Article first published online: 15 JUL 2013
DOI: 10.1111/jgh.12207
© 2013 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd
Journal of Gastroenterology and Hepatology
Special Issue: 7th International Symposium on Alcoholic Liver and Pancreatic Diseases and Cirrhosis. Funding for this conference was made possible (in part) by Grant 5 R13AA20691-02 from the National Institute on Alcohol Abuse and Alcoholism (NIAAA). Guest Editors: Bin Gao and Fu-Sheng Wang
Abstract
Despite major progress in understanding and managing liver disease in the past 30 years, it is now among the top 10 most common causes of death globally. Several risk factors, such as genetics, diabetes, obesity, excessive alcohol consumption, viral infection, gender, immune dysfunction, and medications, acting individually or in concert, are known to precipitate liver damage. Viral hepatitis, excessive alcohol consumption, and obesity are the major factors causing liver injury. Estimated numbers of hepatitis B virus (HBV) and hepatitis C virus (HCV)-infected subjects worldwide are staggering (370 and 175 million, respectively), and of the 40 million known human immunodeficiency virus positive subjects, 4 and 5 million are coinfected with HBV and HCV, respectively. Alcohol and HCV are the leading causes of end-stage liver disease worldwide and the most common indication for liver transplantation in the United States and Europe. In addition, the global obesity epidemic that affects up to 40 million Americans, and 396 million worldwide, is accompanied by an alarming incidence of end-stage liver disease, a condition exacerbated by alcohol. This article focuses on the interactions between alcohol, viral hepatitis, and obesity (euphemistically described here as the Bermuda Triangle of liver disease), and discusses common mechanisms and synergy.The global burden
Liver cirrhosis and hepatocellular carcinoma (HCC) represent end-stage liver disease (ESLD) and thus are associated with mortality. Globally, the incidence and prevalence of liver cirrhosis vary markedly based largely on the causative factors. In the developed world, alcohol, hepatitis C virus (HCV), and nonalcoholic steatohepatitis are the leading causes of cirrhosis, whereas viral hepatitis (especially hepatitis B virus [HBV]) is considered the leading cause in developing countries. Data from 2001 indicate that in developed countries, cirrhosis was the sixth most common cause of death among adults, and in developing countries, it claimed 320 000 lives, ranking as the ninth most common cause of death. In the European Union alone, approximately 29 million individuals suffer from chronic liver disease of whom 170 000 and 47 000 die annually from cirrhosis and liver cancer, respectively.[1] In the United States, approximately 46 700 individuals died from liver cirrhosis and cancer in 2002.[2] HBV and HCV infection are major causes of morbidity and mortality. According to World Health Organization, an estimated 2 billion people have been infected with HBV, and more than 240 million have chronic liver infections worldwide. About 600 000 people die every year from the acute or chronic consequences of HBV infection, which is endemic in China and other parts of Asia, where most people become infected during childhood; 8–10% of the adult population is chronically infected. HBV-induced liver cancer is among the top three causes of death from cancer in men, and a major cause of cancer in women in this region. Globally, cirrhosis attributable to HBV or HCV accounted for 30% and 27%, respectively, and HCC was attributable to HBV (53%) or HCV (25%). Applied to 2002 worldwide mortality estimates, chronic HBV and HCV infections represent 929 000, including 446 000 cirrhosis deaths (HBV: 235 000; HCV: 211 000) and 483 000 liver cancer deaths (HBV: 328 000; HCV: 155 000).[3]
Nonalcoholic fatty liver disease (NAFLD) comprises a wide spectrum of liver damage including steatosis, steatohepatitis, fibrosis, and cirrhosis in patients who do not consume large amount of alcohol.[4] NAFLD is a significant factor for serious liver disease because of its rising prevalence in the general population,[5] and the potential to progress to ESLD and HCC.[6] NAFLD commonly occurs in patients with obesity, diabetes, and hyperlipidemia. In the past two decades, obesity in North America has more than doubled and continues to rise worldwide. In 2005, 8% of men and 12% of women were obese. By 2030, the number of obese adults globally is projected to be 573 million individuals.[7]
The combination of chronic heavy alcohol consumption, viral hepatitis infection, and obesity represent a major assault on liver's health worldwide.
Alcoholic liver disease (ALD)
Alcoholic liver disease (ALD)
Chronic alcohol consumption results in liver disease which varies extensively between individuals in severity and progression for comparable levels of alcohol consumption. This variability could be attributed to variations in the expression and activity of individual isoforms of the alcohol-metabolizing enzymes: alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), but is also influenced by variations in patterns of alcohol intake (binge vs chronic drinking), nutritional status, gender, smoking, or abuse of other drugs. In addition, the onset and severity of ALD is strongly influenced by other comorbid conditions such as obesity or HCV infection. This increase in susceptibility to ALD is not due solely to intrahepatic factors, but may also involve alcohol-induced changes in other tissues, such as adipose tissue, central nervous system, the gut, and the immune system. Factors contributing to alcohol-induced liver disease are thus complex and systemic.[8] The spectrum of ALD includes:
- Fatty liver (hepatic steatosis), characterized histologically by lipid droplets in hepatocytes. This condition is usually reversible upon cessation of alcohol consumption, and thus is thought to be a relatively innocuous side effect of heavy drinking. However, hepatic steatosis often develops in obesity, metabolic syndrome, and type 2 diabetes, clinical conditions that involve significant metabolic defects. Thus, fatty liver by itself reflects a condition of metabolic stress that is a risk factor for the development of more severe forms of liver disease.
- Alcoholic hepatitis, an inflammatory condition characterized by significantly increased serum levels of liver enzymes (alanine aminotranferease and aspartate aminotransferase) and moderate to severe tissue damage, including necrotic foci with neutrophil infiltration. Acute alcoholic hepatitis is a potentially fatal disease that develops in a significant fraction (30–40%) of chronic heavy drinkers.
- Liver fibrosis/cirrhosis, about 10–15% of chronic heavy drinkers proceed to develop fibrosis and cirrhosis.
- HCCs occur in about 2% of cirrhotic patients.
Although factors that facilitate the development of hepatitis and cirrhosis are not well characterized, impairment in the cellular stress defense mechanisms, (e.g. oxidative stress),[9] or derailment of the balance of autocrine or paracrine mediators that are critical in maintaining normal homeostatic conditions are documented. In addition, chronic alcohol consumption interferes with liver regeneration, which under normal conditions is a highly effective repair mechanism that avoids scar tissue formation.
Mechanisms of ALD
Mechanisms of ALD
Various mechanisms have been identified for ALD (Fig. 1) which are involved at various stages of progression.
Figure 2. Hepatitis C virus (HCV), alcohol metabolism, and liver damage. ALD, alcohol dehydrogenase; ALDH, aldehyde dehydrogenase; GSH, glutathione; HCC, hepatocellular carcinoma; IFN, interferon; NAD, nicotinamide adenine dinucleotide; NADH, reduced NAD; NADP, nicotinamide adenine dinucleotide phosphate; RNS, reactive nitrogen species; ROS, reactive oxygen species.
Figure 3. Alcohol, reactive oxygen species (ROS), and mitochondrial dysfunction. CYP2E1, cytochrome P450 2E1; GSH, glutathione; GSSG, oxidized glutathione; H2O2, hydrogen peroxide; MnSOD, manganese superoxide dismutase; NO●, nitric oxide; O2●–, speroxide; ●OH, hydroxyl radical; ONOO–, peroxinitrite.
Figure 1. Known mechanisms of alcoholic liver damage. CB, cannabinoid receptor; ER, endoplasmic reticulum; Fe, Ferrous molecule; HCC, hepatocellular carcinoma; HNE, 4-hydroxynonenal; HSC, hepatic stellate cell; KC, Kupffer cells; LPS, lipopolysaccharide; MAA, malondialdehyde-acetaldehyde adduct; MDA, malondialdehyde; Mt GSH, mitochondrial glutathione; NAD, nicotinamide adenine dinucleotide; NADH, reduced NAD; ROS, reactive oxygen species; TGF, transforming growth factor
Fatty liver
Both intrahepatic and extrahepatic mechanisms are involved in hepatic steatosis:
- a) Intrahepatic factors
Hepatic steatosis due to heavy alcohol consumption has been attributed to a metabolic stress imposed by the fact that the liver is the predominant site of ethanol metabolism. Possible mechanisms include: (i) suppression of mitochondrial fatty acid β-oxidation; (ii) a limitation in the permeability of the outer mitochondrial membrane pore protein voltage-dependent anion-selective channel;[10] (iii) enhancement of hepatic uptake of free fatty acids from the circulation; (iv) increase in de novo synthesis of fatty acids and triglycerides; and (v) derailment of lipoprotein synthesis and secretion.
Chronic alcohol consumption induces a marked increase in cytochrome P450 2E1 (CYP2E1) activity, with a resultant increased demand for nicotinamide adenine dinucleotide phosphate (NADPH), an increased rate of formation of reactive oxygen species (ROS), and a decrease in oxidative stress defense capacity. At the same time, impairment of mitochondrial respiratory capacity caused by defects in the electron transport and ATP synthase complexes results in further increase in ROS formation at the mitochondrial level.[11] The ethanol-induced stress is further exacerbated by defects in the methionine cycle, resulting in a decrease in glutathione (GSH) synthesis, which contributes to the decline in oxidative stress defenses. Importantly, these conditions also reflect an increase in endoplasmic reticulum (ER) stress, a common response do the accumulation of defective proteins.[12] The resulting accumulation of stress conditions in hepatocytes causes an increased susceptibility to cell death signals. Accompanying the structural and functional changes in subcellular organelles, chronic ethanol treatment results in significant changes in the profile of transcription factors that regulate lipid homeostasis in the liver. Ethanol consumption elicits a decrease in peroxisome proliferator-activated receptor (PPAR)-α activity, thereby suppressing the catabolic lipid metabolic pathways, including peroxisomal and mitochondrial fatty acid oxidation. At the same time, ethanol increases the activity of sterol regulatory element-binding protein (SREBP)-1c and SREBP-2, which enhances lipid synthetic pathways. In addition, there has been some evidence that the adenosine monophosphate (AMP)-activated protein kinase (AMPK) is inhibited by ethanol. However, it is difficult to distinguish direct and indirect effects of ethanol. For instance, AMPK activity in the liver is regulated not only by the availability of AMP in the cell, but also responds to extracellular signals, including the adipose tissue derived cytokine adiponectin.
A related regulatory pathway affected by ethanol may involve the deacetylase silent information regulator-1 (SIRT-1), which requires activation by nicotinamide adenine dinucleotide (NAD+). Thus, the change in NAD redox state in the liver during ethanol oxidation may facilitate inhibition of SIRT-1. It has been reported that SIRT-1 activity in the liver of mice is decreased after ethanol treatment.[13] Among the targets of SIRT-1 are several key regulators of lipid metabolism, including the transcriptional coregulators peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). Its deacetylation by SIRT-1 allows it to stimulate gene expression through its interactions with PPAR-α. Furthermore, SREBP-1c is a target for SIRT-1 and its acetylation state may affect its transcriptional activity.
- b) Extrahepatic factors
Lipid metabolism in the liver is integrated with a variety of signals, including circulating hormones, cytokines, nutrition, and other factors that impinge on the intrahepatic processes leading to steatosis. While some of these factors are intrahepatic (e.g. cytokines released from Kupffer cells, endothelial cells, or stellate cells), others are dispatched by remote tissues. Of particular relevance are hormones (e.g. insulin), adiponectin and leptin (secreted from adipose tissue), and stress hormones and satiety factors that act through the hypothalamus or other brain structures to regulate food intake. Chronic ethanol consumption has a notable impact on the synthesis and secretion of several of these factors, in addition to affecting their capacity to impact lipid metabolic pathways in the liver.
Adiponectin, one of the adipokines secreted by adipose tissue to regulate lipid homeostasis, acts on multiple tissues including the liver to sensitize the response to insulin and enhance fatty acid oxidation. In animal experiments, ethanol feeding tends to suppress adiponectin secretion from adipose tissue. However, the effects of ethanol on adiponectin levels may depend on dietary factors such as the content of saturated and unsaturated fat.[14] Whether circulating adiponectin levels are similarly correlated with liver damage in human alcoholics remains unclear.[15]
Insulin plays a dominant role in integrating fatty acid and carbohydrate metabolism in the liver with the energetic needs of other tissues. Nonalcoholic hepatic steatosis that occurs in the metabolic syndrome and type II diabetes is commonly associated with insulin resistance, that is, a decreased capacity to respond to changes in circulating insulin, in multiple tissues including liver and muscle. There is strong evidence that stress responses mediated by free fatty acid accumulation or ER stress result in activation of stress response protein kinases, including protein kinase C and Jun-N-terminal kinase, which affect the intracellular signaling pathways through which insulin exerts its effects.
Alcoholic hepatitis
Alcoholic hepatitis
As described earlier, hepatic steatosis represents a severe condition of increased oxidative stress, ER, and metabolic stress. However, the mechanisms by which such stress conditions can lead to a more severe inflammatory condition remain only partly understood. Increased cell death (by necrosis or apoptosis) sets in motion further pro-inflammatory responses in the liver by producing cytokines and chemokines that help mobilize neutrophils and other inflammatory cells that further enhance liver damage. Also, it appears that overproduction of ROS by the damaged mitochondria could play a salient role. Factors that may be involved in the precipitation of alcoholic hepatitis are briefly discussed later.
Oxidative alcohol metabolism in the liver
Oxidative alcohol metabolism in the liver
Only about 2–10% of the absorbed alcohol is eliminated via the lungs and kidneys; the remaining 90% is metabolized mainly by oxidative pathways in the liver and by nonoxidative pathways in extrahepatic tissues. Oxidative metabolism in the liver results in extensive displacement of the liver's normal metabolic substrates, the production of acetaldehyde and ROS, and an increase in the NADH/NAD+ ratio (Fig. 2).
The major pathway of oxidative metabolism of ethanol in the liver involves multiple isoforms of cytosolic ADH, which results in the production of acetaldehyde. Accumulation of this highly reactive and toxic molecule contributes to liver damage. The oxidation of ethanol is accompanied by the reduction of NAD+ to NADH and, thereby, generates a highly reduced cytosolic environment in hepatocytes. The cytochrome P450 isozymes, including CYP2E1, 1A2, and 3A4, which are predominantly localized to the ER, also contribute to ethanol's oxidation to acetaldehyde in the liver. CYP2E1 is induced by chronic ethanol consumption and assumes an important role in metabolizing ethanol to acetaldehyde at elevated alcohol concentration. It also produces ROS, including hydroxyethyl, superoxide anion, and hydroxyl radicals.
Acetaldehyde, produced by ethanol oxidation, is rapidly metabolized mainly by mitochondrial ALDH2 to form acetate and NADH. Mitochondrial NADH is reoxidized by the electron transport chain (ETC). Most of the acetate resulting from ethanol metabolism escapes the liver to the blood and is eventually metabolized to CO2 by way of the tricarboxylic acid cycle in tissues such as heart, skeletal muscle, and brain, where mitochondria are capable of converting acetate to the intermediate acetyl coenzyme A.
Consequences of alcohol metabolism by oxidative pathways
Consequences of alcohol metabolism by oxidative pathways
- a) Acetaldehyde generation/adduct formation: if accumulated to high concentrations, acetaldehyde can form adducts with DNA and RNA, and decrease DNA repair. It also has the capacity to react with lysine residues on proteins including enzymes, microsomal proteins, microtubules, and affect their function. Formation of protein adducts in hepatocytes may contribute to impaired protein secretion, resulting in hepatomegaly. In addition, acetaldehyde and malondialdehyde (a by-product of lipid peroxidation) can combine and react with lysine residues on proteins, giving rise to stable malondialdehyde-acetaldehyde-protein adducts that are immunogenic and, thus, can contribute to immune-mediated liver damage.
- b) Change in hepatocyte redox state (increase in NADH/NAD+ ratio): both acute and chronic alcohol consumption shift the redox state of the liver to a more reduced level, similar to but more pronounced than the shift observed in diabetes and during starvation. Alcohol metabolism produces a significant increase in the hepatic NADH/NAD+ ratio in both the cytosol and the mitochondria, as evidenced by an increase in the lactate/pyruvate and β-hydroxybutyrate/acetoacetate ratios, respectively, and vastly increases the availability of oxidizable NADH to the ETC in the mitochondria. The liver responds to ethanol exposure in part by increasing the rate of oxygen uptake, which may lead to periods of hypoxia, particularly in the downstream (pericentral) parts of the liver lobule.
- c)Formation of ROS, reactive nitrogen species (RNS), and oxidative stress: Hepatic mitochondria produce ROS through the activity of the ETC as a by-product of oxidative phosphorylation. Normally, a small fraction of electrons entering the ETC can prematurely escape from complexes I and III and directly react with 1–3% of respiratory oxygen molecules to generate the superoxide anion radical, which is then dismutated by the mitochondrial manganese superoxide dismutase into hydrogen peroxide (H2O2). Mitochondrial glutathione peroxidase (GPx) then converts H2O2 into water by using reduced glutathione (GSH) as a cofactor. Thus, most of the ROS generated by the ETC in the normal state are detoxified by the mitochondrial antioxidant defenses. The nondetoxified portion of ROS diffuses out of mitochondria, and affects signal transduction pathways and gene expression, triggering cytokines, hormones, and growth factors, which if excessive may lead to hepatic inflammation, necrosis, and/or apoptosis. In addition, metals (e.g. iron and copper) can further react with H2O2 to produce hydroxyl radicals via the Fenton reaction (Fig. ).
Figure 3. Alcohol, reactive oxygen species (ROS), and mitochondrial dysfunction. CYP2E1, cytochrome P450 2E1; GSH, glutathione; GSSG, oxidized glutathione; H2O2, hydrogen peroxide; MnSOD, manganese superoxide dismutase; NO●, nitric oxide; O2●–, speroxide; ●OH, hydroxyl radical; ONOO–, peroxinitrite.
Nitric oxide (NO), an RNS critical for hepatocyte biology, can interact with peroxides to generate peroxynitrite, which could be detrimental to the liver depending on the amount and duration. NO is produced by inducible nitric oxide synthase which is expressed in all liver cells (i.e. hepatocytes, stellate cells, Kupffer cells, and vascular endothelial cells) and its expression is induced by interleukin (IL)-1β alone or in combination with tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and/or lipopolysaccharide (LPS).
Ethanol-induced oxidative stress has been attributed to a decrease in the NAD+ : NADH ratio, acetaldehyde formation, CYP2E1 induction, hypoxia, cytokine signaling, mitochondrial damage, LPS activation of Kupffer cells, reduction in antioxidants particularly mitochondrial and cytosolic GSH, one electron oxidation of ethanol to 1-hydroxy ethyl radical, and the conversion of xanthine dehydrogenase to xanthine oxidase.
Fibrosis and cirrhosis
Chronic heavy alcohol consumption in the presence of obesity and viral hepatitis could be damaging for the liver. While moderate alcohol consumption was associated with decreased prevalence of steatohepatitis in patients with NAFLD,[46] heavy alcohol consumption is discouraged whether an individual has NAFLD or not. The presence of common mechanisms for liver damage due to viruses, obesity, or chronic heavy alcohol consumption is relevant and may exacerbate damage to the liver when these three conditions exist. Further research is needed to clarify the interaction, if any, between moderate drinking, NAFLD, and viral hepatitis.
References
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Fibrosis and cirrhosis
Fibrosis is a common response of the liver to a chronic inflammatory condition, where hepatic stellate cells (HSC) play a critical (though not exclusive) role.[19] HSCs exist in a quiescent state in the normal liver, but can be activated directly or indirectly in response to apoptotic or necrotic cell death. Cytokines released in the tissue as a result of injury further contribute to HSC activation, resulting in the expression of a myofibroblast phenotype and stimulating the expression of extracellular matrix (ECM) proteins, in particular collagen type 1, which are not normally expressed in the liver. Under conditions of an acute tissue injury, the deposition of collagen fibers is a transient wound-healing response and is followed by fibrinolysis mediated by metalloproteases that are activated as damaged tissue is replaced by newly generated liver cells by the regenerative response. Continuous tissue damage and repair after chronic inflammation, and an imbalance in the normal liver repair mechanisms results in excessive deposition of collagen fibers.[19]
Chronic ethanol consumption can influence this process at multiple levels: (i) enhancement of the pro-inflammatory environment in the liver by stimulating the release of pro-inflammatory cytokines from macrophages and decreasing the activity of protective cell types, including natural killer cells;[20] (ii) enhancement of hepatocyte apoptosis and necrosis in response to oxidative stress and shifting in stress defense signaling pathways; (iii) activation of HSCs and collagen formation (studies on isolated HSCs have demonstrated that ethanol alters their response to transforming growth factor (TGF-β) and IFN-γ through effects on intracellular signaling pathways); and (iv) suppression of the regenerative response to tissue damage that is an essential component of the liver's repair mechanism and thereby facilitates the deposition of scar tissue, which is the hallmark of fibrosis. This is probably accompanied by a suppression of metalloproteases (e.g. by the activation of inhibitor proteins, such as plasminogen activator inhibitor-1 [PAI-1]), which normally would maintain the balance of ECM deposition and resolution to facilitate tissue repair.[21]
Common factors involved in alcohol, obesity, and viral infection
Chronic heavy alcohol consumption, obesity, and viral infection have some common features/mechanisms that may contribute to exacerbation of liver damage when these conditions coexist. Several common mechanisms between two or more of these conditions have been advocated, including oxidative stress, CYP2E1 induction, increased fat synthesis and mobilization, selected gut bacteria, free fatty acids, ER stress, immune response, among others.[22-25] Because of page limitations, only the first two mechanisms (oxidative stress and CYP2E1 induction) will be discussed. Oxidative stress due to alcohol has been discussed earlier.
Obesity and oxidative stress
Obesity involves the accumulation of body fat and is a major risk factor for metabolic syndrome, which is characterized by hyperglycemia, dyslipidemia, and hypertension.[26] Increased oxidative stress in accumulated fat has been reported as a pathogenic mechanism of obesity-associated metabolic syndrome. In nondiabetic humans, systemic oxidative stress correlated positively with fat accumulation and negatively with plasma adiponectin levels. In obese mice, ROS production was selectively increased in adipose tissue, and was accompanied by enhanced expression of NADPH oxidase and decreased expression of anti-oxidative enzymes such as superoxide dismutase in white adipose tissue and GPx in liver.[27] In cultured adipocytes, mitochondrial and peroxisomal oxidation of fatty acids activates NADPH oxidase resulting in increased oxidative stress, which caused increase in messenger RNA (mRNA) expression of inflammatory (PAI-1, TNF-α, IL-6, and monocyte chemotactic protein-1), and suppression of mRNA and secretion of anti-inflammatory (adiponectin, leptin) adipocytokines. Conversely, in obese KKAy mice, treatment with apocynin, an NADPH oxidase inhibitor, reduced ROS production in adipose tissue, increased plasma adiponectin levels, and improved hyperlipidemia and hepatic steatosis. Because oxidative stress underlies the pathophysiology of hepatic steatosis,[28] these results suggest that increased oxidative stress in obese individuals could be further exacerbated by oxidative stress due to chronic heavy alcohol consumption.
Viral infection and oxidative stress
Infection with HCV, in most cases, develops into chronic disease which is manifested by steatosis and fibrosis, as well as HCC. HCV replication induces oxidative stress (Figure 2), which contributes to insulin and interferon resistance, as well as disorders of iron metabolism. Specifically, virus core and nonstructural NS5A proteins increase ROS levels through alteration of calcium homeostasis[29] via a primary effect on the uniporter,[30] and the induction of NADPH oxidase 4.[31] In addition, E1 and E2 and the transmembrane protein NS4B increase ROS generation via ER stress and unfolded protein response,[32, 33] and activates the antioxidant defense regulated by NF-E2-related factor 2.[34] Furthermore, HCV causes mitochondrial damage and induction of double-stranded DNA breaks mediated by NO and ROS, which is abolished by NO and ROS inhibitors.[35] HCV-induced ROS causes hepatic iron accumulation in mice by reducing hepcidin transcription, further magnifying ROS production,[36] and regulating TGF-β1.[37]
CYP2E1, alcohol, and oxidative stress
As mentioned earlier, alcohol-induced oxidative stress is a major mechanism by which ethanol causes liver injury. Of the many suggested pathways by which ethanol induces a state of oxidative stress, induction of CYP2E1 is a central one. Levels of CYP2E1 are increased after acute and chronic alcohol treatment. CYP2E1 generates ROS such as the superoxide anion radical and hydrogen peroxide and, in the presence of iron catalysts, produces the hydroxyl radical, a powerful oxidant (Figure 3). The role of CYP2E1 in chronic ethanol-induced liver injury was studied in wild-type (WT) mice, CYP2E1 knockout (KO) mice and humanized CYP2E1 knockin (KI) mice. Alcohol produced oxidant stress and steatosis in WT mice, but these effects were blunted in the KO mice and restored in the KI mice. These studies show that CYP2E1 contributes to ethanol-induced oxidant stress and liver injury.[38] For a discussion of the biochemical and toxicological properties of CYP2E1 and possible therapeutic implications for treatment of ALD by CYP2E1 inhibitors, the reader is referred to the review article by Lu and Cederbaum.[39]
CYP2E1, obesity, and oxidative stress
Common factors involved in alcohol, obesity, and viral infection
Chronic heavy alcohol consumption, obesity, and viral infection have some common features/mechanisms that may contribute to exacerbation of liver damage when these conditions coexist. Several common mechanisms between two or more of these conditions have been advocated, including oxidative stress, CYP2E1 induction, increased fat synthesis and mobilization, selected gut bacteria, free fatty acids, ER stress, immune response, among others.[22-25] Because of page limitations, only the first two mechanisms (oxidative stress and CYP2E1 induction) will be discussed. Oxidative stress due to alcohol has been discussed earlier.
Obesity and oxidative stress
Obesity involves the accumulation of body fat and is a major risk factor for metabolic syndrome, which is characterized by hyperglycemia, dyslipidemia, and hypertension.[26] Increased oxidative stress in accumulated fat has been reported as a pathogenic mechanism of obesity-associated metabolic syndrome. In nondiabetic humans, systemic oxidative stress correlated positively with fat accumulation and negatively with plasma adiponectin levels. In obese mice, ROS production was selectively increased in adipose tissue, and was accompanied by enhanced expression of NADPH oxidase and decreased expression of anti-oxidative enzymes such as superoxide dismutase in white adipose tissue and GPx in liver.[27] In cultured adipocytes, mitochondrial and peroxisomal oxidation of fatty acids activates NADPH oxidase resulting in increased oxidative stress, which caused increase in messenger RNA (mRNA) expression of inflammatory (PAI-1, TNF-α, IL-6, and monocyte chemotactic protein-1), and suppression of mRNA and secretion of anti-inflammatory (adiponectin, leptin) adipocytokines. Conversely, in obese KKAy mice, treatment with apocynin, an NADPH oxidase inhibitor, reduced ROS production in adipose tissue, increased plasma adiponectin levels, and improved hyperlipidemia and hepatic steatosis. Because oxidative stress underlies the pathophysiology of hepatic steatosis,[28] these results suggest that increased oxidative stress in obese individuals could be further exacerbated by oxidative stress due to chronic heavy alcohol consumption.
Viral infection and oxidative stress
Infection with HCV, in most cases, develops into chronic disease which is manifested by steatosis and fibrosis, as well as HCC. HCV replication induces oxidative stress (Figure 2), which contributes to insulin and interferon resistance, as well as disorders of iron metabolism. Specifically, virus core and nonstructural NS5A proteins increase ROS levels through alteration of calcium homeostasis[29] via a primary effect on the uniporter,[30] and the induction of NADPH oxidase 4.[31] In addition, E1 and E2 and the transmembrane protein NS4B increase ROS generation via ER stress and unfolded protein response,[32, 33] and activates the antioxidant defense regulated by NF-E2-related factor 2.[34] Furthermore, HCV causes mitochondrial damage and induction of double-stranded DNA breaks mediated by NO and ROS, which is abolished by NO and ROS inhibitors.[35] HCV-induced ROS causes hepatic iron accumulation in mice by reducing hepcidin transcription, further magnifying ROS production,[36] and regulating TGF-β1.[37]
CYP2E1, alcohol, and oxidative stress
As mentioned earlier, alcohol-induced oxidative stress is a major mechanism by which ethanol causes liver injury. Of the many suggested pathways by which ethanol induces a state of oxidative stress, induction of CYP2E1 is a central one. Levels of CYP2E1 are increased after acute and chronic alcohol treatment. CYP2E1 generates ROS such as the superoxide anion radical and hydrogen peroxide and, in the presence of iron catalysts, produces the hydroxyl radical, a powerful oxidant (Figure 3). The role of CYP2E1 in chronic ethanol-induced liver injury was studied in wild-type (WT) mice, CYP2E1 knockout (KO) mice and humanized CYP2E1 knockin (KI) mice. Alcohol produced oxidant stress and steatosis in WT mice, but these effects were blunted in the KO mice and restored in the KI mice. These studies show that CYP2E1 contributes to ethanol-induced oxidant stress and liver injury.[38] For a discussion of the biochemical and toxicological properties of CYP2E1 and possible therapeutic implications for treatment of ALD by CYP2E1 inhibitors, the reader is referred to the review article by Lu and Cederbaum.[39]
CYP2E1, obesity, and oxidative stress
As discussed earlier, CYP2E1 is an important factor in liver disease. Several studies suggest that hepatic CYP2E1 activity is increased in patients with nonalcoholic steatohepatitis, chronic alcoholism, or morbid obesity. To study the correlation between obesity and CYP2E1, Emery et al.[40] assessed hepatic CYP2E1 activity—by determining the clearance of chlorzoxazone (CLZ), a CYP2E1-selective probe—in morbidly obese subjects with varying degrees of hepatic steatosis, and normal-weight controls. Obese subjects were evaluated at baseline and 1 year after gastroplasty, a procedure that leads to weight loss. Compared with controls, oral CLZ clearance was elevated approximately threefold in morbidly obese subjects, and was significantly higher among subjects with steatosis involving > 50% of hepatocytes. One year after gastroplasty, the median body mass index decreased by 33%, and total oral CLZ clearance declined by 46%. Thus, hepatic CYP2E1 activity is upregulated in morbidly obese subjects, and the positive association between the degree of steatosis and CYP2E1 activity preoperatively suggests that CYP2E1 induction is related to morbid obesity.[40] Similar results were obtained in genetically obese Zucker rats fed a normal diet (OB) when compared with normal Zucker rats fed a high-fat diet (HF). CYP2E1 induction was greater in both liver and fat of OB rats than in those of HF rats. The induction of CYP2E1 in liver and fat of obese patients may potentially alter the pharmacokinetics of lipophilic drugs metabolized by CYP2E1.[41]
In a recent study, Cederbaum reported that CYP2E1 induction potentiated liver injury in obese mice, and the elevated oxidative stress could be blunted by CYP2E1 inhibitors.[38] In addition, S-Adenosyl-L-methionine decreased oxidative stress, steatosis, liver injury, and mitochondrial dysfunction in the pyrazole-treated obese mice, an important finding with therapeutic implications in obesity-induced metabolic complications.
CYP2E1, HCV, and oxidative stress
CYP2E1 expression in the liver of patients with chronic hepatitis C correlated with the progression of hepatic disease (both lobular inflammation and fibrosis indices), and observed variations were consistent with the preferential distribution of CYP2E1 in the lobular zone.[42] The effect of alcohol metabolism on HCV replication and the antiviral action of IFN was studied in Huh-7 cells that harbor HCV replication and metabolize ethanol via the introduced expression of CYP2E1. Alcohol (up to 100 mmol/L) significantly increased HCV replication, which was dependent on CYP2E1 expression and alcohol-induced oxidative stress, and attenuated the anti-HCV action of IFN.[43] In chronic hepatitis C patients, cross-reactivity between CYP2E1 and specific sequences in HCV-NS5b protein can promote the development of auto-antibodies targeting conformational epitopes on the CYP2E1 surface that might contribute to hepatic injury.[44]
Alcohol's elevation of HCV titer in patients and increase of HCV RNA in replicon cells suggest that HCV replication is increased in the presence and absence of the complete viral replication cycle. Seronello et al.[45] used Huh7 human hepatoma cells that naturally express comparable levels of CYP2E1 as human liver to demonstrate that ethanol, at physiologically relevant concentrations, enhances complete HCV replication. Acetaldehyde, the first metabolite of ethanol, also enhanced HCV replication. They reported that elevated NADH/NAD+ is required for the potentiation of HCV replication by ethanol, and inhibiting CYP2E1 or ALDH suppressed replication. Thus, alteration of cellular NADH/NAD ratio is likely to play a critical role in the potentiation of HCV replication by ethanol (Fig. 4).
CYP2E1 expression in the liver of patients with chronic hepatitis C correlated with the progression of hepatic disease (both lobular inflammation and fibrosis indices), and observed variations were consistent with the preferential distribution of CYP2E1 in the lobular zone.[42] The effect of alcohol metabolism on HCV replication and the antiviral action of IFN was studied in Huh-7 cells that harbor HCV replication and metabolize ethanol via the introduced expression of CYP2E1. Alcohol (up to 100 mmol/L) significantly increased HCV replication, which was dependent on CYP2E1 expression and alcohol-induced oxidative stress, and attenuated the anti-HCV action of IFN.[43] In chronic hepatitis C patients, cross-reactivity between CYP2E1 and specific sequences in HCV-NS5b protein can promote the development of auto-antibodies targeting conformational epitopes on the CYP2E1 surface that might contribute to hepatic injury.[44]
Alcohol's elevation of HCV titer in patients and increase of HCV RNA in replicon cells suggest that HCV replication is increased in the presence and absence of the complete viral replication cycle. Seronello et al.[45] used Huh7 human hepatoma cells that naturally express comparable levels of CYP2E1 as human liver to demonstrate that ethanol, at physiologically relevant concentrations, enhances complete HCV replication. Acetaldehyde, the first metabolite of ethanol, also enhanced HCV replication. They reported that elevated NADH/NAD+ is required for the potentiation of HCV replication by ethanol, and inhibiting CYP2E1 or ALDH suppressed replication. Thus, alteration of cellular NADH/NAD ratio is likely to play a critical role in the potentiation of HCV replication by ethanol (Fig. 4).
Figure 4. Summary of alcohol and HCV interactions. HCV, hepatitis C virus; IFN, interferon; ROS, reactive oxygen species.
Concluding remarks
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