A proteomics study of chemically induced cirrhosis in rat liver revealed the mechanism of thioacelamide hepatotoxicity 1

Chapter Introduction Chapter 1.1 Objective of Our Study In this project, we are interested in studying the changes in protein expression not only from a global but also from a temporal point of view A conventional 2DE-MS proteomics approach was applied to analyze the global protein profiles of livers of rats administered with 300 mg/kg of TAA for 3, and 10 weeks respectively This is followed by 2-DE-MS and identification of differentially regulated proteins This effort will give us an idea of the biochemical changes resulting from the induction of liver cirrhosis upon TAA administration In turn, this will provide a framework for understanding the mechanism of TAA-induced liver cirrhosis The objective of our study is twofold Firstly, the diagnosis of fibrotic liver has been hampered by the unavailability of specific and early biomarkers By using the proteomics approach, we hope to identify as many disease-related proteins as possible and these proteins can serve as potential biomarkers if proved to be disease-specific and found in body fluids Secondly, we wish to tap into the differential display capabilities of the 2-DE-MS approach to help us identify proteins that are differentially expressed in normal compared to the control male Wistar-Furth rat livers Although TAA is an established approach of inducing liver cirrhosis, very little is known about the underlying mechanisms of toxicity and fibrogenesis Therefore, it is illuminating to find proteins or pathways that are Chapter associated with the disease onset process Due to the unbiased, discovery nature of proteomics approach, unexpected or novel findings can be obtained Table 1-1 Outline of our strategy in the study of global protein profiles of TAA administered Wister-Furth rat livers Experiments week TAA week 10 week 2DE Image Analysis Mass Spectrometry Analysis of Results Results groups of male Wistar-Furth were treated with TAA for 3, and 10 weeks respectively The paired controls were left untreated As a result, liver fibrosis and cirrhosis of varying intensity were observed in the three groups of rats These samples could, not only provide us data about the protein expression differences but also temporal protein expression Samples were prepared in buffer compatible with 2DE Triplicates gels were run in 2-DE for each sample to ensure spot reproducibility 2-D gel images were analysed both manually and using PDQuest software to identify spots that were consistently and differentially present between the control and experimental gels Spots of interest were excised from gels, trypsinized and the peptides were analysed on a MALDI-TOF MS for peptide mass fingerprint and the protein identified Once differentially proteins have been identified, proteins or pathways that are involved are deduced Chapter 1.2 Liver - the metabolic center The liver is the largest organ in the body and is essential in keeping the body functioning properly It removes germs and bacteria as well as neutralizes poisons in the blood besides produces immune agents to fight infection It also synthesizes proteins that regulate blood clotting and produces bile to help absorb fats and fat-soluble vitamins In short, it is the metabolic center of our body 1.3 Structural and functional organization of the liver The liver tissue consists of lobules of muralium made of liver cell plates, which extend linearly from the portal tract to the central vein, and the transmural spaces that contain hepatic sinusoids In the liver cell plate, 15 to 25 hepatocytes extend as a monolayer, which is located between the portal tract and the terminal hepatic venule (Fig 1-1A) Within the portal tract, there is a portal venule and a hepatic arteriole, in addition to bile ducts These vessels channel blood into the hepatic sinusoids so that it can perfuse the liver cell plates before finally reaching the hepatic venules where the blood exits to the systemic circulation In contrast to the direction of blood flow, bile flows in the opposite direction, from hepatocytes near the hepatic venules to portal tract bile ducts Chapter A Bile Hepatocytes Sinusoids Portal Tract Blood Hepatic Venules Hepatic artery Portal venule Figure 1-1 B Biliary canaliculi A The basic structural elements of the liver cell plate Only hepatocytes are shown Microvilli Hepatocytes Space of Disse HSC with vitamin A granules Sinusoids Kupffer Cells Fenestrations Endothelial Cells B A traverse view of the liver cell plate The microvilli of hepatocytes extend into the perisinusoidal space of Disse for exchange of solute escape from the sinusoids through the fenestrations Hepatic stellate cells (HSC) are located in the perisinusoidal space Kupffer cells are intra-sinusoidal, participating in the removal of large particles Chapter 1.4 Compositions of the liver The liver consists of two main components; the liver cells and the extracellular space Table 1-1 discloses the relative volumes of different cellular and inter-celullar compartments in a typical liver of a rat, the animal model adopted in our current project These data was derived from stereological morphometric investigations (Blouin et al., 1977) As shown in Table 1-1, hepatocytes, sinusoids, perisinusoidal space of Disse and billiary canaliculi (collectively named lobular parenchyma) make up 96% by volume of a healthy rat liver The remaining 4% are portal triads, the hepatic and central veins (Weibel et al., 1969) Liver cells consist of parenchymal cells (hepatocytes) and non-parenchymal cells (Fig 1-2A) The latter includes endothelial cells, Kupffer cells, hepatic stellate cells (HSCs) (also called Ito cell, fat storing cells or lipocytes), bile duct cells, and pit cells Although non-parenchymal cells not contribute much to the total volume of the liver, they occupy (Blouin et al., 1977) 26.5% of the total cell membrane surface and 55% of the total fat droplet volume On the other hand, the extracellular space is made up of the perisinusoidal space of Disse, the sinusoidal lumen, billiary canaliculi and extracellular matrix (ECM) proteins Hepatic sinusoids, where Kupffer cells are located, lead the mixed blood of the terminal branches of the hepatic artery and portal vein to the central vein Billiary canaliculi, where bile is accumulated and drained, is formed by the invaginations of the adjacent plasma membranes of two hepatocytes (Fig 1-1B) The space of Disse contains many cytoplasmic dendritic projections and cell bodies of hepatic stellate cells (HSCs), Chapter abundant microvilli and hepatocytes, nerve endings and a non-electron dense complex ECMs Table 1-2 Composition of a healthy rat liver (from Blouin et al., 1977) Relative Volume Relative number of cells Extracellular space compartment Sinusoidal lumen 10.6% Disse Space 4.9% Billiary canaculi 0.4% Relative part of cells in total liver volume Hepatocytes 78.0% 60% Non-hepatocytes 6.3% Sinusoidal endothelial cells 2.8% 19% Kupffer cells 2.1% 15% Hepatic stellate cells 1.4% 6% Chapter A LIVER CELLS Parenchymal Nonparenchymal i.) Endothelial Cells ii.) Kupffer Cells iii.) Stellate Cells iv.) Pit Cells v.) i.) Hepatocytes Bile Duct cells B Presumed embryological origin Epithelial i.) Hepatocytes ii.) Bile Duct Cells Mesenchymal Hematopoietic i.) Endothelial Cells i.) Kupffer Cells ii.) Hepatic Stellate Cells ii.) Pit Cells Figure 1-2A The different cell types in the liver Figure 1-2B The presumed embryological origins of different cell types in the liver Upon activation, hepatic stellate cells (HSCs) can actually undergo transition from mesenchymal cells to more contractile myofibroblast-like cells This is an important feature of liver fibrosis (Adapted from Kaplowitz, 1996) Chapter 1.4.1 Liver cells 1.4.1.1 Parenchymal cells 1.4.1.1.1 Hepatocytes Hepatocytes are important effector cells that play key roles in the majority of liver functions Among others, they are responsible for glucogenolysis and gluconeogenesis of the liver, besides being involved in the inactivation of toxic ammonia in the urea cycle They also synthesize serum proteins such as albumins, components of the complement system and acute-phase proteins The hepatocytes are involved in the synthesis of many classes of lipoproteins as well as the catabolism of blood-derived cholesterol-enriched proteins Therefore, they also contribute to the metabolism of exogenous and endogenous lipids They are well-equipped for oxidative stress too and are thus capable of detoxification of some endo- and exogenous substances Besides, the production of bile components such as bile acids, cholesterol, phospholipids and conjugated bilirubin takes place in the hepatocytes After liver resection or severe liver injury, the parenchymal cells have phenomenal capacity for proliferation to restore organ mass The process of liver regeneration was reported to be regulated by extracellular factors as well as by many substances released by neighbouring nonparenchymal cells (Fausto, 2000) Chapter 1.4.1.2 Nonparenchymal cells 1.4.1.2.1 Sinusoidal endothelial cells Endothelial cells of the liver sinusoids (SECs) are structurally and functionally unique from other endothelial cells Not only they lack basement membrane, they are also enclosed by the cytoplasmic processes of the hepatic stellate cells (HSCs) The SECs form a filtration barrier between the sinusoidal lumen and the hepatocytes (Fig 11B), controlling the exchange of various substances between the two compartments via the dynamic changes of the fenestrae size Due to the presence of numerous plasma membrane receptors, SECs possess a large pinocytotic and endocytic capacity (Wisse, 1972) In fact, a key physiological function of SEC is the receptor-mediated endocytosis of circulating collagen, hyaluronic acid, fibronectin, laminin, nidogen and chondroitin sulphate proteoglycan (Smedsrod et al., 1994) They also perform immunological functions by acting as antigen-presenting cells (APCs) as they constitutively express MHC class I and II (Rubinstein et al., 1986), CD4, CD11, CD54 and CD106 necessary for the presentations of antigens to T cells 1.4.1.2.2 Kupffer cells Kupffer cells are located within the lumen of the sinusoids, whose double lining is contributed by the cellular extensions of the Kupffer cells themselves (Motta, 1984) Being the resident macrophages of the liver, Kupffer cells constitute the largest population of macrophages in the mammalian body (Bouwens et al., 1986) They are Chapter excised and subjected to mass spectrometric analysis to identify the differentially expressed proteins There are currently several technical challenges in 2-DE First, hydrophobic and large proteins are usually not separated well due to their poor solubility and size Second, with 2-DE, it is difficult to visualize the low abundance proteins This is especially true for body fluids such as serum and cerebrospinal fluid where the bulk of the protein content consists of serum albumins and globulins Third, due to the inherent variation in these biological samples, it is difficult to define normal protein expression patterns that can be compared with the diseased state Finally, quantification of protein spots depends heavily on image analysis, that in turn, relies on a good staining agent that is both sensitive and possesses extended linear dynamic range These properties are currently absent in Coommasie blue and silver stain Although fluorescent stain is capable of overcoming these two shortcomings, it is extremely expensive Recently, commercially Differential In-gel Electrophoresis (DIGE) was introduced In this method, two pools of proteins are labeled with different fluorescent dyes The labeled proteins are mixed in equal amount and then separated in the same 2-D gel, instead of in different gels, as in the conventional approach The advantages of using DIGE compared to conventional 2D gels are twofold First, due to inherent sensitivity of fluorescent dyes, an increased number of differences can be detected, including the smallest differences Besides, it also eliminates gel-to-gel variation because the different samples are separated together in a single gel 41 Chapter pH Condition A -ve 10 +ve Reduction and Alkylation solubilize proteins SDS-PAGE Isoelectric focusing (IEF) Condition B • • • • Staining Silver Coomassie Flourescent Autoradiography Mass spectrometric identification of spots as shown in Figure Excision of spots of interest Condition A Condition B Image Analysis Figure 1-5 Workflow in the conventional differential display proteomics using 2DE-MS (Adapted from Pandey et al., 2000) 42 Chapter Pooled internal standards Label with Cy2 Cy2 Protein Condition A Label with Cy3 Overlay Cy3 Protein Condition B Label with Cy5 Cy5 Mix labeled extracts 2DE Gel imaging with fluorescent imager Image analysis and quantification with proprietary software Figure 1-6 shows differential gel electrophoresis (DIGE) 43 Chapter 1.8.3.1.1.2 Quantitative mass spectrometry Mass spectrometry is extremely useful for identifying proteins (Eng et al., 1994), and analyzing their modifications However, the intensity of a mass spectrum is not readily correlated with the amount of analyte present in the sample Therefore, changes in protein expression level are usually quantified from the staining intensities of proteins on gels Recently, the introduction of stable isotopic nuclei (13C, 15 N and 2H) methods, originally applied in small-molecule analysis, into MS-based proteomics has allowed relative changes in proteins to be determined This approach involves biological or chemical incorporation of a stable isotope derivative in one of two states to be compared Stable isotope incorporation changes the mass of the peptides by a predictable amount The peak ratio between the underivatized and derivatized sample can then be determined with mass spectrometry and this ratio reflects the relative changes in protein expression between the two states In an example of biological incorporation of stable isotopic nuclei, microbes were grown on normal or 15N media (Lahm et al., 2000) The samples were then mixed and peak ratios determined in MS analysis of gel separated proteins However, 15 N labeling is only suitable for yeasts and microbes since 15N enriched media are expensive The same principles have been applied in a variety of ways, including measurement of the relative amount of phosphorylation using stable isotopes labeling in whole cells (Oda et al., 1999) 44 Chapter Recently, stable isotope labeling with amino acids in cell culture (SILAC) was introduced (Ong et al., 2002) This method uses 13C substituted essential amino acids, such as leucine and arginine for incorporation into mammalian cell lines So far, this has been the first description of biological incorporation of isotopic labels in mammalian systems and is extremely useful for medical proteomics In chemical incorporation, a technique has been developed (Gygi and Rist et al., 1999), termed isotopic coded affinity tags (ICAT), for quantitative mass spectrometry The ICAT reagent is a tri-functional molecule containing a biotin tag, a linker sequence containing either eight deuterium atoms (heavy reagent) or eight hydrogen atoms (light reagent), and a group reactive to cysteine residues In ICAT, proteins from the first cell populations are labeled with the heavy reagent, whereas those from second cell populations are labeled with the light reagent Equal quantities of each protein sample are combined and digested with trypsin, and cysteine-labeled peptides are isolated with an avidin column Mass spectrometry is used to analyze the paired atomic masses for each peptide, and after further fragmentation, to determine their amino acid sequences Thus, peptides can be quantified and the corresponding genes identified 45 Cell State Cell State All cysteines labeled with light ICAT containing biotin tags All cysteines labeled with heavy ICAT containing biotin tags Combine, optionally fractionate and proteolyze Affinity isolation (biotin-avidin) of ICAT labeled peptides Analysis by LC/MS Quantitate relative protein levels by measuring peak ratios Peptide Peptide Peptide Peptide 100 Relative Abundance Retention time Identify proteins by sequence information (MS/MS scan) 100 Relative Abundance Mass/Charge Figure 1-7 shows the ICAT strategy where proteins isolated from state cells are treated with the light reagent (control sample) while proteins isolated from state cells are labeled with the heavy reagent The two samples corresponding to the two different cell states are then mixed and digested with trypsin The ICAT-labeled peptides are then separated from the other peptides by affinity chromatography using an avidin column Indeed, the biotin group on the ICAT reagent has very strong affinity for the avidin stationary phase After elution of the bound peptides from the affinity media, a separation by miniaturized LC coupled to MS/MS capabilities allows both the quantitation and the identification of proteins contained in the original samples.(Adapted from Gygi et al., 1999) Chapter 1.8.3.1.1.3 Protein chips Protein chips borrow the strength of the current semi-conductor manufacturing technology (Walter et al., 2000) It is now possible to array nearly the entire complement of proteins produced from a library of cDNA clones onto a surface to probe for small molecule interactions In this approach, “bait” proteins such as antibodies are printed in an array format onto special, chemically treated surfaces The chip can then be used to screen for binding partners with protein sample of interest for specific interactions This approach has been used to study antibody-antigen interactions (De Wildt et al., 2000), protein-protein interactions (Haab et al., 2000) and protein-small molecule interactions (MacBeath et al., 2000) Another commercial instrument couples protein chips and MALDI-TOF, and is named Surface-enhanced Laser Desorption/Ionization – Time of Flight (SELDI-TOF) system (Hutchens and Yip, 1993) In this system, the chips are made of chromatographic materials with different protein-capturing properties For example, an ion-exchange material is used to capture proteins with charges The chips are first incubated with the proteins This is followed by intensive washing to remove non-specific interaction Bound proteins are then subjected to MALDI-TOF MS to obtain the mass spectra for protein and peptide samples Recently, patterns that distinguish between cancer patients and normal subjects with remarkable accuracy have been reported for ovarian (Petricoin et al., 2002) and prostate cancer (Wright et al., 1999) 47 Chapter 1.8.3.2 Structural Proteomics Structural proteomics entails the systematic understanding of the structural basis for protein interactions and functions Currently, X-ray crystallography and nuclear magnetic resonance (NMR) are the most widely-used techniques for structural determination of proteins and protein complexes, although new electron microscopy techniques such as cryo-electron microscopy and 2-D electron microscopy are gaining popularity So far, X-ray crystallography is the most accurate method for the determination of protein structures In this method, protein crystals are subjected to strong beam of Xrays produced in the synchrotrons The X-ray interacts with the electrons in the crystals to produce an electron-density map of the molecule The electron-density map is then used to decipher the molecular structure of the protein However, X-ray crystallography requires milligram quantities of a pure and mono-disperse protein which can form highquality crystals for acquisition of high-resolution data Besides, there are no universal rules for the crystallization conditions of different proteins Therefore, it is necessary to screen a wide range of conditions such as pH, salt protein concentration and co-factors for successful crystallization In contrast to X-ray crystallography, nuclear magnetic resonance (NMR) determines protein structure in aqueous solution In this method, the inter-atomic distances between specific protons are measured in a known amino acid sequence, of usually less than 200 residues In NMR, the inter-proton distances are determined 48 Chapter through space, using nuclear Overhauser effect spectroscopy (NOESY), or through bonds, by correlated spectroscopy (COSY) When these distances are coupled with covalent bond distances and angles, group polarity, chirality and van der Waals radii, the 3-D structure of a protein can be solved with the help of computation However, interproton measurement is imprecise and they are insufficient to imply a unique structure Currently, electron microscopy (EM) such as cryo-EM has become more popular in protein structure determination In cryo-EM, electrons are scattered by the charge on the nucleus screened by the electron shell of atoms, but nevertheless the electron scattering density closely resembles that seen by X-rays 1.8.3.3 Protein-protein interactions (Cell mapping proteomics) Protein-protein interaction is another important facet of proteomics Other than abundance, structure and localization, the interacting partner of a protein can indicate biological function and thus can be exploited for therapeutic purposes This is because when a protein of unknown function interacts with one of known function, they usually participate in the same or related cellular functions This concept has been termed guiltby-association (Oliver, 2000) The mapping of protein-protein interaction networks would be very valuable for understanding the biology of the cell and this area is called cell mapping proteomics 49 Chapter 1.8.3.3.1 Purification of protein complexes Using the affinity purification approach, biochemists have performed studies on protein-protein interaction by purifying the entire multi-protein complex Among these affinity methods, glutathione-S-transferase (GST)-fusion proteins, antibodies, peptides, DNA and RNA can be used as handles to co-purify specifically-bound cellular targets A protein of interest can also be tagged with an epitope such as Myc-, HA- or Flag-tags and overexpressed in cells This is followed by co-immunoprecipitation with its interaction partners with an antibody against the epitope Pull-down protein complexes can then be separated by gels and have the components identified with mass spectrometry or westernblot 1.8.3.3.2 Yeast two-hybrid system The yeast two-hybrid system is a genetic method to study protein-protein interactions It is based on the fact that when the DNA-binding domain of yeast transcription factors comes into close contact with the activation domain, increased transcription of a set of genes is induced It was first reported in 1989 (Fields et al., 1989) when it was initially designed as an assay to detect interaction between two known proteins but was later developed as a screening assay to find partners for a protein of interest (Chien et al., 1991) In the yeast two-hybrid system, open reading frames (ORFs) are fused to the DNA-binding or the activation domain of GAL4 When the proteins encoded by two 50 Chapter ORFs interact in the nucleus of the yeast cell, transcription of the reporter genes will increase as a result Subsequently, once a positive interaction is detected, the ORF is identified by sequencing the relevant clones Yeast two-hybrid is a simple yet highthroughput method for screening protein-protein interactions However, this method is frequently plagued by the presence of false positives (proteins that interact nonspecifically) and false negatives (protein-protein interactions that are undetected in a classical yeast-two-hybrid approach) 1.8.3.3.3 Phage display In phage display, bacteriophage particles are used to express a particular peptide or protein fused to a capsid protein It can then be used to screen for peptide epitopes, peptide ligands or even enzyme substrates The phage-displayed polypeptide can be selected through interaction with a target using affinity chromatography and further characterized by amplification and sequencing of the corresponding gene Generally, combinatorial peptide libraries or cDNA libraries are used in most phage-display-based studies 51 Chapter 1.8.3.3.4 Fluorescent resonance energy transfer (FRET) As the name suggests, fluorescence resonance energy transfer (FRET) (Wu et al., 1994; Clegg, 1995) refers to a phenomenon whereby a higher-energy donor fluorophore is stimulated and then it transfer the energy to the lower-energy acceptor fluorophore, causing it to emit photons This happens when two fluorophores with overlapping emission/adsorption spectra are within 100 Angstrom of one another, and their transition dipoles are appropriately oriented FRET has been made possible by the availability of green fluorescent protein (GFP) and the development of spectral derivatives of it (Tsien, 1998) so as to detect protein interactions in real time in living cells In such experiments, one protein is fused to a FRET donor, the other to a FRET acceptor The proteins are expressed inside cells, and their interaction is monitored by fluorescence microscopy or light spectroscopy Currently, use of FRET is limited by the low signal-to-noise ratios of the two available mutant GFP pairs usable for FRET, by rapid photo-bleaching of GFP and mutant GFPs, and most importantly, by the fact that FRET only works over distances up to ~100 Angstrom 1.8.4 Proteomics and the study of diseases The use of 2-DE has been associated with diseases or toxicology studies This area of research aims to contribute to the identification of proteins, via protein expression profiling, that are associated with certain diseases It is called disease proteomics or 52 Chapter clinical proteomics Clinical proteomics is set to contribute to the understanding of the disease processes, as well as finding disease biomarkers and therapeutic targets that can help in the diagnosis and prognosis of diseases 1.8.4.1 Biomarker discovery Currently, the use of proteomics approaches in disease marker discovery, predominantly for cancer, has been a hot of pursuit in clinical proteomics Among the most intensive are the attempts to identify cancer-associated proteins using 2-dimensional gel coupled to mass spectrometry (2DE-MS) For instance, proteomics approach has been used to study cancers such as hepatocellular carcinoma (Seow et al., 2000), bladder carcinoma (Celis et al., 2000), breast carcinoma (Franzen et al., 1996), renal cell carcinoma (Sarto et al., 1997) and lung carcinoma (Okuzawa et al., 1994) In these projects, disease associated proteins have been identified in cell/tissue extracts Further studies will be required to determine if these proteins can also be detected in biological fluids such as plasma or serum Another increasingly popular approach is to use SELDITOF to identify the serum proteins Microlitre of serum from many samples is applied on a surface of a protein-binding plate, with properties to bind a class of proteins The bound proteins are then treated and analysed by MALDI The mass spectra patterns obtained for different samples reflect the protein and peptide content of the samples Patterns that distinguish between cancer patients and normal subjects with remarkable accuracy have been reported in several types of cancer These include prostate cancer (Wright et al., 1999) and ovarian cancer (Petricoin et al., 2002) 53 Chapter 1.8.4.2 Study of infectious diseases Proteome analysis of virulent microorganisms can be used to elucidate their associating virulence factors and antigens These targets are important for diagnosis, therapy and protection The earliest documented proteomics efforts to analyze pathogenic microorganisms was performed over 20 years ago, in which, protein expression patterns of Escherichia Coli were characterized by Fred Neidhardt (Van Bogelen et al., 1999) under different growth conditions Currently, microorganisms that are studied using the proteomics approach are Plasmodium falciparum, Borrelia burgdorferi and Toxoplasma gondii 1.8.4.3 Toxicology 2-DE is highly sensitive for screening toxicity or to probe toxic mechanisms A common approach is to use 2-D gels to compare proteins that are expressed following treatment with a given drug with those present in untreated conditions Changes in biochemical pathways can be identified via observed changes in protein expressions When proteomic signatures for known toxic compounds are sufficiently compiled, it will be possible to use it to assess the toxicity of novel compounds For example, 2-DE and NMR have been applied to study glomerular nephrotoxicity in the rat following exposure to puramycinaminonucleoside (Cutler et al., 1999) This is done by monitoring the proteins in urine As a result of this study, a detailed understanding of the nature and progression of the proteinuria associated with 54 Chapter glomerular nephrotoxicity was achieved In a second example, a rabbit model study of lead toxicity has identified a number of differentially expressed proteins following increased lead exposure, of which several molecules, provisionally identified as glutathione-S-transferase variants, may be developed into valuable markers of lead toxicity in humans (Kanitz et al., 1999) 55 ... containing biotin tags All cysteines labeled with heavy ICAT containing biotin tags Combine, optionally fractionate and proteolyze Affinity isolation (biotin-avidin) of ICAT labeled peptides Analysis... (Cell mapping proteomics) Protein-protein interaction is another important facet of proteomics Other than abundance, structure and localization, the interacting partner of a protein can indicate... 14 Chapter 1. 5 Liver fibrosis and cirrhosis Liver cirrhosis accounts for more than 27 000 deaths in the USA per year, making it the ninth leading cause of death (Grant et al., 19 91) In Singapore,