Key words

1 Introduction

Chronic liver failure is the process of progressive destruction and regeneration of liver parenchyma leading to fibrosis and cirrhosis and would be responsible for an estimated 10.4, 7.3 and 5.3 deaths per 100,000 population in Europe, United States, and Canada, respectively [1–3]. The end-stage process of liver degeneration and failure, or cirrhosis, is the final common pathway of most forms of liver disease. Chronic liver failure may be caused by several conditions including, among others, alcohol, virus, non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steato-hepatitis (NASH), biliary disease as well as metabolic disorders. Many complications may arise from chronic liver failure, including portal hypertension, impaired metabolic capacity, synthesis dysfunction, malnutrition, ascites, hepatorenal syndrome, increased risk for the development of hepatocellular carcinoma as well as hepatic encephalopathy (Fig. 16.1).

Fig. 16.1
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Etiologic factors of chronic liver failure and resulting complications (NAFLD non-alcoholic fatty liver disease, NASH non-alcoholic steato-hepatitis)

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Hepatic encephalopathy, which is observed in approximately 80 % of patients with chronic liver failure [4–6], is a debilitating neuropsychiatric complication of liver disease. Characterized by a constellation of symptoms, including cognitive, psychiatric, and motor disturbances, hepatic encephalopathy can progress to coma and death. Hepatic encephalopathy encompasses several clinical signs such as asterixis, stupor, seizures, and coma; its severity is usually graded with the West Haven Criteria (Table 16.1) [7]. However, in order to address the universal concern about the accuracy of the West Haven scale in diagnosing the severity of overt hepatic encephalopathy, the International Society for Hepatic Encephalopathy and Nitrogen metabolism (ISHEN), the official authority for issuing updates on terminology regarding hepatic encephalopathy, has recently proposed a revised classification (Fig. 16.2) [8]. Overall, hepatic encephalopathy has a significant impact on patients’ quality of life and on their ability to function daily. Furthermore, hepatic encephalopathy leads to a poor prognosis and a greater risk of mortality [9]. This chapter will summarize the involvement of oxidative stress in the pathogenesis of hepatic encephalopathy, its consequences, and its potential role in therapeutic strategy.

Table 16.1 Grading of hepatic encephalopathy according to the West Haven criteria
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Fig. 16.2
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Proposed ISHEN classification [8]. (HE hepatic encephalopathy, MHE minimal HE, OHE overt HE)

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2 Pathogenesis of Hepatic Encephalopathy

The pathophysiologic basis of hepatic encephalopathy is multifactorial and remains unclear. However, there is general agreement that ammonia plays a key role [10]. Ammonia accumulates in the brain in chronic liver failure leading to impaired bioenergetics [10, 11], altered neurotransmission [11–13], activation of peripheral benzodiazepine receptors [12, 14], leading to the synthesis of neurosteroids [15] as well as glutamate-mediated excitotoxicity [16, 17] and excessive production of glutamine [18, 19]. However, in the setting of chronic liver failure, the correlation between ammonia and severity of hepatic encephalopathy remains inconclusive [20, 21], suggesting that other pathogenic factors may be implicated. In recent years, oxidative stress has also been suggested to be part of the pathophysiologic cascade in hepatic encephalopathy. Among the factors responsible for oxidative stress development in the setting of hepatic encephalopathy are ammonia, manganese, intracellular calcium, mitochondrial permeability transition (MPT), electron transport chain, N-methyl d-aspartate (NMDA) receptors, peripheral benzodiazepine receptor, nuclear factor-Kappa B, inflammation, and glutamine. Data from the literature suggests that the relationship between these factors and oxidative stress in the pathogenesis of hepatic encephalopathy is complex. However, a detailed review of these factors in relation to hepatic encephalopathy is beyond the scope of this chapter. In the present chapter, these factors will be tackled emphasizing the implication of ammonia and manganese-induced oxidative stress in relation with hepatic encephalopathy.

3 Oxidative Stress in Hepatic Encephalopathy

Oxidative stress, a condition in which the production of free radicals is far in excess of their rate of detoxification by endogenous mechanisms [22], refers to a state in which tissue and cellular redox balance is altered towards a more oxidizing environment [23, 24]. Precisely, oxidative stress results from an imbalance between the generation of reactive oxygen species and the cellular antioxidant defense capacity, potentially able to affect molecular structure and function. Reactive oxygen species include, among other, hydrogen peroxide (H2O2), hydroxyl radical (•OH), superoxide anion (O2 •−), and peroxynitrite (ONOO−). These reactive oxygen intermediates, which play important roles in cell signaling [25], are highly reactive due to the presence of unpaired valence shell electrons and are constantly produced during oxygen metabolism. However, in excess, reactive oxygen species are very harmful to the cell, for their reaction with cellular structures and macromolecules, and lead to cellular dysfunction. Indeed, oxidative stress affects major cellular components, including lipids, proteins, and DNA. In addition, the brain is absolutely dependent upon oxidative metabolism for cell survival and, being a highly aerobic tissue accounting for 20 % of total oxygen consumed by the body, is prone to dysfunction due to oxidative stress.

The neuropathology of hepatic encephalopathy in chronic liver failure reveals primarily changes in astrocytes (glial cells of the central nervous system) including cell swelling which consequently leads to brain edema [26–28]. Specifically, hepatic encephalopathy resulting from chronic liver failure reflects the clinical manifestation of a low-grade cerebral edema that develops after exhaustion of the volume-regulatory capacity of the astrocytes in response to ammonia and other hepatic encephalopathy-precipitating factors (e.g., inflammation, infection, hyponatremia) [29, 30]. As a consequence, astrocyte swelling triggers a complex signaling cascade which relies on NMDA receptor activation and elevation of intracellular calcium concentration, which result in increased formation of reactive oxygen species through activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and nitric oxide synthase (NOS) [29, 31]. Since oxidative stress in turn promotes astrocyte swelling, a self-amplifying signaling loop between osmotic- and oxidative stress is proposed [31, 32]. Among the consequences of this oxidative stress response are protein-tyrosine nitration, oxidation of RNA, and activation of Zn2+-dependent transcription [31–35].

3.1 Ammonia-Induced Oxidative Stress in Hepatic Encephalopathy

Evidence for the involvement of oxidative stress in hepatic encephalopathy initially arose from the observation that Alzheimer type II astrocytes, a distinctive neuropathologic finding in brains of patient with hepatic encephalopathy, contain large amounts of lipofuscin pigments [36], consisting of peroxidized lipids [37]. Excessive amounts of lipofuscin pigments were also detected in ammonia-treated astrocyte cultures [37, 38]. Subsequently, more than 20 years ago, O’Connor and Costell [39] postulated that oxidative stress is implicated in the pathophysiologic mechanisms responsible for hepatic encephalopathy. They reported that hyperammonemic mice displayed evidence of lipid peroxidation in the brain [39]. Since these findings have been reported, substantial evidence from cell cultures and animal studies for an important role of hyperammonemia and oxidative stress in the pathogenesis of hepatic encephalopathy has been reported.

3.1.1 In Vitro Evidence of Ammonia-Induced Oxidative Stress in Hepatic Encephalopathy

Much of the evidence for a role of oxidative stress in ammonia neurotoxicity has been derived from cell culture studies [40]. Evidence suggests a close interrelation between astrocyte swelling, a characteristic feature of hepatic encephalopathy, and the production of reactive oxygen species leading to oxidative stress [41]. Specifically, astrocytes have been suggested to be a major source of reactive oxygen species under simulated hepatic encephalopathy conditions [41, 42]. Indeed, astrocytes exposed to a pathophysiological concentration of ammonia were found to stimulate the production of free radicals and reactive oxygen species. In hypoosmotically treated cultured rat astrocytes, a NMDA receptor-dependent elevation of the intracellular calcium concentration was identified to be essential to swelling-dependent reactive oxygen species generation [43]. Ammonia-induced free radical generation via the activation of NADPH oxidase was observed in cultured astrocytes [42–44], while astrocyte swelling triggers a p47(phox)-dependent NADPH oxidase-catalyzed reactive oxygen species production [31]. In addition, in astrocyte cultures exposed to ammonia, increased heme oxygenase-1 (HO-1) [44] and inducible NOS (iNOS) expression as well as nitric oxide production were identified [34, 45]. Ammonia has also been shown to significantly increase soluble guanylyl cyclase [46], a source of nitric oxide, and decrease cellular glutathione (GSH) level [47], a major endogenous antioxidant, in cultured astrocytes. Furthermore, natriuretic peptides, which are known to attenuate the production of reactive oxygen species in other systems [47, 48], were shown to reduce the accumulation of reactive oxygen species in ammonia-treated cultured astrocytes [49]. Altogether, these studies suggest that oxidative stress is induced by astrocytes in conditions associated with increased levels of ammonia.

Another factor by which ammonia and oxidative stress have been related to hepatic encephalopathy is the MPT, a calcium-dependent process characterized by the opening of the permeability transition pore (PTP) in the inner mitochondrial membrane. Oxidative stress triggers the induction of the MPT and, as a consequence, there is an increased permeability to protons, ions, and other solutes <1,500 Da [50], leading to a collapse of the mitochondrial inner membrane potential. Loss of the mitochondrial membrane potential results in osmotic swelling of the mitochondrial matrix, movement of metabolites across the inner membrane, defective oxidative phosphorylation, cessation of ATP synthesis, and the generation of reactive oxygen species. It was reported that, in cultured astrocytes, oxidative stress would be involved in the induction of the MPT by ammonia, suggesting that oxidative stress and the subsequent induction of the MPT contribute to the pathogenesis of hepatic encephalopathy [51, 52]. Also, treatment of cultured astrocytes with ammonia caused a significant dissipation of the mitochondrial membrane potential as well as an increase in the mitochondrial permeability to 2-deoxyglucose. Both of these changes were blocked by cyclosporin A, a MPT inhibitor. Similarly, ammonia caused a decrease in the mitochondrial calcein fluorescence (an index of the MPT), which was also blocked by cyclosporin A [53].

3.1.2 In Vivo Evidence of Ammonia-Induced Oxidative Stress in Hepatic Encephalopathy

Evidence for the implication of oxidative stress in the pathogenesis of hepatic encephalopathy has also been reported in studies in experimental animals. For example, NOS activity has been shown to be increased in the brains of portacaval-shunted rats [54]. iNOS and neuronal NOS (nNOS) protein expression is also increased in the brains of these animals [22, 55, 56], whereas brain endothelial NOS (eNOS) protein expression is increased in thioacetamide (TAA)-induced cirrhosis in the rat [57]. It was also reported that astrocyte swelling stimulated the production of cerebral nitric oxide in ammonia-treated rats [58]. An increase in HO-1 mRNA expression as well as a decrease in copper/zinc-superoxide dismutase (Cu/Zn SOD) gene expression have been reported in the brains of portacaval-shunted rats [59]. Protein tyrosine nitration, a consequence of oxidative stress, was also demonstrated in the cerebral cortex of these animals [34, 56].

In a rat model of chronic liver failure (portacaval anastomosis), neurons were immunoreactive to nNOS, whereas iNOS was expressed in pyramidal-like cortical neurons and perivascular astrocytes [56]. In the same animals, nitrotyrosine immunoreactivity was found in pyramidal-like cortical neurons and in perivascular astrocytes. It was also demonstrated that nNOS and iNOS are produced in the Purkinje (neuronal) cells and Bergmann glial cells in rats following portacaval anastomosis [55]. TAA-cirrhotic rats showed nNOS immunoreactivity in stellate and basket neurons and eNOS immunoreactivity in perivascular glial cells of the white matter [57]. In the same experimental model of chronic liver failure, eNOS was located in Purkinje cell bodies and vessels endothelial cells [57]. Taken together, these data suggest that neuronal, glial, and endothelial cells are all sources of free radicals and nitric oxide in hepatic encephalopathy indicating that oxidative stress in hepatic encephalopathy is a multicellular phenomenon.

Systemically, the relationship between hyperammonemia and oxidative stress differs from that depicted in the brain. Indeed, in another model of chronic liver failure and hyperammonemia/hepatic encephalopathy, the bile-duct ligated rat, Bosoi et al. [60] observed the presence of systemic oxidative stress and cerebral edema. The authors suggested that systemic oxidative stress might be an important “first hit,” which, followed by increases in ammonia, leads to the onset of brain edema [60]. In a similar model of cirrhosis, an increase in lipid peroxidation and reduction in antioxidant enzymes in the cerebral cortex and cerebellum were reported [61]. Interestingly, the administration of N-acetylcysteine exerted a protective effect through the attenuation of oxidative stress [61].

3.1.3 Human Studies

While considerable evidence indicates the presence of oxidative stress markers in experimental models of hepatic encephalopathy, documentation of oxidative stress in humans is limited. Increased amount of lipofuscin pigments was found in brains of patients with hepatic encephalopathy [36, 40]. Elevated blood levels of reactive oxygen species were also identified in patients with hepatic encephalopathy resulting from chronic alcohol consumption, which was associated with decrease antioxidant capacity [62]. Increased SOD activity, thiobarbituric acid reactive substances, and decreased catalase activities were observed in cirrhotic children [63]. Increased nitric oxide after transjugular intrahepatic portosystemic shunt insertion in patients with cirrhosis was also reported [64]. Furthermore, postmortem cortical brain tissue samples from patients with cirrhosis dying with or without hepatic encephalopathy were analyzed and compared with brains from patients without liver disease [35]. The results indicate that hepatic encephalopathy in patients with cirrhosis is associated with oxidative stress, protein tyrosine nitration, and RNA oxidation [35].

In postmortem human brain tissue obtained from autopsies of patients with cirrhosis and hepatic encephalopathy, a whole human genome microarray approach revealed altered expression of genes related to oxidative stress [65]. Specifically, expression levels of genes involved in oxidative stress defense, such as HO-1, selenoprotein-V, peroxiredoxin-4, and peroxisome proliferator-activated receptor α (PPARα), were elevated in patients with cirrhosis with hepatic encephalopathy but not in patients with cirrhosis without hepatic encephalopathy, when compared with controls [65]. Taken together, these data strongly suggest a role for oxidative stress in the pathogenesis of hepatic encephalopathy in patients with chronic liver failure and indicate that cerebral oxidative stress is a hallmark of hepatic encephalopathy in patients with liver failure.

3.2 Manganese-Induced Oxidative Stress in Hepatic Encephalopathy

Manganese is an essential trace element found in a variety of biological tissues and is necessary for normal functioning of several physiological processes including amino acid, lipid, protein, and carbohydrate metabolism [66]. Manganese is also an important component of a number of cerebral enzymes, in particular, glutamine synthetase, an ammonia detoxifying enzyme. At low levels, manganese binds with superoxide dismutase to form MnSOD, an important mitochondrial antioxidant enzyme [67]. However, when excessive, manganese contributes to neurological abnormalities such as parkinsonism and dystonia [68]. Occupational exposure to excessive manganese levels leads to neurotoxicity, referred to as manganism, which resembles Parkinson’s disease [69]. Chronic exposure of various cell types to manganese was shown to induce oxidative stress [70–72].

Manganese has also been implicated in the pathogenesis of hepatic encephalopathy [73]. Manganese highly accumulates in astrocytes [74, 75], which renders these cells more vulnerable to its toxicity. Consistent with this vulnerability, manganese has been shown to decrease antioxidant capacity [72] and generate oxidative stress [72, 76, 77], which are prevented by pre-treatment with N-acetylcysteine [78]. Manganese also brings about mitochondrial dysfunction [79, 80], including decreased energy production [72] and the induction of the MPT [81], and causes histopathological changes in astrocytes (Alzheimer type II change) [82]. Interestingly, morphologic and functional changes after exposure of astrocytes to manganese are similar to those observed after ammonia treatment. Cultured astrocytes exposed to ammonia (5 mM) or manganese acetate (100 mM) were shown to increase both free radicals production and l-arginine uptake (a precursor of nitric oxide), and such effects were synergized when manganese was co-treated with ammonia [77, 83]. Similarly, exposure of primary cortical astrocytes to a low concentration of manganese (10 μM) was shown to potentiate interferon-gamma and tumor necrosis factor-alpha-induced expression of iNOS mRNA and protein along with an increased production of nitric oxide [84]. The potentiating effect was a consequence of the activation of soluble guanylate cyclase and mitogen-activated protein kinase (MAPK) signaling pathways [84]. Cultured astrocytes exposed to manganese were also shown to inhibit glutamate uptake by a process involving oxidative stress [85, 86]. Additionally, it was demonstrated that treatment of rats with manganese chloride led to an increase in manganese level in brain that was accompanied by the development of pathological changes similar to those seen in hepatic encephalopathy (Alzheimer type II astrocytosis), and such changes were significantly reduced when rats were treated with antioxidant N-actetylcysteine. In primary rat cortical neurons exposed to manganese, an increase in biomarkers of oxidative damage (F(2)-isoprostanes), which was prevented by pretreatment with the antioxidant Trolox (hydrophilic analog of vitamin E), was reported [87]. These results were confirmed in mice exposed to manganese [87]. Finally, it was recently demonstrated that manganese leads to an increase in markers of oxidative stress in rat brain chronically exposed to manganese [88]. These studies suggest that manganese contributes to oxidative stress in hepatic encephalopathy and that such effect is exacerbated in the presence of ammonia.

Patients with chronic liver failure and those who had surgically created portal-systemic shunts have elevated plasma and brain manganese levels, most likely reflecting the combined effects of hepatocellular failure, impaired biliary excretion, and the presence of portal-systemic shunting of blood [89]. This may lead to selective manganese accumulation in the globus pallidus, caudate nucleus, and putamen, and the adjacent areas of the basal ganglia manifest as hyperintensity of these brains areas on T1-Magnetic Resonance Imaging [89, 90]. Indeed, elevated manganese levels were found in the globus pallidus obtained at autopsy from patients with chronic liver failure [90, 91]. Disturbances of manganese homeostasis may partly account for the cognitive impairment associated with chronic liver failure [92].

4 Antioxidant Strategies for the Treatment of Hepatic Encephalopathy

Treatment of experimental animals suffering from hyperammonemia and hepatic encephalopathy with antioxidants (e.g., ascorbate, alpha-tocopherol, dimethylsulfoxide) was shown to have beneficial effects by improving antioxidant status as well as their clinical condition [93, 94]. The antioxidant melatonin was shown to reduce blood and brain ammonia level as well as attenuate brain lipid peroxidation in rats after TAA injection [95]. Additionally, increased malondialdehyde levels and decreased glutathione peroxidase, catalase, and SOD activities were found in the hippocampal tissue of rats with portal hypertension (a model of low-grade hepatic encephalopathy), and such effects were reversed when rats were treated with curcumin, a known antioxidant [96]. Rats treated with morin (3,4,7,2′,4′-pentahydroxyflavone), a flavonol, were shown to be protected against oxidative stress in brains of chronic hyperammonemic rats [97]. It was recently demonstrated that guanosine, a nucleoside exhibiting antioxidant properties [98, 99], was neuroprotective in a rat model of chronic hepatic encephalopathy by reducing oxidative stress markers in the brain [100].

The antioxidant N-acetylcysteine has proven useful in reducing brain edema in acute liver failure [101] and in the management of patients with acute liver failure [102–105]. In addition, N-acetylcysteine was shown to delay the progression of encephalopathy in azoxymethane-induced acute liver failure in mice, as well as to reduce brain water content [101]. Finally, hypothermia, which has been shown to improve brain edema in animals and humans with acute liver failure, is also known to reduce free radical production [106]. Interestingly, N-acetylcysteine was able to ameliorate spatial memory and motor coordination deficits observed experimental chronic liver failure (bile-duct ligated rats) [61]. N-Acetylcysteine supplementation decreased lipid peroxidation and was also able to restore the activity of antioxidant enzymes as well as structural deficits observed in the cortex and cerebellum of cirrhotic animals with hepatic encephalopathy. Together, these data clearly demonstrate that the protective effect of N-acetylcysteine in experimental hepatic encephalopathy is mediated through attenuation of oxidative stress, suggesting a therapeutic role for N-acetylcysteine in patients afflicted with hepatic encephalopathy.

5 Conclusion

Oxidative stress has evolved in recent years as a major pathogenetic factor in hepatic encephalopathy and experimental evidence for oxidative stress in brain in experimental models of hepatic encephalopathy due to chronic liver failure is increasing. Indeed, several reports suggest that oxidative stress participates in the pathophysiologic cascade responsible for hepatic encephalopathy. While the factors responsible for oxidative stress formation in hepatic encephalopathy remain incompletely understood, it appears that ammonia and manganese would be partly responsible for the production of reactive oxygen species. Although increased oxidative stress in hepatic encephalopathy resulting from chronic liver failure has been demonstrated by some groups, its consequences are not fully established. Additional studies on the role of oxidative stress in chronic hepatic encephalopathy are warranted. Increased oxidative stress has been documented in several studies and antioxidants were shown to be protective against ammonia-induced astrocyte swelling and cerebral edema in liver failure. Antioxidant therapy such as N-acetylcysteine is already being used in the management of acute liver failure and its complications. Other antioxidants could prove to be valuable adjuncts to traditional hepatic encephalopathy therapies, such as ammonia-lowering strategies, in the context of chronic liver failure. Further studies are needed in order to assess these possibilities.