Does alcohol directly stimulate pancreatic fibrogenesis? Studies with rat pancreatic stellate cells (original) (raw)
Related papers
Pancreatology, 2007
caused by PDGF, which was prevented by antioxidant N-acetyl-L -cysteine, ROS scavenger tiron, and the NADPH oxidase inhibitor diphenylene iodium. The effects of PDGF on NADPH oxidase activity and DNA synthesis were prevented in PaSCs isolated from the pancreas of mice with a genetic deficiency of p47 phox . Conclusions: Ethanol causes proliferation of stellate cells by augmenting the activation of the cell's NADPH oxidase system stimulated by PDGF. These results provide new insights into the mechanisms of alcohol-induced fibrosing disorders.
Alcoholism-clinical and Experimental Research, 2009
Background: Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are major enzymes responsible for metabolism of ethanol. Genetic polymorphisms of ADH1B, ADH1C, and ALDH2 occur among racial populations. The metabolic effect and metabolites contribute to pathogenesis of pancreatic injury. The goal of this study was to determine the functional expressions and cellular localization of ADH and ALDH families in human pancreas.Methods: Fifty five surgical specimens of normal pancreas as well as 15 samples each for chronic pancreatitis and pancreatic cancer from archival formalin-fixed paraffin-embedded tissue specimens were investigated. Class-specific antibodies were prepared by affinity chromatographies from rabbit antisera raised against recombinant human ADH1C1, ADH4, ADH5, ADH7, ALDH1A1, ALDH2, and ALDH3A1. The isozyme expression patterns of ADH/ALDH were identified by isoelectric focusing, and the activities were assayed spectrophotometrically. The protein contents of ADH/ALDH isozymes were determined by immunoblotting, and the cellular localizations were detected by immunohistochemistry and histochemistry.Results: At 33 mM ethanol, pH 7.5, the activities were significantly different between allelic phenotypes of ADH1B. The activity of ALDH2-inactive phenotypes was slightly lower than ALDH2-active phenotypes at 200 μM acetaldehyde. The protein contents were in the following decreasing order: ALDH1A1, ALDH2, ADH1, and ADH5. ADH1B was detected in the acinar cells and ADH1C in the ductular, islet, and stellate cells. The expression of ADH1C appeared to be increased in the activated pancreatic stellate cells in chronic pancreatitis and pancreatic cancer.Conclusions: Alcohol dehydrogenase and ALDH family members are differentially expressed in the various cell types of pancreas. ADH1C may play an important role in modulation of activation of pancreatic stellate cells.
Ethanol-induced cytotoxicity in rat pancreatic acinar AR42J cells: Role of fatty acid ethyl esters
Alcohol and Alcoholism, 2007
Aims: To understand the mechanism(s) of alcoholic pancreatitis and role of fatty acid ethyl esters (FAEEs, nonoxidative metabolites of ethanol) in ethanol-induced pancreatic injury. Methods: A time-and concentration-dependent synthesis of FAEEs and the cytotoxicity of ethanol and its predominant fatty acid esters were studied in rat pancreatic tumour (AR42J) cells in cultures. Role of FAEEs in ethanol-induced cytotoxicity was investigated by measuring the synthesis of FAEEs, injury markers and apoptosis in cells incubated simultaneously with ethanol and FAEE synthase inhibitor, 3-benzyl-6-chloro-2-pyrone. The cells were pre-incubated with caspase-3 inhibitor (N-acetyl-DEVD-CHO) to measure the effect of caspase-3 inhibition on ethanol-induced apoptosis. Results: The levels of FAEEs synthesized in cell cultures incubated with 800 mg% ethanol for 6 h were ∼10-fold higher (60 nmol/25 × 10 6 cells) than those in cells incubated with 100 mg% ethanol (5.4 nmol/25 × 10 6 cells). Ethanol exposure resulted in a concentration-dependent apoptosis (10, 12 and 13% at 200, 400 and 800 mg% ethanol, respectively, vs 5% in controls). A similar concentration-dependent apoptosis was also found in the cells incubated with ethyl oleate (one of the predominant FAEEs reported in alcoholic patients). Inhibition of FAEE synthesis and resultant apoptosis was found in the cells incubated simultaneously with pancreatic FAEE synthase inhibitor and ethanol. Ethanol-induced apoptosis was significantly inhibited in cells pre-incubated with caspase-3 inhibitor. Conclusions: These results support our hypothesis that ethanol-induced cytotoxicity in AR42J cells is mediated by the non-oxidative metabolite(s) of ethanol, and caspase-3 mediated apoptosis could be one of the mechanisms involved in ethanol-induced pancreatic injury.
The Journal of Pathology, 2000
Chronic alcoholic pancreatitis (CAP) is characterized by progressive pancreatic ®brosis and loss of the acinar cell mass, but the pathogenesis of pancreatic ®brosis in the human is poorly understood. It has been recently suggested that lipid peroxidation-derived aldehydes such as 4hydroxynonenal (HNE) are involved in tissue damage and ®brosis in other organs. The aim of this study was to evaluate the role of oxidative stress in the development of alcohol-induced pancreatic ®brosis in humans, and to assess the contribution of pancreatic periacinar stellate cells (PSC) in the in vivo synthesis of extracellular matrix components during CAP. Lipid peroxidation was evaluated in tissue specimens obtained from patients with CAP who underwent surgical procedures, by immunohistochemistry using a monoclonal antibody directed against HNE±protein adducts. Immunohistochemical determination of collagen type I, a-smooth muscle actin (a-SMA), and the b subunit of human platelet-derived growth factor (PDGF-Rb) was also performed. In addition, the tissue mRNA expression of procollagen I, PDGF-Rb, and transforming growth factor-b1 (TGF-b1) was evaluated by in situ hybridization. In CAP, increased formation of HNE±protein adducts was evident in acinar cells adjacent to the interlobular connective tissue that stained positively for collagen type I. HNE staining was absent in normal pancreas. Several non-parenchymal periacinar cells (PSC) underlay the HNE-stained acinar cells. Those PSC stained positively for a-SMA and PDGF-Rb and showed active synthesis of procollagen type I by in situ expression of the speci®c mRNAs. The pattern of expression of PDGF-Rb mRNA re¯ected that observed in immunostaining, showing increased amounts of transcripts in PSC. TGF-b1 mRNA expression was increased in CAP, but transcripts were found in several cell types including PSC, acinar, and ductal cells. These results indicate that signi®cant lipid peroxidation phenomena occur in CAP and that they are associated with active synthesis of collagen by PSC.
Exposure of precision-cut rat liver slices to ethanol accelerates fibrogenesis
AJP: Gastrointestinal and Liver Physiology, 2010
Ethanol metabolism in the liver induces oxidative stress and altered cytokine production preceding myofibroblast activation and fibrogenic responses. The purpose of this study was to determine how ethanol affects the fibrogenic response in precision-cut liver slices (PCLS). PCLS were obtained from chow-fed male Wistar rats (200–300 g) and were cultured up to 96 h in medium, 25 mM ethanol, or 25 mM ethanol and 0.5 mM 4-methylpyrazole (4-MP), an inhibitor of ethanol metabolism. Slices from every time point (24, 48, 72, and 96 h) were examined for glutathione (GSH) levels, lipid peroxidation [thiobarbituric acid-reactive substance (TBARS) assay], cytokine production (ELISA and RT-PCR), and myofibroblast activation [immunoblotting and immunohistochemistry for smooth muscle actin (SMA) and collagen]. Treatment of PCLS with 25 mM ethanol induced significant oxidative stress within 24 h, including depletion of cellular GSH and increased lipid peroxidation compared with controls ( P < 0....
Toxicology and Applied Pharmacology, 2010
Pancreatitis caused by activation of digestive zymogens in the exocrine pancreas is a serious chronic health problem in alcoholic patients. However, mechanism of alcoholic pancreatitis remains obscure due to lack of a suitable animal model. Earlier, we reported pancreatic injury and substantial increases in endogenous formation of fatty acid ethyl esters (FAEEs) in the pancreas of hepatic alcohol dehydrogenase (ADH)-deficient (ADH −) deer mice fed 4% ethanol. To understand the mechanism of alcoholic pancreatitis, we evaluated dose-dependent metabolism of ethanol and related pancreatic injury in ADH − and hepatic ADH-normal (ADH +) deer mice fed 1, 2 or 3.5% ethanol via Lieber-DeCarli liquid diet daily for 2 months. Blood alcohol concentration (BAC) was remarkably increased and the concentration was ~1.5-fold greater in ADH − vs. ADH + deer mice fed 3.5% ethanol. At the end of the experiment, remarkable increases in pancreatic FAEEs and significant pancreatic injury indicated by the presence of prominent perinuclear space, pyknotic nuclei, apoptotic bodies and dilation of glandular ER were found only in ADH − deer mice fed 3.5% ethanol. This pancreatic injury was further supported by increased plasma lipase and pancreatic cathepsin B (a lysosomal hydrolase capable of activating trypsinogen), trypsinogen activation peptide (by-product of trypsinogen activation process) and glucose-regulated protein 78 (endoplasmic reticulum stress marker). These findings suggest that ADH-deficiency and high alcohol levels in the body are the key factors in ethanol-induced pancreatic injury. Therefore, determining how this early stage of pancreatic injury advances to inflammation stage could be important for understanding the mechanism(s) of alcoholic pancreatitis.
Experimental and Molecular Pathology, 2018
The single most common cause of chronic pancreatitis (CP, a serious inflammatory disease) is chronic alcohol abuse, which impairs hepatic alcohol dehydrogenase (ADH, a major ethanol oxidizing enzyme). Previously, we found ~5 fold greater fatty acid ethyl esters (FAEEs), and injury in the pancreas of hepatic ADH deficient (ADH −) vs. hepatic normal ADH (ADH +) deer mice fed 3.5g% ethanol via liquid diet daily for two months. Therefore, progression of ethanolinduced pancreatic injury was determined in ADH − deer mice fed ethanol for four months to delineate the mechanism and metabolic basis of alcoholic chronic pancreatitis (ACP). In addition to a substantially increased blood alcohol concentration and plasma FAEEs, significant degenerative changes, including atrophy and loss of acinar cells in some areas, ultrastructural changes evident by such features as swelling and disintegration of endoplasmic reticulum (ER) cisternae and ER stress were observed in the pancreas of ethanol-fed ADH − deer mice vs. ADH + deer mice. These changes are consistent with noted increases in pancreatic injury markers (plasma lipase, pancreatic trypsinogen activation peptide, FAEE synthase and cathepsin B) in ethanol-fed ADH − deer mice. Most importantly, an increased levels of pancreatic glucose regulated protein (GRP) 78 (a prominent ER stress marker) were found to be closely associated with increased phosphorylated eukaryotic initiation factor (eIF) 2α signaling molecule in PKR-like ER kinase branch of unfolded protein response (UPR) as compared to X box binding protein 1S and activating transcription factor (ATF)6-50kDa protein of inositol requiring enzyme 1α and ATF6 branches of UPR, respectively, in ethanol-fed ADH − vs. ADH + deer mice. These results along with findings on plasma FAEEs, and pancreatic histology and injury markers suggest a metabolic basis of ethanol-induced pancreatic injury, and provide new avenues to understand metabolic basis and molecular mechanism of ACP.
Molecular pharmacology, 1996
Chronic pancreatitis is characterized by inflammation and fibrosis leading to tissue destruction; in industrialized nations, alcohol abuse is the cause of 70-80% of cases of pancreatitis in adults. The purpose of the current work was to determine whether free radical adducts are produced by the pancreas during the early phases of chronic exposure to ethanol. Accordingly, rats were chronically fed ethanol using the model of continuous enteral infusion developed by Tsukamoto et al.[Am. J. Physiol. 247: R595-R599 (1984)]. Histological evaluation revealed only mild acinar steatosis and spotty necrosis after 4 weeks of alcohol treatment; the pancreatic enzymes lipase and amylase were not elevated. Furthermore, no fibrosis was detected, nor were there differences in pancreatic collagen alpha 1(l) mRNA levels between the dietary control and ethanol-treated groups. After 4 weeks, rats were injected with the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (1 g/kg intravenously), and ...