Rodent models of alcoholic liver disease: Of mice and men

Abstract

Alcoholic liver disease (ALD) is a major cause of acute and chronic liver disease worldwide. The progressive nature of ALD is well described; however, the complex interactions under which these pathologies evolve remain to be fully elucidated. Clinically there are no clear biomarkers or universally accepted, effective treatment strategies for ALD. Experimental models of ALD are an important component in identifying underlying mechanisms of alcohol-induced injury to develop better diagnostic markers, predictors of disease progression, and therapeutic targets to manage, halt, or reverse disease progression. Rodents remain the most accessible model for studying ALD pathology. Effective rodent models must mimic the natural history of ALD while allowing examination of complex interactions between multiple hepatic, and non-hepatic, cell types in the setting of altered metabolic or oxidative/nitrosative stress, inflammatory responses, and sensitivity to cytotoxic stress. Additionally, mode and duration of alcohol delivery influence hepatic response and present unique challenges in understanding disease pathology. This review provides an overview of rodent models of ALD, their strengths and weaknesses relative to human disease states, and provides insight of the potential to develop novel rodent models to simulate the course of human ALD.

Introduction

Alcoholic liver disease (ALD) is a significant cause of morbidity and mortality globally (Beier, Arteel, & McClain, 2011; Gao & Bataller, 2012; Jeong & Gao, 2008; Nath & Szabo, 2009), and refers to a spectrum of hepatic pathologies resulting from acute/binge or chronic alcohol exposure/abuse for which disease progression develops in a dose and time dependent manner (Diehl, 2002; Nath & Szabo, 2009). Alcohol abuse is a leading factor in mortality from liver disease and increases the risk for a wide range of adverse health effects (Beier et al., 2011; Gao & Bataller, 2012; Gerke, Hapke, Rumpf, & John, 1997; Siegmund, Haas, Schneider, & Singer, 2003). In the United States, the Centers for Disease Control and Prevention estimate that ≈50% of people aged 18 or older drink alcohol regularly, and of these, 5% are classified as heavy drinkers (≥30 g alcohol/day) and 15% binge drink (≥5 drinks consumed on a single occasion) (CDC, 2009; Mann, Smart, & Govoni, 2003; Naimi et al., 2003).

The toxic effects of alcohol are exerted on multiple organs; however, the liver, as the primary site of alcohol metabolism, is a major target of injury (Karinch, Martin, & Vary, 2008; Lieber, 2000, 2005). Both chronic and acute drinking deliver unique pathological consequences that affect liver disease and injury, and almost half of all end-stage liver disease in the United States is attributed to alcohol abuse (Beier et al., 2011; Gao & Bataller, 2012). Currently there are no effective, universally accepted therapies for treatment of ALD (Arteel, 2010; Diehl, 2002). Understanding the pathology of ALD is impeded by the complexity of interactions between alcohol (including amount, duration, type consumed) and different hepatic cell types. The effect(s) of alcohol is/are further complicated by host genetics, variability in immunological and metabolic responses, nutritional status, and the presence/absence of comorbid factors such as smoking, and obesity (Nath & Szabo, 2009; Tsukamoto, Machida, Dynnyk, & Mkrtchyan, 2009).

Significant progress has been made in developing animal models with which to investigate mechanisms of ALD initiation and progression. To be effective, ALD models should replicate the etiology and natural history of the human disease. In human ALD pathology the “classic” pattern of liver injury begins with hepatomegaly and alcoholic steatosis (fatty liver), an event that occurs in ≈90% of heavy alcohol users. Of the patients that develop alcohol-induced steatosis, 10–35% progress to steatohepatitis (fatty liver with inflammation), and a further 35–40% go on to develop fibrosis and cirrhosis (end-stage liver disease) (Arteel, 2010; Mann et al., 2003; Nath & Szabo, 2009; Sozio & Crabb, 2008; Szabo & Bala, 2010). Cirrhosis, the most serious form of ALD, is directly associated with duration and amount of alcohol consumed (estimated to be ≈30 g/day for 10 years) (Mann et al., 2003), and is the leading risk factor for subsequent development of hepatocellular carcinoma (HCC) (McKillop & Schrum, 2009).

During early stages of ALD, alcohol consumption diverts metabolic pathways toward hepatic triglyceride accumulation (Lieber, 1991; Sozio & Crabb, 2008). Lipid accumulation leads to increased derangement of metabolic function and increases hepatic sensitivity to toxins (de la Hall et al., 2001; Siegmund et al., 2003). This is an important prerequisite during the development of inflammation (Ramaiah, Rivera, & Arteel, 2004). Metabolic disturbances also contribute to impaired nutrient absorption and distribution, effects that are dependent on both amount of alcohol consumed, and patterns of consumption.

Chronic, long-term alcohol consumption and metabolism is associated with metabolic derangements and changes in nicotinamide adenine dinucleotide (NAD/NADH) ratios favoring accumulation of reducing equivalents in the liver (NADH). Changes in NAD/NADH ratio contribute to hepatic accumulation of triglycerides and depress the citric acid cycle. Alcohol also affects mitochondrial membrane function, metabolic demand, and generation of reactive oxygen species (ROS), a factor exacerbated by alcohol-dependent induction of cytochrome P450 2E1 (CYP2E1) (Lieber, 2005; Lu & Cederbaum, 2008). The toxic responses and injury associated with acute/binge drinking are mediated by amount and rate of alcohol consumption. During acute alcohol consumption, the majority of alcohol is metabolized by successive oxidation reactions, first via alcohol dehydrogenase (ADH) to acetaldehyde, which is in turn oxidized by acetaldehyde dehydrogenase (ALDH) to acetate and water (Lieber, 2005). Acetaldehyde is a toxic intermediate that is usually processed efficiently to prevent accumulation. However, chronic long-term alcohol ab(use) leads to CYP2E1 enzyme induction, which also generates acetaldehyde as the first metabolite during alcohol oxidation (Lieber, 2005; Lu & Cederbaum, 2008). Cytochrome P450 enzymes require oxygen for their catalytic function and reactions mediated by CYP2E1 are poorly coupled, leading to incomplete oxygen reduction and formation of oxyradicals and the partially reduced hydroxyethyl radical, thereby increasing oxidative stress responses (Albano, 2006; Lieber, 2005; Lu & Cederbaum, 2008). In addition, CYP2E1 is associated with increased alcohol tolerance observed in individuals that chronically abuse alcohol and plays a significant role in activation of pro-carcinogens to carcinogens (Dey & Cederbaum, 2006; Lieber, 2005; Lu & Cederbaum, 2008).

In addition to changes in hepatic parenchymal cell (hepatocyte) physiology and function, human ALD is characterized by a state of chronic hepatic inflammation. Alcohol leads to alterations in the gastrointestinal mucosa by disrupting epithelial tight junctions allowing for bacterial endotoxin (lipopolysaccharide; LPS) translocation into the liver via the portal vein (Beck, Morris, & Buell, 1986; Siegmund et al., 2003; Szabo & Bala, 2010). Hepatic response to gut derived endotoxin is a critical step in the development of ALD (Rao, 2009). Presence of LPS in the liver activates innate immune responses primarily via Kupffer cell sensitization (Szabo & Bala, 2010; Thurman, 1998; Yin et al., 1999) leading to intrahepatic inflammation and the production of tumor necrosis factor α (TNFα) and other pro-inflammatory cytokines (Jeong & Gao, 2008; Szabo & Bala, 2010). Additionally innate immune signaling is a mediator of tissue and organ homeostasis regulating proliferation and apoptosis of intestinal epithelial cells, and modulating liver regeneration after loss of liver mass (Seki & Schnabl, 2012). Aberrant regulation of immune system signaling may trigger harmful, inflammatory responses that contribute to tissue and organ injuries, fibrosis, and carcinogenesis (Seki & Schnabl, 2012). Collectively, these cytokine cascades combine to orchestrate liver injury, largely through neutrophil recruitment, although other inflammatory cell types (natural killer, natural killer T-lymphocytes, T-lymphocytes, and dendritic cells) also become activated, each serving unique roles in hepatic injury, repair and remodeling (Gao et al., 2011). Finally, prolonged alcohol-induced changes to liver function, hepatic circulation, and immune responses lead to hepatic stellate cell activation from a quiescent, lipid/vitamin A storing phenotype, to a pro-mitogenic, collagen producing state (de la Hall et al., 2001; Karaa, Thompson, McKillop, Clemens, & Schrum, 2008). Initially, collagen deposition is localized to perivenular and pericellular regions, resulting in portal tract-septal fibrosis that surrounds apoptotic/necrotic hepatocytes (Michalak et al., 2003), and the eventual formation of fibrous septae and scar tissue that encompasses regenerative hepatocytes (Diehl, 2002; Karaa et al., 2008; McKillop, Moran, Jin, & Koniaris, 2006; Ramaiah et al., 2004).

In developing effective models it is important to consider the goal of the intended study, the benefits to scientific research, and the potential limitations with respect to the contribution of the biochemical and pathophysiological consequences of acute or chronic alcohol consumption to organs, tissues and cells, and the role of genetic variability inherent to the population. As is evident from the preceding summary, while multiple factors contribute to ALD progression, there are three essential characteristics related to ALD pathology that must be considered in developing effective disease models. 1. Changes in hepatic metabolism, and particularly changes that lead to accumulation of lipids and/or depletion of essential nutrients, and enhanced hepatotoxicity of alcohol with formation of ROS. 2. Activation of innate inflammatory immune responses, Kupffer cell activation and associated induction of pro-inflammatory cytokines, and migration of infiltrating neutrophils. 3. Increased hepatic damage resulting from persistent inflammatory immune responses leading to stellate cell activation and collagen deposition, combined with compensatory hepatic regeneration.

In addition to these factors increasing evidence suggests other factors may be equally important in considering models of ALD. For example, alcohol induces changes in the gut microbiome that may influence innate and inflammatory immune responses (Seki & Schnabl, 2012; Son, Kremer, & Hines, 2010; Szabo & Bala, 2010; Yan et al., 2011). Indeed gut sterilization by antibiotics or alteration of gut microbiota using probiotics effectively reduces hepatic injury in experimental ALD (Miller & Spicer, 2012; Yan et al., 2011). Additionally, as the liver is a sexually dimorphic organ, the effect(s) of alcohol on liver pathology in males and females is an important factor in hepatic disease pathology (Ellefson et al., 2011; Ramaiah et al., 2004). For instance, while females are more susceptible to initial injury following alcohol consumption they are less likely to progress to cirrhosis and HCC (Frezza et al., 1990; Iimuro et al., 1997; Ikejima et al., 1998; Saunders, Davis, & Williams, 1981; Sorensen et al., 1984).

To date, no rodent model of ALD has been described that effectively replicates human alcoholic hepatitis with progression to fibrosis or cirrhosis without the addition of a secondary insult (e.g. iron, high fat diet, vitamin supplementation, LPS injection) (de la Hall et al., 2001; Karaa et al., 2008; Nanji & French, 2003). The aim of the current review is to provide a summary of the rodent models of ALD currently in use, the relative merits and potential drawbacks associated with these models, and the possible application of newer models being developed that may overcome some, or all, of these limitations.