, USA.
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Rui BB, Chen H, Jang L, Li Z, Yang JM, Xu WP, Wei W. Melatonin Upregulates the Activity of AMPK and Attenuates Lipid Accumulation in Alcohol-induced Rats. Alcohol Alcohol 2015; 51:11-9. [PMID: 26564773 DOI: 10.1093/alcalc/agv126] [Citation(s) in RCA: 43] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2015] [Accepted: 09/28/2015] [Indexed: 12/14/2022] Open
Abstract
AIMS Melatonin is supposed to be an effective hepatoprotective agent. The effects and mechanisms of melatonin on alcoholic fatty liver (AFL) have not been well explored. The aim of this study was to investigate the preventive and therapeutic effects of melatonin on alcohol-induced fatty liver rats. METHODS The AFL rats were induced by intragastric infusion of alcohol plus a high-fat diet for 6 weeks, and melatonin (10, 20, 40 mg/kg) was administered by gastric perfusion. We also established fatty acid overload cell model in HepG2 cells to investigate the effect of melatonin on AMP-activated protein kinase (AMPK) activity. RESULTS The results showed that melatonin (20 and 40 mg/kg) administration significantly reduced alcohol-induced hepatic steatosis with lowering activities of serum alanine aminotransferase, aspartate aminotransferase and levels of serum and hepatic triglyceride. The activity of superoxide dismutase was increased and the content of malondialdehyde was decreased in liver homogenates of rats treated with melatonin. Melatonin increased the phosphorylation of AMPK in the liver tissues of alcohol-induced rats as well. Additionally, in vitro studies showed that melatonin increased the expression of melatonin1A receptor (MT1R), whereas luzindole, a receptor antagonist of melatonin, had no effect on its expression. In addition, melatonin reduced the levels of adenosine 3',5'-cyclic monophosphate (cAMP) and increased the phosphorylation of AMPK, and melatonin treatment could markedly reverse these effects. CONCLUSION In conclusion, melatonin could protect against liver injury caused by alcohol gastric perfusion. The effect may be related to alleviating lipid peroxidation and upregulating the activity of AMPK mediated by MT1R signaling pathway.
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Affiliation(s)
- Bei-bei Rui
- Anhui No. 2 Province People's Hospital, Hefei, Anhui 200041, China
| | - Hao Chen
- Institute of Clinical Pharmacology, Anhui Medical University, Key Laboratory of Anti-inflammatory and Immune Medicine, Ministry of Education, Hefei 230032, China
| | - Lei Jang
- Anhui No. 2 Province People's Hospital, Hefei, Anhui 200041, China
| | - Zhen Li
- Anhui No. 2 Province People's Hospital, Hefei, Anhui 200041, China
| | - Jing-mo Yang
- Anhui Provincial Cancer Hospital, Hefei, Anhui 230001, China
| | - Wei-ping Xu
- Anhui Medical University affiliated Provincial Hospital, Hefei, Anhui 230001, China
| | - Wei Wei
- Institute of Clinical Pharmacology, Anhui Medical University, Key Laboratory of Anti-inflammatory and Immune Medicine, Ministry of Education, Hefei 230032, China
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Carr RM, Correnti J. Insulin resistance in clinical and experimental alcoholic liver disease. Ann N Y Acad Sci 2015; 1353:1-20. [PMID: 25998863 DOI: 10.1111/nyas.12787] [Citation(s) in RCA: 47] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Alcoholic liver disease (ALD) is the number one cause of liver failure worldwide; its management costs billions of healthcare dollars annually. Since the advent of the obesity epidemic, insulin resistance (IR) and diabetes have become common clinical findings in patients with ALD; and the development of IR predicts the progression from simple steatosis to cirrhosis in ALD patients. Both clinical and experimental data implicate the impairment of several mediators of insulin signaling in ALD, and experimental data suggest that insulin-sensitizing therapies improve liver histology. This review explores the contribution of impaired insulin signaling in ALD and summarizes the current understanding of the synergistic relationship between alcohol and nutrient excess in promoting hepatic inflammation and disease.
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Affiliation(s)
- Rotonya M Carr
- Division of Gastroenterology, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
| | - Jason Correnti
- Division of Gastroenterology, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
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Fatty liver-induced changes in stereotypic behavior in rats and effects of glucagon-like peptide-1 analog on stereotypy. Kaohsiung J Med Sci 2014; 30:447-52. [PMID: 25224767 DOI: 10.1016/j.kjms.2014.05.007] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2013] [Revised: 04/26/2014] [Accepted: 02/25/2014] [Indexed: 02/06/2023] Open
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Mathews S, Xu M, Wang H, Bertola A, Gao B. Animals models of gastrointestinal and liver diseases. Animal models of alcohol-induced liver disease: pathophysiology, translational relevance, and challenges. Am J Physiol Gastrointest Liver Physiol 2014; 306:G819-23. [PMID: 24699333 PMCID: PMC4024729 DOI: 10.1152/ajpgi.00041.2014] [Citation(s) in RCA: 98] [Impact Index Per Article: 8.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Over the last four decades, chronic ethanol feeding studies in rodents using either ad libitum feeding or intragastric infusion models have significantly enhanced our understanding of the pathogenesis of alcoholic liver disease (ALD). Recently, we developed a chronic plus binge alcohol feeding model in mice that is similar to the drinking patterns of many alcoholic hepatitis patients: a history of chronic drinking and recent excessive alcohol consumption. Chronic+binge ethanol feeding synergistically induced steatosis, liver injury, and neutrophil infiltration in mice, which may be useful for the study of early alcoholic liver injury and inflammation. Using this chronic+binge model, researchers have begun to identify novel mechanisms that participate in the pathogenesis of alcoholic liver injury, thereby revealing novel therapeutic targets. In this review article, we briefly discuss several mouse models of ALD with a focus on the chronic+binge ethanol feeding model.
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Affiliation(s)
| | | | | | | | - Bin Gao
- Laboratory of Liver Diseases, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, Maryland
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Erbaş O, Solmaz V, Aksoy D, Yavaşoğlu A, Sağcan M, Taşkıran D. Cholecalciferol (vitamin D 3) improves cognitive dysfunction and reduces inflammation in a rat fatty liver model of metabolic syndrome. Life Sci 2014; 103:68-72. [DOI: 10.1016/j.lfs.2014.03.035] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2013] [Revised: 03/13/2014] [Accepted: 03/29/2014] [Indexed: 01/14/2023]
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The association between seizure predisposition and inflammation in a rat model of fatty liver disease. Neurol Sci 2014; 35:1441-6. [DOI: 10.1007/s10072-014-1778-5] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2014] [Accepted: 03/27/2014] [Indexed: 12/18/2022]
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Detection of impaired cognitive function in rat with hepatosteatosis model and improving effect of GLP-1 analogs (exenatide) on cognitive function in hepatosteatosis. ScientificWorldJournal 2014; 2014:946265. [PMID: 24741367 PMCID: PMC3967460 DOI: 10.1155/2014/946265] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2013] [Accepted: 01/22/2014] [Indexed: 12/21/2022] Open
Abstract
The aims of the study were to evaluate (1) detection of cognitive function changing in rat with hepatosteatosis model and (2) evaluate the effect of GLP-1 analog (exenatide) on cognitive function in hepatosteatosis. In the study group, 30% fructose was given in nutrition water to perform hepatosteatosis for 8 weeks to 18 male rats. Six male rats were chosen as control group and had normal nutrition. Fructose nutrition group were stratified into 3 groups. In first group (n = 6), intracerebroventricular (ICV) infusion of exenatide (n = 6) was given. ICV infusion of NaCl (n = 6) was given to second group. And also, the third group had no treatment. And also, rats were evaluated for passive avoidance learning (PAL) and liver histopathology. Mean levels of latency time were statistically significantly decreased in rats with hepatosteatosis than those of normal rats (P < 0.00001). However, mean level of latency time in rats with hepatosteatosis treated with ICV exenatide was statistically significantly increased than that of rats treated with ICV NaCl (P < 0.001). Memory performance falls off in rats with hepatosteatosis feeding on fructose (decreased latency time). However, GLP-1 ameliorates cognitive functions (increased latency time) in rats with hepatosteatosis and releated metabolic syndrome.
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Liu Y, Meyer C, Xu C, Weng H, Hellerbrand C, ten Dijke P, Dooley S. Animal models of chronic liver diseases. Am J Physiol Gastrointest Liver Physiol 2013; 304:G449-G468. [PMID: 23275613 DOI: 10.1152/ajpgi.00199.2012] [Citation(s) in RCA: 153] [Impact Index Per Article: 12.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Chronic liver diseases are frequent and potentially life threatening for humans. The underlying etiologies are diverse, ranging from viral infections, autoimmune disorders, and intoxications (including alcohol abuse) to imbalanced diets. Although at early stages of disease the liver regenerates in the absence of the insult, advanced stages cannot be healed and may require organ transplantation. A better understanding of underlying mechanisms is mandatory for the design of new drugs to be used in clinic. Therefore, rodent models are being developed to mimic human liver disease. However, no model to date can completely recapitulate the "corresponding" human disorder. Limiting factors are the time frame required in humans to establish a certain liver disease and the fact that rodents possess a distinct immune system compared with humans and have different metabolic rates affecting liver homeostasis. These features account for the difficulties in developing adequate rodent models for studying disease progression and for testing new pharmaceuticals to be translated into the clinic. Nevertheless, traditional and new promising animal models that mimic certain attributes of chronic liver diseases are established and being used to deepen our understanding in the underlying mechanisms of distinct liver diseases. This review aims at providing a comprehensive overview of recent advances in animal models recapitulating different features and etiologies of human liver diseases.
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Affiliation(s)
- Yan Liu
- Department of Medicine II, Section Molecular Hepatology-Alcohol Associated Diseases, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany
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Gyamfi D, Everitt HE, Tewfik I, Clemens DL, Patel VB. Hepatic mitochondrial dysfunction induced by fatty acids and ethanol. Free Radic Biol Med 2012; 53:2131-2145. [PMID: 23010494 DOI: 10.1016/j.freeradbiomed.2012.09.024] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/02/2012] [Revised: 09/14/2012] [Accepted: 09/15/2012] [Indexed: 02/08/2023]
Abstract
Understanding the key aspects of the pathogenesis of alcoholic fatty liver disease particularly alterations to mitochondrial function remains to be resolved. The role of fatty acids in this regard requires further investigation due to their involvement in fatty liver disease and obesity. This study aimed to characterize the early effects of saturated and unsaturated fatty acids alone on liver mitochondrial function and during concomitant ethanol exposure using isolated liver mitochondria and VA-13 cells (Hep G2 cells that efficiently express alcohol dehydrogenase). Liver mitochondria or VA-13 cells were treated with increasing concentrations of palmitic or arachidonic acid (1 to 160 μM) for 24 h with or without 100 mM ethanol. The results showed that in isolated liver mitochondria both palmitic and arachidonic acids significantly reduced state 3 respiration in a concentration-dependent manner (P<0.001), implicating their ionophoric activities. Increased ROS production occurred in a dose-dependent manner especially in the presence of rotenone (complex I inhibitor), which was significantly more prominent in arachidonic acid at 80 μM (+970%, P<0.001) than palmitic acid (+40%, P<0.01). In VA-13 cells, ethanol alone and both fatty acids (40 μM) were able to decrease the mitochondrial membrane potential and cellular ATP levels and increase lipid formation. ROS production was significantly increased with arachidonic acid (+110%, P<0.001) exhibiting a greater effect than palmitic acid (+39%, P<0.05). While in the presence of ethanol, the drop in the mitochondrial membrane potential, cellular ATP levels, and increased lipid formation were further enhanced by both fatty acids, but with greater effect in the case of arachidonic acid, which also correlated with significant cytotoxicity (P<0.001). This study confirms the ability of fatty acids to promote mitochondrial injury in the development of alcoholic fatty liver disease.
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Affiliation(s)
- Daniel Gyamfi
- Department of Biomedical Sciences, School of Life Sciences, University of Westminster, 115 New Cavendish Street, London, W1W 6UW, UK
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Zhou JY, Jiang ZA, Zhao CY, Zhen Z, Wang W, Nanji AA. Long-term binge and escalating ethanol exposure causes necroinflammation and fibrosis in rat liver. Alcohol Clin Exp Res 2012; 37:213-22. [PMID: 23009062 DOI: 10.1111/j.1530-0277.2012.01936.x] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2011] [Accepted: 06/10/2012] [Indexed: 01/17/2023]
Abstract
BACKGROUND To investigate whether "binge" and escalating alcohol exposure in the rat influences the development of pathological liver injury. METHODS Time courses for the formation of eicosanoids by cyclooxygenase (COX), oxidative stress and nitrosative stress production, expression of hypoxia-inducible factor 1 (HIF-1), cytokines, hepatic tissue necroinflammation, and fibrosis were assessed in rats during 16 weeks of daily alcohol gavage. RESULTS In this model of binge and escalating levels of alcohol, hepatic steatosis, necrosis, and inflammation as well as fibrosis were increased over the 16-week period. The levels of COX-2, oxidative stress, nitrosative stress, HIF-1, proinflammatory mediators (tumor necrosis factor-α, interleukin 1(β) [IL-1(β) ], IL-6), and procollagen-I were increased over the 16-week period. The content of IL-10 in rat serum increased at the end of 4 and 8 weeks but decreased thereafter and was significantly decreased at 12 and 16 weeks. CONCLUSIONS A rat model of alcoholic liver disease (ALD) with long-term binge and escalating ethanol exposure was developed. Our data support the hypothesis that enhanced eicosanoid production by COX, oxidative stress and nitrosative stress, HIF-1, and the imbalance between pro- and anti-inflammatory cytokines plays an important role in the pathogenesis of ALD.
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Affiliation(s)
- Jun-Ying Zhou
- Department of Infectious Disease , Third Hospital, Hebei Medical University, Shijiazhuang, China.
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Lachenmeier DW, Kanteres F, Rehm J. Epidemiology-based risk assessment using the benchmark dose/margin of exposure approach: the example of ethanol and liver cirrhosis. Int J Epidemiol 2010; 40:210-8. [PMID: 20819784 DOI: 10.1093/ije/dyq150] [Citation(s) in RCA: 37] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023] Open
Abstract
BACKGROUND A novel approach to derive a threshold dose with respect to alcohol-related harm, the benchmark dose (BMD) methodology, is introduced to provide a basis for evidence-based drinking guidelines. This study is the first to calculate a BMD for alcohol exposure using epidemiological cohort data. With this BMD we will be able to calculate the margin of exposure (MOE) for alcohol consumption, which can be used for comparative risk assessment and applied to setting public health policy. METHODS Benchmark dose-response modelling of epidemiological data gathered during a recent systematic review and meta-analysis of alcohol consumption as a risk factor for liver cirrhosis morbidity and mortality. RESULTS For a benchmark response (BMR) of 1.5%, the resulting BMD values were 30.9 g/day for males and 29.7 g/day for females; the corresponding lower one-sided confidence values were 25.7 and 27.2 g/day, respectively. The intake scenario for the Canadian population resulted in an MOE of 1.23. Intake scenarios for individuals as based on the Canadian drinking guidelines led to MOE values between 0.96 and 1.91. Using an uncertainty factor of 10, the acceptable daily intake for alcohol would be 2.6 g/day. CONCLUSIONS The BMD approach was feasible in developing evidence-based guidelines for low-risk drinking. As our calculated MOEs result around unity (i.e. 1) for moderate drinking, it is evident that the current guidelines correspond very well to low risk on the dose-response curve. The BMD methodology therefore validates current guidelines. The results again highlight the health risk associated with alcohol consumption.
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Zhang R, Hu Y, Yuan J, Wu D. Effects of Puerariae radix extract on the increasing intestinal permeability in rat with alcohol-induced liver injury. JOURNAL OF ETHNOPHARMACOLOGY 2009; 126:207-214. [PMID: 19735712 DOI: 10.1016/j.jep.2009.08.044] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/14/2009] [Revised: 08/21/2009] [Accepted: 08/30/2009] [Indexed: 05/28/2023]
Abstract
AIM OF THE STUDY Puerariae radix, as an edible plant, has been used for centuries in China to treat alcohol-related problems, including alcoholic liver disease (ALD). However, the mechanisms of Puerariae radix on the liver-protective effect have not been fully explored. Because an increased intestinal permeability is a major factor for ALD, the present study investigates whether Puerariae radix extract (PRE) inhibits ALD through prevention of alterations in intestinal permeability. MATERIALS AND METHODS We used an animal model of chronic alcohol-induced liver injury that is associated with increased intestinal permeability. Male Wistar rats were given increasing alcohol doses from 2 g/kg/d to 8 g/kg/d and alcohol plus PRE via intragastric feeding for 10 weeks. Chronic alcohol exposure caused an elevation in serum alanine aminotransferase (ALT) as well as aspartate aminotransferase (AST) levels and a decrease in superoxide dismutase (SOD) activity, and hepatic damages including steatosis, inflammation, and necrosis, determined by serum enzymatic analysis and morphological analysis, respectively. The damage to small intestine induced by chronic alcohol treatment was examined by intestinal histological, immunohistochemical analysis, and permeability assays. RESULTS Alcohol-induced hepatic pathological changes, elevations in ALT and AST, and a decrease in SOD activity were significantly inhibited in PRE treated animals. The inhibitory effect of PRE on alcohol-induced liver injury was associated with suppression of alcohol induced the increase of intestinal permeability. CONCLUSIONS The results showed that this beneficial effect of PRE on ALD could be partly explained by improving intestinal barrier dysfunction induced by alcohol.
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Affiliation(s)
- Rongrong Zhang
- Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
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Bardag-Gorce F, Oliva J, Dedes J, Li J, French BA, French SW. Chronic ethanol feeding alters hepatocyte memory which is not altered by acute feeding. Alcohol Clin Exp Res 2009; 33:684-92. [PMID: 19170665 PMCID: PMC2811268 DOI: 10.1111/j.1530-0277.2008.00885.x] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
BACKGROUND Gene expression changes in the liver after acute binge drinking may differ from the changes seen in chronic ethanol feeding in the rat. The changes in gene expression after chronic ethanol feeding may sensitize the liver to alcohol-induced liver damage, which is not seen after acute binge drinking. METHODS To test this hypothesis, gene microarray analysis was performed on the livers of rats (n = 3) fed an acute binge dose of ethanol (6 g/kg body wt) and killed at 3 and 12 hours after ethanol by gavage. The gene microarrays were compared with those made on the liver of rats from a previous study, in which the rats were fed ethanol by intragastric tube for 1 month (36% of calories derived from ethanol). RESULTS Microarray analysis data varied between the acute and chronic models in several important respects. Growth factors increased mainly in the chronic alcohol fed rat. Changes in enzymes involved in oxidative stress were noted only with chronic ethanol feeding. Gene expression of fat metabolism was increased only with chronic ethanol feeding. Most importantly, epigenetic related enzymes and acetylation and methylation of histones changed only after chronic ethanol feeding. CONCLUSIONS The results support the concept that chronic ethanol ingestion induces altered gene expression as a result of changes in epigenetic mechanisms, where acetylation and methylation of histones were altered.
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Affiliation(s)
- F Bardag-Gorce
- Department of Pathology, Harbor-UCLA Medical Center, Torrance, California 90509, USA
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Ethanol diversely alters palmitate, stearate, and oleate metabolism in the liver and pancreas of rats using the deuterium oxide single tracer. Pancreas 2009; 38:e47-52. [PMID: 19248221 PMCID: PMC4662660 DOI: 10.1097/mpa.0b013e318199fea4] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
OBJECTIVE To determine tissue-specific effects of alcohol on fatty acid synthesis and distribution as related to functional changes in triglyceride transport and membrane formation. METHODS Tissue fatty acid profile and de novo lipogenesis were determined in adult male Wistar rats after 5 weeks of ethanol feeding using deuterated water and gas chromatography/mass spectrometry. Liver and pancreas fatty acid profiles and new synthesis fractions were compared with those from control rats on an isocaloric diet. RESULTS Fatty acid ratios in the liver indicated that there was a more than 2-fold accumulation of stearate to that of palmitate, with an apparent decrease in oleate content. On the other hand, in the pancreas, there was a 17% decrease in the stearate-to-palmitate ratio, whereas the oleate-to-palmitate ratio was increased by 30%. The fractions of deuterium-labeled palmitate and stearate were substantially reduced in the liver and pancreas of the alcohol-treated animals. Deuterium labeling of oleate was reduced in the liver but not in the pancreas, consistent with the oleate/stearate ratios in these tissues. CONCLUSIONS Long-term alcohol exposure results in opposite effects on the desaturase activity in the liver and pancreas, limiting fatty acid transport in the liver but promoting the exocrine function of the pancreas.
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Abstract
Liver damage leads to an inflammatory response and to the activation and proliferation of mesenchymal cell populations within the liver which remodel the extracellular matrix as part of an orchestrated wound-healing response. Chronic damage results in a progressive accumulation of scarring proteins (fibrosis) that, with increasing severity, alters tissue structure and function, leading to cirrhosis and liver failure. Efforts to modulate the fibrogenesis process have focused on understanding the biology of the heterogeneous liver fibroblast populations. The fibroblasts are derived from sources within and out with the liver. Fibroblasts expressing alpha-smooth muscle actin (myofibroblasts) may be derived from the transdifferentiation of quiescent hepatic stellate cells. Other fibroblasts emerge from the portal tracts within the liver. At least a proportion of these cells in diseased liver originate from the bone marrow. In addition, fibrogenic fibroblasts may also be generated through liver epithelial (hepatocyte and biliary epithelial cell)-mesenchymal transition. Whatever their origin, it is clear that fibrogenic fibroblast activity is sensitive to (and may be active in) the cytokine and chemokine profiles of liver-resident leucocytes such as macrophages. They may also be a component driving the regeneration of tissue. Understanding the complex intercellular interactions regulating liver fibrogenesis is of increasing importance in view of predicted increases in chronic liver disease and the current paucity of effective therapies.
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Nieto N. Ethanol and fish oil induce NFkappaB transactivation of the collagen alpha2(I) promoter through lipid peroxidation-driven activation of the PKC-PI3K-Akt pathway. Hepatology 2007; 45:1433-45. [PMID: 17538965 DOI: 10.1002/hep.21659] [Citation(s) in RCA: 52] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
UNLABELLED To analyze whether fish oil, as a source of polyunsaturated fatty acids from the n-3 series, could synergize with ethanol to promote collagen I upregulation in vivo, collagen alpha2(I) promoter-betaGal (COL1A2-betaGal) transgenic mice were fed a diet enriched in fish oil in the presence of ethanol (ethanol group) or dextrose (control group). Ethanol-fed mice showed mild steatosis, increased alanine aminotransferase (ALT), aspartate aminotransferase (AST), nonsterified fatty acids, and plasma alcohol levels along with elevated cytochrome P450 2E1 activity, lipid peroxidation end products, and low glutathione (GSH) levels, which suggested enhanced oxidant stress and liver injury. Increased transactivation of the COL1A2 promoter assessed by betaGal activity was shown in vivo and by transfection with deletion constructs for the collagen alpha1(I) promoter (COL1A1) and COL1A2 promoters in vitro. Transcriptional regulation of both COL1A1 and COL1A2 promoters was validated by nuclear in vitro transcription run-on, northern blot analysis, and quantitative polymerase chain reaction, which was followed by the subsequent upregulation of collagen I protein with no changes in matrix metalloproteinase 13 (MMP 13). To further analyze the potential mechanism for collagen I upregulation, an in vitro coculture model was designed with primary stellate cells seeded on the bottom plate of a Boyden chamber and the rest of the liver cells plated on a cell culture insert, and fish oil or fish oil plus ethanol were added. The combination of fish oil plus ethanol increased nuclear factor kappaB binding to the COL1A2 promoter both in vivo and in the cocultures and also resulted in increased phosphorylation of protein kinase C, activation of PI3 kinase, and phosphorylation of Akt. The in vitro addition of vitamin E prevented such activation and collagen I increase. Furthermore, inhibitors of all 3 kinases blocked the increase in collagen I and NFkappaB binding to the COL1A2 promoter; the latter was also prevented by vitamin E. CONCLUSION These results suggest that fish oil (mainly n-3 polyunsaturated fatty acids [PUFAs]) can synergize with ethanol to induce collagen I, transactivating the COL1A2 promoter through a lipid peroxidation-PKC-PI3K-Akt-NFkappaB-driven mechanism in the absence of overt steatosis and inflammation.
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Affiliation(s)
- Natalia Nieto
- Department of Medicine, Division of Liver Diseases, Mount Sinai School of Medicine, 1425 Madison Avenue, New York, NY 10029, USA.
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Yuan G, Gong Z, Li J, Li X. Ginkgo biloba extract protects against alcohol-induced liver injury in rats. Phytother Res 2007; 21:234-8. [PMID: 17154234 DOI: 10.1002/ptr.2054] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Ginkgo biloba extract has been shown to be hepatoprotective. However, its benefits when used in alcoholic liver injury have not been demonstrated. This study investigated the effects of Ginkgo biloba extract on alcohol-induced liver injury in a chronic alcohol plus fish oil gavage model in rats. Liver injury was evaluated histologically and by serum alanine aminotransferase (ALT). Liver tumor necrosis factor-alpha (TNF-alpha) protein levels, malondialdehyde (MDA) and glutathione (GSH) contents were determined. TNF-alpha mRNA expression in the liver was analysed by reverse transcriptase-polymerase chain reaction (RT-PCR). Rats given fish oil plus alcohol developed macrovesicular and microvesicular steatosis, spotty necrosis and mild inflammation in the liver and elevated serum ALT levels, which were accompanied by increased MDA contents, reduced GSH contents and elevated TNF-alpha protein and mRNA expressions in the liver. Supplementation with Ginkgo biloba extract (200 mg/kg, orally) improved the liver injury, blunted the rises of MDA contents and TNF-alpha expression, and restored the GSH content in the liver. Ginkgo biloba extract protects against alcohol-induced liver injury, and the mechanism may involve a reduction of lipid peroxidation, prevention of GSH depletion and inhibition of TNF-alpha expression in the liver.
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Affiliation(s)
- Guangjin Yuan
- Department of Infectious Diseases, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province, China.
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Watabiki T, Okii Y, Tokiyasu T, Yoshimura S, Yoshida M, Akane A, Shikata N, Tsubura A. Long-Term Ethanol Consumption in ICR Mice Causes Mammary Tumor in Females and Liver Fibrosis in Males. Alcohol Clin Exp Res 2006. [DOI: 10.1111/j.1530-0277.2000.tb00025.x] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Affiliation(s)
| | - Yutaka Okii
- Department of Legal Medicine; Kansai Medical University; Moriguchi Japan
| | - Takuma Tokiyasu
- Department of Legal Medicine; Kansai Medical University; Moriguchi Japan
| | - Sumitaka Yoshimura
- Department of Legal Medicine; Kansai Medical University; Moriguchi Japan
| | - Manabu Yoshida
- Department of Legal Medicine; Kansai Medical University; Moriguchi Japan
| | - Atsushi Akane
- Department of Legal Medicine; Kansai Medical University; Moriguchi Japan
| | - Nobuaki Shikata
- Department of Pathology 2; Kansai Medical University; Moriguchi Japan
| | - Airo Tsubura
- Department of Pathology 2; Kansai Medical University; Moriguchi Japan
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25
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Uesugi T, Froh M, Gäbele E, Isayama F, Bradford BU, Ikai I, Yamaoka Y, Arteel GE. Contribution of angiotensin II to alcohol-induced pancreatic fibrosis in rats. J Pharmacol Exp Ther 2004; 311:921-8. [PMID: 15316086 DOI: 10.1124/jpet.104.071324] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
The mechanisms by which alcohol causes pancreatic fibrosis remain unknown. Recent studies have demonstrated that angiotensin II contributes to the development of fibrosis in liver, kidney, and heart injury. Here, the effects of angiotensin-converting enzyme inhibitor (captopril) and angiotensin II receptor antagonist (losartan) on alcohol-induced pancreatic fibrosis were examined in an intragastric ethanol-feeding model. Male rats were fed a high-fat liquid diet with either ethanol (16-20 g/kg/day) or isocaloric maltose-dextrin (control) for 4 weeks. Subgroups daily received captopril (60 mg/kg/day), losartan (3 mg/kg/day), or no additional agent included in liquid diets. Mean urine alcohol concentrations in all groups fed ethanol were more than 270 mg/dl and not significantly different. Dietary alcohol caused diffuse gland atrophy and interlobular and intralobular fibrosis with mild structural distortion in the pancreas, an effect that was blunted by captopril or losartan treatment. Alcohol also increased the number of alpha-smooth muscle actin-positive cells and transforming growth factor-beta mRNA expression in the pancreas. These increases were blunted significantly by captopril or losartan treatment. These data suggest that angiotensin II contributes to the development of chronic alcohol-induced pancreatic fibrosis through its stimulation of transforming growth factor-beta expression.
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Affiliation(s)
- Takehiko Uesugi
- Department of Pharmacology and Toxicology, 1307 Research Tower, HSC, University of Louisville School of Medicine, Louisville, KY 40292, USA
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26
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Nanji AA. Role of different dietary fatty acids in the pathogenesis of experimental alcoholic liver disease. Alcohol 2004; 34:21-5. [PMID: 15670661 DOI: 10.1016/j.alcohol.2004.08.005] [Citation(s) in RCA: 66] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2004] [Revised: 08/11/2004] [Accepted: 08/11/2004] [Indexed: 12/30/2022]
Abstract
Both dietary fatty acids and alcohol play an important role in the pathogenesis of alcoholic liver disease. Findings of studies in rats show a steatogenic role for dietary fat. A role for polyunsaturated fatty acids in alcoholic liver disease is supported by results of studies, which show that pathologic changes occur only in rats fed ethanol with polyunsaturated fatty acids. The mechanisms through which the fatty acids promote alcoholic liver disease include enhanced oxidative stress, production of endotoxin, and increased expression of proinflammatory cytokines.
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Affiliation(s)
- Amin A Nanji
- Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical Center, 3400 Spruce Street, Founders 7.103, Philadelphia, PA 19104, USA.
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27
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Nanji AA, Dannenberg AJ, Jokelainen K, Bass NM. Alcoholic liver injury in the rat is associated with reduced expression of peroxisome proliferator-alpha (PPARalpha)-regulated genes and is ameliorated by PPARalpha activation. J Pharmacol Exp Ther 2004; 310:417-24. [PMID: 15016835 DOI: 10.1124/jpet.103.064717] [Citation(s) in RCA: 81] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
Alcoholic liver disease is associated with a state of hepatic fatty acid overload. We examined the effect of ethanol and different types of dietary fat on the expression of mRNA for liver fatty acid binding protein (L-FABP), peroxisome proliferator-activated receptor-alpha (PPARalpha), and peroxisomal fatty acyl CoA oxidase (FACO). Four groups of rats (n = 5) were fed intragastrically, a liquid diet with or without ethanol (10-16 g/kg/day) for 4 weeks. Pair-fed controls received isocaloric amounts of dextrose. The source of fat was either corn oil or fish oil. Ethanolfed rats developed fatty liver, necrosis, and inflammation; the changes were more severe in the fish oil-ethanol (FE) rats. PPARalpha mRNA levels were not different between groups, although there was a trend toward increased levels in ethanol-fed rats. We calculated L-FABP/PPARalpha and FACO/PPARalpha ratios as a measure of FACO and L-FABP up-regulation relative to PPARalpha expression. Both FACO/PPARalpha and L-FABP/PPARalpha ratios were significantly decreased in FE rats. However, only L-FABP/PPARalpha was decreased in corn oil plus ethanol rats. Also, the level of L-FABP/mRNA correlated inversely with the degree of fatty liver in ethanol-fed rats. Since expression of PPARalpha response genes was impaired in ethanol-fed rats, we determined whether activation of PPARalpha would normalize the PPARalpha response and prevent the pathological changes in ethanol-fed rats. Treatment with clofibrate, a PPARalpha-activating ligand, led to a marked decrease in fatty liver and complete abrogation of necroinflammatory changes in FE rats. Also, nuclear factor kappaB activation and up-regulation of tumor necrosis factor-alpha and cyclooxygenase-2 was also abolished in clofibrate-treated rats. We conclude that adaptive gene regulation of FACO and L-FABP by PPARalpha is impaired in ethanol-fed rats and that treatment with clofibrate, a PPARalpha ligand, prevents alcohol-induced pathological liver injury, possibly by reversing the above changes.
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Affiliation(s)
- Amin A Nanji
- Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104-4283, USA.
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Feierman DE, Melinkov Z, Nanji AA. Induction of CYP3A by ethanol in multiple in vitro and in vivo models. Alcohol Clin Exp Res 2003. [PMID: 12824820 DOI: 10.1111/j.1530-0277.2003.tb04424.x] [Citation(s) in RCA: 45] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
BACKGROUND Cytochrome P-450 3A (CYP3A) is responsible for the metabolism of numerous therapeutic agents. The content of CYP3A seems to be affected by ethanol ingestion. Because ethanol is used widely, its potential interaction with CYP3A is of great interest. The effects of ethanol on CYP3A content and activity were assessed in different in vivo and in vitro models. METHODS Rats fed either the Lieber-DeCarli ethanol-containing diet or an ethanol and liquid diet via the intragastric tube feeding method were used. Additionally, HepG2 cell lines that constitutively and stably express human CYP3A4 were constructed to study ethanol interactions with CYP3A4. RESULTS In all models tested, ethanol induced CYP3A activity and content, as assessed by the metabolism of fentanyl, a sensitive and specific CYP3A substrate, and Western blot analysis, respectively. In the CYP3A4-expressing HepG2 cell line, incubation with ethanol caused a dose-dependent increase in CYP3A4 activity. Ethanol also increased messenger RNA levels of CYP3A4. In the HepG2-CYP3A4 line, incubation with cycloheximide caused a decrease in fentanyl metabolism secondary to a decrease in CYP3A4 levels; this decrease was prevented by coincubation of cycloheximide with ethanol. CONCLUSIONS Ethanol induced CYP3A activity and content both in vitro and in vivo. There may be multiple mechanisms of induction of CYP3A4 by ethanol, including stabilization of messenger RNA and protein. Ethanol-induced increases in both the protein level and activity of CYP3A4 may play a role that might be of pathophysiological or clinical significance.
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Affiliation(s)
- Dennis E Feierman
- Department of Anesthesiology, The Mount Sinai Medical Center, New York, New York 10029-6574, USA.
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McKim SE, Uesugi T, Raleigh JA, McClain CJ, Arteel GE. Chronic intragastric alcohol exposure causes hypoxia and oxidative stress in the rat pancreas. Arch Biochem Biophys 2003; 417:34-43. [PMID: 12921777 DOI: 10.1016/s0003-9861(03)00349-7] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
The effect of chronic enteral ethanol on pancreatic hypoxia was investigated using the hypoxia marker, pimonidazole. Male Wistar rats were fed an ethanol-containing diet for 3 weeks using an enteral model shown to cause pancreatic damage; pimonidazole (120 mg/kg i.v.) was injected 1h before sacrifice. Pimonidazole and 4-hydroxynonenal (an index of lipid peroxidation) adducts were detected immunochemically. Breathing air with low oxygen content (8% O(2)) for 1h increased pimonidazole adduct accumulation approximately 2-fold in pancreata of nai;ve rats, confirming that this technique will detect increases in hypoxia in pancreata. Pancreata of rats fed ethanol began to show signs of damage after 3 weeks. Ethanol feeding also significantly increased pimonidazole adducts in pancreas approximately 2-fold (1 or 3 weeks of ethanol produced similar values). Concomitant with increasing hypoxia in the pancreas, alcohol also caused a significant increase in 4-hydroxynonenal adducts, indicative of increased oxidative stress. These results indicate that chronic ethanol causes hypoxia at the cellular level in the pancreas in vivo; further, the data support the hypothesis that hypoxia is involved in mechanisms of chronic alcoholic pancreatitis.
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Affiliation(s)
- Stephen E McKim
- Laboratory of Hepatobiology and Toxicology, Department of Pharmacology, University of North Carolina, Chapel Hill, NC, USA
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Preedy VR, McIntosh A, Bonner AB, Peters TJ. Ethanol dosage regimes in studies of ethanol toxicity: influence of nutrition and surgical interventions. Addict Biol 2003; 1:255-62. [PMID: 12893464 DOI: 10.1080/1355621961000124866] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
Abstract
In this review we consider some of the practical facets of acute and chronic drug regimes. In particular, we illustrate our arguments with specific reference to alcohol and draw attention to methodological constraints that might alter biochemical or physiological processes. These includes the imposition of nutritional abnormalities and surgical procedures. These two areas are highlighted because there is evidence to show that they have marked influences on metabolic parameters, exemplified by tissue protein synthesis. In general, there is no controversy as to methods for acute studies with alcohol, although some reports have failed to investigate whether intravenous, intragastric or intraperitoneal regimes more accurately mimic the clinical situation. With regard to chronic feeding regimes, evidence is provided to support the argument that, currently, the most appropriate feeding protocol ensures that both control and treated groups receive identical amounts of nutrients albeit with differences in the calories apportioned to ethanol, which is substituted by isocaloric glucose or other carbohydrates in controls. However, there are still other methods being employed: these are subject to misinterpretation as the principle of ensuring that control and ethanol-fed rats receive identical amounts of nutrients, is ignored. In this review we also draw attention to the fact that surgical procedures, which are often employed to facilitate the measurement of body parameters (for example, implantation of cannulae), themselves alter tissue metabolism. The importance of this relates to the concept of metabolic superimposition, which implicates interacting responses when two or more stresses (i.e. surgery and drug administration) are superimposed.
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Affiliation(s)
- V R Preedy
- Department of Clinical Biochemistry, King's College School of Medicine and Dentistry, London, UK
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Feierman DE, Melinkov Z, Nanji AA. Induction of CYP3A by ethanol in multiple in vitro and in vivo models. Alcohol Clin Exp Res 2003; 27:981-8. [PMID: 12824820 DOI: 10.1097/01.alc.0000071738.53337.f4] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
BACKGROUND Cytochrome P-450 3A (CYP3A) is responsible for the metabolism of numerous therapeutic agents. The content of CYP3A seems to be affected by ethanol ingestion. Because ethanol is used widely, its potential interaction with CYP3A is of great interest. The effects of ethanol on CYP3A content and activity were assessed in different in vivo and in vitro models. METHODS Rats fed either the Lieber-DeCarli ethanol-containing diet or an ethanol and liquid diet via the intragastric tube feeding method were used. Additionally, HepG2 cell lines that constitutively and stably express human CYP3A4 were constructed to study ethanol interactions with CYP3A4. RESULTS In all models tested, ethanol induced CYP3A activity and content, as assessed by the metabolism of fentanyl, a sensitive and specific CYP3A substrate, and Western blot analysis, respectively. In the CYP3A4-expressing HepG2 cell line, incubation with ethanol caused a dose-dependent increase in CYP3A4 activity. Ethanol also increased messenger RNA levels of CYP3A4. In the HepG2-CYP3A4 line, incubation with cycloheximide caused a decrease in fentanyl metabolism secondary to a decrease in CYP3A4 levels; this decrease was prevented by coincubation of cycloheximide with ethanol. CONCLUSIONS Ethanol induced CYP3A activity and content both in vitro and in vivo. There may be multiple mechanisms of induction of CYP3A4 by ethanol, including stabilization of messenger RNA and protein. Ethanol-induced increases in both the protein level and activity of CYP3A4 may play a role that might be of pathophysiological or clinical significance.
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Affiliation(s)
- Dennis E Feierman
- Department of Anesthesiology, The Mount Sinai Medical Center, New York, New York 10029-6574, USA.
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Nanji AA, Jokelainen K, Tipoe GL, Rahemtulla A, Thomas P, Dannenberg AJ. Curcumin prevents alcohol-induced liver disease in rats by inhibiting the expression of NF-kappa B-dependent genes. Am J Physiol Gastrointest Liver Physiol 2003; 284:G321-7. [PMID: 12388178 DOI: 10.1152/ajpgi.00230.2002] [Citation(s) in RCA: 193] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Induction of NF-kappaB-mediated gene expression has been implicated in the pathogenesis of alcoholic liver disease (ALD). Curcumin, a phenolic antioxidant, inhibits the activation of NF-kappaB. We determined whether treatment with curcumin would prevent experimental ALD and elucidated the underlying mechanism. Four groups of rats (6 rats/group) were treated by intragastric infusion for 4 wk. One group received fish oil plus ethanol (FE); a second group received fish oil plus dextrose (FD). The third and fourth groups received FE or FD supplemented with 75 mg. kg(-1). day(-1) of curcumin. Liver samples were analyzed for histopathology, lipid peroxidation, NF-kappaB binding, TNF-alpha, IL-12, monocyte chemotactic protein-1, macrophage inflammatory protein-2, cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and nitrotyrosine. Rats fed FE developed fatty liver, necrosis, and inflammation, which was accompanied by activation of NF-kappaB and the induction of cytokines, chemokines, COX-2, iNOS, and nitrotyrosine formation. Treatment with curcumin prevented both the pathological and biochemical changes induced by alcohol. Because endotoxin and the Kupffer cell are implicated in the pathogenesis of ALD, we investigated whether curcumin suppressed the stimulatory effects of endotoxin in isolated Kupffer cells. Curcumin blocked endotoxin-mediated activation of NF-kappaB and suppressed the expression of cytokines, chemokines, COX-2, and iNOS in Kupffer cells. Thus curcumin prevents experimental ALD, in part by suppressing induction of NF-kappaB-dependent genes.
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Affiliation(s)
- Amin A Nanji
- Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104-4283, USA.
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McKim SE, Konno A, Gäbele E, Uesugi T, Froh M, Sies H, Thurman RG, Arteel GE. Cocoa extract protects against early alcohol-induced liver injury in the rat. Arch Biochem Biophys 2002; 406:40-6. [PMID: 12234488 DOI: 10.1016/s0003-9861(02)00425-3] [Citation(s) in RCA: 57] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Oxidants have been shown to be involved in alcohol-induced liver injury. This study was designed to determine whether cocoa flavonoid extract, composed mostly of epicatechin and epicatechin oligomers, protects against early alcohol-induced liver injury in rats. Male Wistar rats were fed high-fat liquid diets with or without ethanol (10-14 g/kg per day) and cocoa extract (400 mg/kg per day) continuously for 4 weeks using an enteral feeding protocol. Mean body weight gains ( approximately 4 g/day) were not significantly different between treatment groups. Cocoa extract did not affect average daily urine ethanol concentrations ( approximately 200mg/dL). After 4 weeks, serum alanine amino transferase levels of the ethanol group were increased nearly fourfold (110+/-16 IU/L) compared to control values (35+/-3 IU/L); this effect of ethanol was blocked by cocoa extract (60+/-6 IU/L). Additionally, enteral ethanol caused severe fat accumulation, mild inflammation, and necrosis in the liver; cocoa extract significantly blunted these changes. Increases in liver TNFalpha protein levels caused by ethanol were completely blocked by cocoa extract. Further, ethanol significantly increased the accumulation of protein adducts of 4-hydroxynonenal, a product of lipid peroxidation serving as an index of oxidative stress; again this was counteracted by the addition of cocoa extract. These results indicate that dietary flavanols such as those found in cocoa can prevent early alcohol-induced liver injury.
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Affiliation(s)
- Stephen E McKim
- Laboratory of Hepatobiology and Toxicology, Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27514, USA
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Abstract
Methionine catabolism occurs mostly in the liver through the formation of S-adenosylmethionine (SAM) in a reaction catalyzed by methionine adenosyltransferase (MAT). S-adenosylmethionine is the principal biologic methyl donor, a precursor for polyamines, and in liver, it is also a precursor for reduced glutathione (GSH). Liver-specific and non-liver-specific MAT are products of two different genes, MAT1A and MAT2A, respectively. Mature liver expresses MAT1A, whereas MAT2A is expressed in extrahepatic tissues and induced during liver growth and de-differentiation. The type of MAT expressed by the cell affects the steady-state SAM level, DNA methylation, and growth rate. This has been demonstrated further by using the MAT1A knockout mouse model in which hepatic SAM and GSH levels decrease, the liver becomes larger and more susceptible to injury, and steatohepatitis develops spontaneously. Altered methionine metabolism in alcoholic liver disease results in decreased transmethylation and transsulfuration, changes that may play important pathogenic roles. Major changes include a relative switch in MAT expression; decreased hepatic SAM, GSH, and DNA methylation levels; decreased homocysteine metabolism; and hyperhomocysteinemia. Consequences of hepatic DNA hypomethylation include increased expression of c-myc and DNA strand break accumulation. One possible consequence of hyperhomocysteinemia is increased fibrogenesis. Abnormal methionine metabolism may also occur in Kupffer cells, which express both forms of MAT. If SAM levels also decrease in these cells, this may contribute to the induction of tumor necrosis factor (TNF) expression and release. In summary, altered hepatic methionine metabolism can have serious consequences that affect not only hepatocytes, but also hepatic stellate and Kupffer cells. These changes can lead to impaired antioxidant defense, altered gene expression, promotion of fibrogenesis, and even hepatocarcinogenesis.
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Affiliation(s)
- Shelly C Lu
- USC Liver Disease Research Center, USC-UCLA Research Center for Alcoholic Liver and Pancreatic Diseases, Keck School of Medicine USC, Los Angeles, CA 90033, USA.
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Sachan DS, Yatim AM, Daily JW. Comparative effects of dietary corn oil, safflower oil, fish oil and palm oil on metabolism of ethanol and carnitine in the rat. J Am Coll Nutr 2002; 21:233-8. [PMID: 12074250 DOI: 10.1080/07315724.2002.10719215] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/26/2022]
Abstract
OBJECTIVE This study was launched to determine comparative effects of corn oil (CO), safflower oil (SO), fish oil (FO) and palm oil (PO) on carnitine status and ethanol metabolism in rats. METHODS Twenty-four male Sprague-Dawley rats (300 g bw) were randomly divided into four groups (n = 6) and fed a semisynthetic diets containing fat as oils listed above. Blood and 24 hour urine samples were collected before and after dietary treatment and acute ethanol administration. Samples were analyzed for blood-ethanol concentration (BEC) and carnitine species. RESULTS The diets containing FO and PO retarded ethanol metabolism compared to the diets containing CO and SO. The effect of these dietary fats on carnitine species in plasma and urine was varied before and after dietary treatment and following a single oral ethanol dose. The liver carnitine content was higher in the PO group after dietary and ethanol treatment. CONCLUSION It is concluded that attenuation of ethanol clearance was related to unique fatty acid makeup of the oils that in part may be attributed to the composite ratio of saturated to unsaturated fatty acids in the oils.
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Affiliation(s)
- Dileep S Sachan
- Department of Nutrition, Agricultural Experiment Station, University of Tennessee, Knoxville 37996-1900, USA.
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Arteel GE, Uesugi T, Bevan LN, Gäbele E, Wheeler MD, McKim SE, Thurman RG. Green tea extract protects against early alcohol-induced liver injury in rats. Biol Chem 2002; 383:663-70. [PMID: 12033455 DOI: 10.1515/bc.2002.068] [Citation(s) in RCA: 52] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/01/2023]
Abstract
Oxidants have been shown to be involved in alcohol-induced liver injury. This study was designed to test the hypothesis that the antioxidant polyphenolic extract of green tea, comprised predominantly of epigallocatechin gallate, protects against early alcohol-induced liver injury in rats. Male Wistar rats were fed high-fat liquid diets with or without ethanol (10-14 g kg(-1) day(-1)) and green tea (300 mg kg(-1) day(-1)) continuously for 4 weeks using an intragastric enteral feeding protocol. Mean body weight gains (approximately 4 g/day) were not significantly different between treatment groups, and green tea extract did not the affect average concentration or the cycling of urine ethanol concentrations (0-550 mg dl(-1) day(-1)). After 4 weeks, serum ALT levels were increased significantly about 4-fold over control values (35+/-3 IU/l) by enteral ethanol (114+/-18); inclusion of green tea extract in the diet significantly blunted this increase (65+/-10). Enteral ethanol also caused severe fatty accumulation, mild inflammation, and necrosis in the liver. While not affecting fat accumulation or inflammation, green tea extract significantly blunted increases in necrosis caused by ethanol. Furthermore, ethanol significantly increased the accumulation of protein adducts of 4-hydroxynonenal, a product of lipid peroxidation and an index of oxidative stress; green tea extract blocked this effect almost completely. TNFalpha protein levels were increased in liver by alcohol; this phenomenon was also blunted by green tea extract. These results indicate that simple dietary antioxidants, such as those found in green tea, prevent early alcohol-induced liver injury, most likely by preventing oxidative stress.
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Affiliation(s)
- Gavin E Arteel
- Department of Pharmacology, University of North Carolina at Chapel Hill, 27599-7365, USA
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Nanji AA, Jokelainen K, Fotouhinia M, Rahemtulla A, Thomas P, Tipoe GL, Su GL, Dannenberg AJ. Increased severity of alcoholic liver injury in female rats: role of oxidative stress, endotoxin, and chemokines. Am J Physiol Gastrointest Liver Physiol 2001; 281:G1348-56. [PMID: 11705739 DOI: 10.1152/ajpgi.2001.281.6.g1348] [Citation(s) in RCA: 103] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Alcoholic liver injury is more severe and rapidly developing in women than men. To evaluate the reason(s) for these gender-related differences, we determined whether pathogenic mechanisms important in alcoholic liver injury in male rats were further upregulated in female rats. Male and age-matched female rats (7/group) were fed ethanol and a diet containing fish oil for 4 wk by intragastric infusion. Dextrose isocalorically replaced ethanol in control rats. We analyzed liver histopathology, lipid peroxidation, cytochrome P-450 (CYP)2E1 activity, nonheme iron, endotoxin, nuclear factor-kappa B (NF-kappa B) activation, and mRNA levels of cyclooxygenase-1 (COX-1) and COX-2, tumor necrosis factor-alpha (TNF-alpha), monocyte chemotactic protein-1 (MCP-1), and macrophage inflammatory protein-2 (MIP-2). Alcohol-induced liver injury was more severe in female vs. male rats. Female rats had higher endotoxin, lipid peroxidation, and nonheme iron levels and increased NF-kappa B activation and upregulation of the chemokines MCP-1 and MIP-2. CYP2E1 activity and TNF-alpha and COX-2 levels were similar in male and female rats. Remarkably, female rats fed fish oil and dextrose also showed necrosis and inflammation. Our findings in ethanol-fed rats suggest that increased endotoxemia and lipid peroxidation in females stimulate NF-kappa B activation and chemokine production, enhancing liver injury. TNF-alpha and COX-2 upregulation are probably important in causing liver injury but do not explain gender-related differences.
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Affiliation(s)
- A A Nanji
- Department of Pathology, The University of Hong Kong and Queen Mary Hospital, Hong Kong.
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Nanji AA, Lau GK, Tipoe GL, Yuen ST, Chen YX, Thomas P, Lan HY. Macrophage migration inhibitory factor expression in male and female ethanol-fed rats. J Interferon Cytokine Res 2001; 21:1055-62. [PMID: 11798463 DOI: 10.1089/107999001317205187] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023] Open
Abstract
Macrophage migration inhibitory factory (MIF) regulates macrophage accumulation at sites of injury and can promote the inflammatory response. We studied MIF expression in the intragastric feeding rat model for alcoholic liver injury. Male and age-matched female rats were fed ethanol or dextrose with fish oil. Two groups of male rats were fed medium-chain triglycerides with ethanol or dextrose. Analysis of liver histopathology, lipid peroxidation, endotoxin, mRNA, and immunohistochemistry for MIF, tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) were carried out. Male and female rats fed fish oil and ethanol showed necroinflammatory liver injury and had the highest expression of MIF, TNF-alpha, and IFN-gamma in the liver. Decreased levels of MIF protein were seen in rats with higher endotoxin levels, suggesting that preformed MIF is released into the circulation. MIF is an important mediator of the inflammatory response in alcoholic liver disease and a potential therapeutic target.
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Affiliation(s)
- A A Nanji
- Department of Pathology and Center for the Study of Liver Diseases, The University of Hong Kong and Queen Mary Hospital, Hong Kong.
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Wyatt TA, Sisson JH. Chronic ethanol downregulates PKA activation and ciliary beating in bovine bronchial epithelial cells. Am J Physiol Lung Cell Mol Physiol 2001; 281:L575-81. [PMID: 11504683 DOI: 10.1152/ajplung.2001.281.3.l575] [Citation(s) in RCA: 67] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Previously, we reported that ethanol (EtOH) stimulates a rapid increase in ciliary beat frequency (CBF) of bovine bronchial epithelial cells (BBEC). Agents activating cAMP-dependent protein kinase (PKA) also stimulate CBF. EtOH stimulates BBEC CBF through cyclic nucleotide kinase activation. However, EtOH-stimulated CBF is maximal by 1 h and subsides by 6 h, returning to baseline by 24 h. We hypothesized that the loss of EtOH-stimulated CBF was a result of downregulation of PKA activity. To determine the PKA activation state in response to EtOH, ciliated BBEC were stimulated for 0--72 h with various concentrations of EtOH and assayed for PKA. EtOH (100 mM) treatment of the cells for 1 h increased PKA activity threefold over unstimulated controls. PKA activity decreased with increasing time from 6 to 24 h. When BBEC were preincubated with 100 mM EtOH for 24 h, the stimulation of PKA by isoproterenol or 8-bromo-cAMP was abrogated. EtOH desensitizes BBEC to PKA-activating agents, suggesting that EtOH rapidly stimulates, whereas long-term EtOH downregulates, CBF via PKA in BBEC.
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Affiliation(s)
- T A Wyatt
- Research Service, Department of Veterans Affairs Medical Center, Omaha, Nebraska 68105, USA.
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40
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Eagon PK, Elm MS, Tadic SD, Nanji AA. Downregulation of nuclear sex steroid receptor activity correlates with severity of alcoholic liver injury. Am J Physiol Gastrointest Liver Physiol 2001; 281:G342-9. [PMID: 11447013 DOI: 10.1152/ajpgi.2001.281.2.g342] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Chronic ethanol ingestion in rats and humans results in significant alterations in sex steroid levels and expression of sex hormone-dependent phenotype. In this study, we used the intragastric feeding model in male rats to determine hepatic sex hormone receptor activity under circumstances of chronic ethanol exposure and differing degrees of liver injury induced by type of dietary fat. Pathological analysis and quantitation of hepatic androgen receptor (AR) and estrogen receptor (ER) activity, serum sex hormones, and sex hormone-responsive protein and mRNA expression were performed. The activity of the physiologically relevant nuclear form of both AR and ER was significantly decreased with ethanol and correlated inversely with the severity of liver injury. Serum testosterone levels, as well as expression of an androgen-dependent hepatic mRNA, were decreased by ethanol and progressive liver injury. Serum estradiol increased with liver injury. We postulate that these changes in receptor activity may be due to the oxidative stress, reduced cellular energy, and/or altered cytokine milieu known to occur in this model.
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Affiliation(s)
- P K Eagon
- Veterans Affairs Medical Center, and Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.
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41
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Kono H, Nakagami M, Rusyn I, Connor HD, Stefanovic B, Brenner DA, Mason RP, Arteel GE, Thurman RG. Development of an animal model of chronic alcohol-induced pancreatitis in the rat. Am J Physiol Gastrointest Liver Physiol 2001; 280:G1178-86. [PMID: 11352811 DOI: 10.1152/ajpgi.2001.280.6.g1178] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
This study was designed to develop an animal model of alcoholic pancreatitis and to test the hypothesis that the dose of ethanol and the type of dietary fat affect free radical formation and pancreatic pathology. Female Wistar rats were fed liquid diets rich in corn oil (unsaturated fat), with or without a standard or high dose of ethanol, and medium-chain triglycerides (saturated fat) with a high dose of ethanol for 8 wk enterally. The dose of ethanol was increased as tolerance developed, which allowed approximately twice as much alcohol to be delivered in the high-dose group. Serum pancreatic enzymes and histology were normal after 4 wk of diets rich in unsaturated fat, with or without the standard dose of ethanol. In contrast, enzyme levels were elevated significantly by the high ethanol dose. Increases were blunted significantly by dietary saturated fat. Fibrosis and collagen alpha1(I) expression in the pancreas were not detectable after 4 wk of enteral ethanol feeding; however, they were enhanced significantly by the high dose after 8 wk. Furthermore, radical adducts detected by electron spin resonance were minimal with the standard dose; however, the high dose increased carbon-centered radical adducts as well as 4-hydroxynonenal, an index of lipid peroxidation, significantly. Radical adducts were also blunted by approximately 70% by dietary saturated fat. The animal model presented here is the first to demonstrate chronic alcohol-induced pancreatitis in a reproducible manner. The key factors responsible for pathology are the amount of ethanol administered and the type of dietary fat.
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Affiliation(s)
- H Kono
- Laboratory of Hepatobiology and Toxicology, Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7365, USA
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Abstract
Oxidants have been shown to be involved in alcohol-induced liver injury. Moreover, 2-phenyl-1,2-benzisoselenazole-3(2H)-one (ebselen), an organoselenium compound and glutathione peroxidase mimic, decreases oxidative stress and protects against stroke clinically. This study was designed to test the hypothesis that ebselen protects against early alcohol-induced liver injury in rats. Male Wistar rats were fed high-fat liquid diets with or without ethanol (10-16 g/kg/d) continuously for up to 4 weeks using the intragastric enteral feeding protocol developed by Tsukamoto and French. Ebselen (50 mg/kg twice daily, intragastrically) or vehicle (1% tylose) was administered throughout the experiment. Mean urine ethanol concentrations were not significantly different between treatment groups, and ebselen did not affect body weight gains or cyclic patterns of ethanol concentrations in urine. After 4 weeks, serum ALT levels were increased significantly about 4-fold over control values (37 +/- 5 IU/l) by enteral ethanol (112 +/- 7 IU/l); ebselen blunted this increase significantly (61 +/- 8 IU/l). Enteral ethanol also caused severe fatty accumulation, mild inflammation, and necrosis in the liver (pathology score: 4.3 +/- 0.3). In contrast, these pathological changes were blunted significantly by ebselen (pathology score: 2.5 +/- 0.4). While there were no significant effects of either ethanol or ebselen on glutathione peroxidase activity in serum or liver tissue, ebselen blocked the increase in serum nitrate/nitrite caused by ethanol. Furthermore, ethanol increased the activity of NF-kappaB over 5-fold, the number of infiltrating neutrophils 4-fold, and the accumulation of 4-hydroxynonenal over 5-fold. Ebselen blunted all of these effects significantly. These results indicate that ebselen prevents early alcohol-induced liver injury, most likely by preventing oxidative stress, which decreases inflammation.
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Affiliation(s)
- H Kono
- Laboratory of Hepatobiology and Toxicology, Department of Pharmacology, University of North Carolina at Chapel Hill, 27599-7365, USA
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French SW. Intragastric ethanol infusion model for cellular and molecular studies of alcoholic liver disease. J Biomed Sci 2001; 8:20-7. [PMID: 11173972 DOI: 10.1007/bf02255967] [Citation(s) in RCA: 69] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
The intragastric alcohol infusion rat model (IAIRM) of alcoholic liver disease (ALD) has been utilized in various laboratories to study various aspects of ALD pathogenesis including oxidative stress, cytokine upregulation, hypoxic damage, apoptosis, ubiquitin-proteasome pathway and CYP2E1 induction. The basic value of the model is that it produces pathologic changes which resemble ALD including microvesicular and macrovesicular fat, megamitochondria, apoptosis, central lobular and pericellular fibrosis, portal fibrosis, bridging fibrosis, central necrosis, and mixed inflammatory infiltrate including PMNs and lymphocytes. The model is valuable because the diet and ethanol intake are totally under the control of the investigator. A steady state can be maintained with high or low blood alcohol levels for long periods. The cycling of the blood alcohol levels, when a constant infusion rate of alcohol is maintained, simulates binge drinking. Using this model the importance of dietary fat, especially the degree of saturation of the fatty acids on the induction of liver pathology, has been documented. The role of endotoxin, the Kupffer cell, TNFalpha, and NADPH oxidase have been demonstrated. The importance of 2E1 in oxidative stress induction has been shown using inhibitors of the isozyme. The importance of dietary iron in the pathogenesis of cirrhosis has been documented. Acetaldehyde has been shown to play a role in preventing liver pathology by preventing NFkappaB activation. Using the model, to maintain high blood alcohol levels is found to be necessary to demonstrate proteasomal peptidase inhibition. Ubiquitin synthesis is also inhibited at high blood alcohol levels in the IAIRM model. Oxidized proteins accumulate in the liver at high blood alcohol levels. Neoantigens derived from protein adducts formed with products of oxidation induce autoimmune mechanisms of liver injury. Thus, in many ways the model has revolutionized our understanding of the pathogenesis of ALD.
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Affiliation(s)
- S W French
- Department of Pathology, Harbor-UCLA Medical Center, 1000 W. Carson St., Torrance, CA 90509, USA.
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Lu SC, Huang ZZ, Yang H, Mato JM, Avila MA, Tsukamoto H. Changes in methionine adenosyltransferase and S-adenosylmethionine homeostasis in alcoholic rat liver. Am J Physiol Gastrointest Liver Physiol 2000; 279:G178-85. [PMID: 10898761 DOI: 10.1152/ajpgi.2000.279.1.g178] [Citation(s) in RCA: 123] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
Liver-specific and non-liver-specific methionine adenosyltransferase (MAT) are products of two genes, MAT1A and MAT2A, respectively, that catalyze the formation of S-adenosylmethionine (SAM). We previously showed that MAT2A expression was associated with more rapid cell growth. Changes in MAT expression have not been examined in animal models of alcoholic liver injury, which is the focus of the current study. After rats were fed intragastrically with ethanol and high fat for 9 wk, the mRNA level of both MAT forms doubled but only the protein level of MAT2A increased. Although liver-specific MAT activity did not change, it was 32% lower after one and 68% lower after eight weekly enteral doses of lipopolysaccharide. Hepatic levels of methionine, SAM, and DNA methylation fell by approximately 40%. c-myc was hypomethylated, and its mRNA level increased. Genome-wide DNA strand break increased. Thus in the prefibrotic stage of alcoholic liver injury, there is already a switch in MAT expression, global DNA hypomethylation, increased c-myc expression, and genome-wide DNA strand break. These changes may be important in predisposing this liver disease to malignant degeneration.
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Affiliation(s)
- S C Lu
- Division of Gastrointestinal and Liver Diseases, University of Southern California-University of California, Los Angeles 90033, USA
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45
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Horie Y, Kato S, Ohki E, Tamai H, Yamagishi Y, Ishii H. Hepatic Microvascular Dysfunction in Endotoxemic Rats After Acute Ethanol Administration. Alcohol Clin Exp Res 2000. [DOI: 10.1111/j.1530-0277.2000.tb02041.x] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023]
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Kono H, Wheeler MD, Rusyn I, Lin M, Seabra V, Rivera CA, Bradford BU, Forman DT, Thurman RG. Gender differences in early alcohol-induced liver injury: role of CD14, NF-kappaB, and TNF-alpha. Am J Physiol Gastrointest Liver Physiol 2000; 278:G652-61. [PMID: 10762620 DOI: 10.1152/ajpgi.2000.278.4.g652] [Citation(s) in RCA: 102] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
The purpose of this study was to determine whether early alcohol-induced liver injury (ALI) in females is associated with changes in CD14 on Kupffer cells, activation of hepatic nuclear factor (NF)-kappaB, and expression of tumor necrosis factor (TNF)-alpha mRNA. Male and female rats were given high-fat control or ethanol-containing diets for 4 wk using the intragastric enteral protocol. Physiological parameters were similar in both genders. Ethanol was increased as tolerance developed with higher blood levels than previously observed, resulting in a fourfold increase in aspartate aminotransferase (males 389 +/- 47 IU/l vs. females 727 +/- 66 IU/l). Hepatic pathology developed more rapidly and was nearly twofold greater and endotoxin levels were significantly higher in females after ethanol. Also, expression of CD14 on Kupffer cells was 1.5-fold greater and binding of transcription factor NF-kappaB in hepatic nuclear extracts and TNF-alpha mRNA expression were threefold greater in females. These data are consistent with the hypothesis that elevated endotoxin after ethanol triggers more activation of Kupffer cells via enhanced CD14 expression in females. NF-kappaB is activated in this process, leading to increases in TNF-alpha mRNA expression in the liver and more severe liver injury in females. It is concluded that gender differences in ALI are dependent on endotoxin and a signaling cascade leading to TNF-alpha.
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Affiliation(s)
- H Kono
- Laboratory of Hepatobiology and Toxicology, Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina 27599-7365, USA
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47
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Kono H, Enomoto N, Connor HD, Wheeler MD, Bradford BU, Rivera CA, Kadiiska MB, Mason RP, Thurman RG. Medium-chain triglycerides inhibit free radical formation and TNF-alpha production in rats given enteral ethanol. Am J Physiol Gastrointest Liver Physiol 2000; 278:G467-76. [PMID: 10712267 DOI: 10.1152/ajpgi.2000.278.3.g467] [Citation(s) in RCA: 37] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
This study determined whether free radical formation by the liver, tumor necrosis factor (TNF)-alpha production by isolated Kupffer cells, and plasma endotoxin are affected by dietary saturated fat. Rats were fed enteral ethanol and corn oil (E-CO) or medium-chain triglycerides (E-MCT) and control rats received corn oil (C-CO) or medium-chain triglycerides (C-MCT) for 2 wk. E-CO rats developed moderate fatty infiltration and slight inflammation; however, E-MCT prevented liver injury. Serum aspartate aminotransferase levels, gut permeability, and plasma endotoxin doubled with E-CO but were blunted approximately 50% with E-MCT. In Kupffer cells from E-CO rats, intracellular calcium was elevated by lipopolysaccharide (LPS) in a dose-dependent manner. In cells from E-MCT rats, increases were blunted by approximately 40-50% at all concentrations of LPS. The LPS-induced increase in TNF-alpha production by Kupffer cells was dose dependent and was blunted by 40% by MCT. E-CO increased radical adducts and was reduced approximately 50% by MCT. MCT prevent early alcohol-induced liver injury, in part, by inhibition of free radical formation and TNF-alpha production by inhibition of endotoxin-mediated activation of Kupffer cells.
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Affiliation(s)
- H Kono
- Laboratory of Hepatobiology and Toxicology, Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill 27599-7365, USA
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Enomoto N, Ikejima K, Bradford BU, Rivera CA, Kono H, Goto M, Yamashina S, Schemmer P, Kitamura T, Oide H, Takei Y, Hirose M, Shimizu H, Miyazaki A, Brenner DA, Sato N, Thurman RG. Role of Kupffer cells and gut-derived endotoxins in alcoholic liver injury. J Gastroenterol Hepatol 2000; 15 Suppl:D20-5. [PMID: 10759216 DOI: 10.1046/j.1440-1746.2000.02179.x] [Citation(s) in RCA: 93] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
The hepatotoxic effects of alcohol have been described in detail, but factors responsible for its hepatotoxicity have only partially been characterized. For example, it is known that chronic ethanol ingestion increases hepatotoxicity and produces fatty liver, hepatitis and cirrhosis. However, acute ethanol consumption reduces endotoxin hepatotoxicity. It now appears that Kupffer cells participate in several aspects of these phenomena. Previously, most studies on the effects of alcohol on liver function have focused chiefly on the hepatocyte. Recently, attention has been directed towards the effect of ethanol ingestion on Kupffer cell function, which is stimulated by gut-derived endotoxins (lipopolysaccharides) via mechanisms dependent on increased gut permeability and the possible relationship between Kupffer cells and alcohol-induced liver injury. Here we will review new evidence for the proposal that Kupffer cells and endotoxins play a pivotal role in hepatotoxicity following alcohol exposure, based on studies using the continuous intragastric enteral feeding model developed by Tsukamoto and French and an acute model developed by us.
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Affiliation(s)
- N Enomoto
- Department of Pharmacology, University of North Carolina at Chapel Hill, USA
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Yin M, Wheeler MD, Kono H, Bradford BU, Gallucci RM, Luster MI, Thurman RG. Essential role of tumor necrosis factor alpha in alcohol-induced liver injury in mice. Gastroenterology 1999; 117:942-52. [PMID: 10500078 DOI: 10.1016/s0016-5085(99)70354-9] [Citation(s) in RCA: 537] [Impact Index Per Article: 20.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
BACKGROUND & AIMS Tumor necrosis factor (TNF)-alpha is associated with increased mortality in alcoholics, but its role in early alcohol-induced liver injury is not fully understood. Recently, it was shown that injury induced by the enteral alcohol delivery model of Tsukamoto and French was reduced by antibodies to TNF-alpha. To obtain clear evidence for or against the hypothesis that TNF-alpha is involved, we studied TNF receptor 1 (TNF-R1, p55) or 2 (TNF-R2, p75) knockout mice. METHODS Long-term enteral alcohol delivery was modified for male gene-targeted mice lacking TNF-R1 and TNF-R2. Animals were given a high-fat liquid diet continuously with either ethanol or isocaloric maltose-dextrin as a control for 4 weeks. RESULTS Ethanol elevated serum levels of alanine aminotransferase nearly 3-fold in wild-type and TNF-R2 knockout mice but not in TNF-R1 knockout mice. Likewise, ethanol caused severe liver injury in wild-type mice (pathology score, 5.5 +/- 0.6) and TNF-R2 knockout mice (pathology score, 5.0 +/- 0.4), but not in TNF-R1 knockout mice (pathology score, 0.8 +/- 0.4; P < 0.001). CONCLUSIONS Long-term ethanol feeding caused liver injury in wild-type and TNF-R2 knockout mice but not in TNF-R1 knockout mice, providing solid evidence in support of the hypothesis that TNF-alpha plays an important role in the development of early alcohol-induced liver injury via the TNF-R1 pathway. Moreover, the long-term enteral ethanol feeding technique we described for the first time for knockout mice provides a useful new tool for alcohol research.
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Affiliation(s)
- M Yin
- Laboratory of Hepatobiology, Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
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50
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Lu SC, Huang ZZ, Yang JM, Tsukamoto H. Effect of ethanol and high-fat feeding on hepatic gamma-glutamylcysteine synthetase subunit expression in the rat. Hepatology 1999; 30:209-14. [PMID: 10385658 DOI: 10.1002/hep.510300134] [Citation(s) in RCA: 51] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/14/2023]
Abstract
Glutathione (GSH) is important in antioxidant defense. A major determinant of the rate of GSH synthesis is the activity of the rate-limiting enzyme, gamma-glutamylcysteine synthetase (GCS). A heavy (HS) and light subunit (LS) make up GCS; oxidative stress regulates both transcriptionally. Cis-acting elements important for the oxidative stress-induced transcriptional up-regulation of both subunits are antioxidant response element (ARE) and activator protein-1 (AP-1). Nuclear factor-kappaB (NF-kappaB) may also regulate the heavy subunit. Chronic ethanol ingestion causes oxidative stress, increases AP-1 expression, and depletes hepatic GSH. Data conflict regarding GSH synthesis and are lacking regarding GCS subunit gene expression. We examined the effect of chronic ethanol ingestion on ARE, AP-1, and NF-kappaB activity and GCS subunit expression. Male Wistar rats were fed an ethanol and high-fat (28.7% cal) diet intragastrically for 9 weeks. Liver GSH level fell by 40%, although GCS activity doubled. GCS-HS mRNA level doubled, whereas GCS-LS mRNA level remained unchanged. Electrophoretic mobility shift assay (EMSA) showed that binding to ARE, AP-1, and NF-kappaB probes all increased. In conclusion, chronic ethanol ingestion increased GCS-HS expression and GCS activity by activating cis-acting elements important for transcriptional up-regulation of GCS-HS. GCS-LS mRNA level remained unchanged despite activation of ARE and AP-1, suggesting that negative transcriptional factors may be involved or the mRNA may be unstable. Despite induction in GCS activity, GSH level fell because of alterations in the other factors important in determining the steady-state GSH level.
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Affiliation(s)
- S C Lu
- Division of Gastroenterology and Liver Diseases, USC Liver Disease Research Center, USC School of Medicine, Los Angeles, CA, USA.
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