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Free Radical Research, November 2013; 47(11): 869–880 © 2013 Informa UK, Ltd. ISSN 1071-5762 print/ISSN 1029-2470 online DOI: 10.3109/10715762.2013.837577

REVIEW ARTICLE

Special issue on “Oxidative stress and redox signaling in the gastrointestinal tract and related organs” for Free Radical Research Involvement of free radicals and oxidative stress in NAFLD/NASH Y. Sumida1, E. Niki1,2, Y. Naito1 & T. Yoshikawa1

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1Department of Gastroenterology and Hepatology, Kyoto Prefectural University of Medicine, Kyoto, Japan, and 2National Institute of Advanced Industrial Science and Technology, Health Research Institute, Ikeda, Osaka, Japan

Abstract Non-alcoholic fatty liver disease (NAFLD) is now the most common liver disease affecting high proportion of the population worldwide. NAFLD encompasses a large spectrum of conditions ranging from fatty liver to non-alcoholic steatohepatitis (NASH), which can progress to cirrhosis and cancer. NAFLD is considered as a multifactorial disease in relation to the pathogenic mechanisms. Oxidative stress has been implicated in the pathogenesis of NAFLD and NASH and the involvement of reactive oxygen species (ROS) has been suggested. Many studies show the association between the levels of lipid oxidation products and disease state. However, often neither oxidative stress nor ROS has been characterized, despite oxidative stress is mediated by multiple active species by different mechanisms and the same lipid oxidation products are produced by different active species. Further, the effects of various antioxidants have been assessed in human and animal studies, but the effects of drugs are determined by the type of active species, suggesting the importance of characterizing the active species involved. This review article is focused on the role of free radicals and free radical-mediated lipid peroxidation in the pathogenesis of NAFLD and NASH, taking characteristic features of free radical-mediated oxidation into consideration. The detailed analysis of lipid oxidation products shows the involvement of free radicals in the pathogenesis of NAFLD and NASH. Potential beneficial effects of antioxidants such as vitamin E are discussed. Keywords: Antioxidant, fatty liver, lipid peroxidation, steatohepatitis, vitamin E Abbreviations: 8-isoP, 8-isoprostaglandin F2α; AAPH, 2,2′-azobis(2-amidinopropane) dihydrochloride; CYP, cytochrome P450; DG, diacylglycerol; FFA, free fatty acid; H(P)ETE, hydro(pero)xyeicosatetraenoic acid; H(P)ODE, hydro(pero)xyoctadecadienoic acid; Keap1-Nrf2, Kelch-like ECH-associated protein 1- nuclear erythroid 2-related factor 2; HO-1, hemeoxygenase-1; LXR, liver X receptor; MDA, malondialdehyde; NAFL, non-alcoholic fatty liver; NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis; PL, phospholipid; RCT, randomized controlled trial; ROS, reactive oxygen species; SOD, superoxide dismutase; TBARS, thiobarbituric acid reactive substances; TG, triacylglycerol; TNF-α, tumor necrosis factor-α TRX, thioredoxin; UDCA, ursodeoxycholic acid

Introduction Non-alcoholic fatty liver disease (NAFLD), a hepatic manifestation of metabolic syndrome, is now the most common liver disorder affecting high proportion of the population worldwide. The incidence of NAFLD is increasing with increasing two major risk factors, obesity and Type-2 diabetes due to lifestyle and diet, but it is noteworthy that this disease is being recognized in children as well as adults. The characteristic feature of NAFLD is an excessive accumulation of fat, notably triglyceride, in the liver and it encompasses a large spectrum from benign steatosis to non-alcoholic steatohepatitis (NASH), liver cirrhosis, liver failure, and even hepatocellular carcinoma [1–3]. Under these circumstances, liver fatty acid synthesis from glucose is stimulated, redirecting toward triglyceride formation [4].

Specific alterations in hepatic lipid composition have been observed by lipidomic analysis in the plasma from NASH patients [5]. In a practice guideline published by the American Association for the Study of Liver Diseases (AASLD) [6], the definition of NAFLD requires that (a) there is evidence of hepatic steatosis, either by imaging or by histology and (b) there are no causes for secondary hepatic fat accumulation such as significant alcohol consumption, use of steatogenic medication or hereditary disorders. NAFLD is histologically further categorized into non-alcoholic fatty liver (NAFL) and NASH. NAFL is defined as the presence of hepatic steatosis with no evidence of hepatocellular injury in the form of ballooning of the hepatocytes. NASH is defined as the presence of hepatic steatosis and inflammation with hepatocyte injury (ballooning) with or without fibrosis.

Correspondence: Etsuo Niki, National Institute for Advanced Industrial Science and Technology, Health Research Institute, 1 - 8-31 Midorigaoka, Ikeda, Osaka 563 - 8577, Japan. Tel: ⫹ 81-72-751-9991. Fax: ⫹ 81-72-751-9964. E-mail: [email protected] and Yuji Naito, Molecular Gastroenterology and Hepatology, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kamigyo-ku, Kyoto 602-8566, Japan. Tel: ⫹ 81-75-251-5519; Fax: ⫹ 81-75-251-710. E-mail: [email protected] (Received date: 28 April 2013; Accepted date: 20 August 2013; Published online: 17 September 2013)

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870 Y. Sumida et al. NAFLD and NASH are multifactorial diseases and oxidative stress has been implicated in their pathogenesis, although a causal relationship or a pathogenic link between NAFLD/NASH and oxidative stress has not been established [2,4,7]. The role of oxidative stress in liver steatosis production and its progression to inflammation leading to steatohepatitis has been discussed in relation to alterations in metabolic and pro-inflammatory transcription factors expression. Increased levels of reactive oxygen species (ROS) and lipid oxidation products and decreased levels of antioxidant enzymes such as superoxide dismutase (SOD) and catalase and antioxidant compounds such as glutathione have been observed in patients of NAFLD/NASH compared with those observed in the healthy subjects [8]. It was reported that the levels of α-tocopherol, lutein, zeaxanthin, lycopene, and β-carotene were significantly decreased in NASH patients compared with those of controls [9]. Statistically high levels of reactive carbonyl species were observed in plasma of NAFLD subjects compared with those observed in healthy subjects [10]. However, the oxidative stress and ROS have rarely been characterized, despite oxidative stress is mediated by multiple active species by different mechanisms and different oxidants produce the same lipid oxidation products. Some oxidation is mediated by enzymes while others by non-enzymatic oxidants. The non-enzymatic oxidation proceeds either by free radical mechanisms or by non-radical mechanisms. It is noteworthy that the effects of drugs are determined by the type of active species and reaction mechanisms. Thus, it is important to analyze specific oxidation products in order to determine the responsible active species. This review article is focused on the role of free radicals and oxidative stress in the pathogenesis of NAFLD/NASH and potential beneficial effects of radical-scavenging antioxidants. NAFLD/NASH, oxidative stress, and free radicals Many human and animal studies have observed the association between disease state of NAFLD/NASH and biomarkers of oxidative stress or lipid oxidation [8,11–17]. Lipid oxidation products are useful biomarkers for oxidative stress in vivo, since the mechanisms of lipid oxidation is now well understood and their physiological levels in human fluids and tissues are high enough to be identified and quantified with gas chromatography–mass spectrometry (GC/MS) or high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) [18,19]. It may be noted that thiobarbituric acid reactive substances (TBARS) and malondialdehyde (MDA), both used frequently as biomarker of lipid oxidation, may be formed by multiple oxidants and are not suitable for analysis of active species in lipid oxidation. Lipids are oxidized in vivo by multiple oxidants [20]. Some are enzymes such as lipoxygenase, cyclooxygenase, and cytochrome P450, while others are non-enzymatic free radicals and non-radical oxidants such as hypochlorite and singlet oxygen. These oxidants oxidize unsaturated

fatty acids and cholesterol to give the same products. For example, hydroperoxyeicosatetraenoic acids are produced from arachidonic acid, one of the major polyunsaturated fatty acids (PUFA) in vivo, by lipoxygenase, singlet oxygen, and free radicals. They are readily reduced to the corresponding hydroxyeicosatetraenoic acids (HETE) by glutathione peroxidases and selenoprotein P. HETEs are also produced by the oxidation of arachidonic acid with cytochrome P450 (CYP). Numerous regio-, stereo- and enantio-isomers of HETEs are detected in human samples and their identification is important to know the responsible oxidants. Interestingly, lipoxygenase and CYP oxidize arachidonic acid to give specifically 12(S)-cis,trans-HETE and 12(R)cis,trans-HETE, respectively [21], while free radicalmediated lipid oxidation termed lipid peroxidation gives racemic 12-cis,trans- and 12-trans,trans-HETE, respectively [20]. Although the enzymatic lipid oxidation does not always proceed with complete enantio-specificity [22], the detailed analysis of lipid oxidation products enables us to identify the responsible oxidant. It should be noted that trans,trans-hydroxyfatty acids such as HODE and HETE are specific products of free radical-mediated lipid peroxidation [19,20,23]. Isoprostanes, a series of prostaglandin-like compounds formed by a free radical-mediated peroxidation of arachidonic acid independent of cyclooxygenase, are also specific marker of free radical oxidation [19]. It is advised to measure isoprostanes by LC–MS or GC–MS, rather than ELISA. It was reported that 9- and 13-hydroxyoctadecadienoic acid (HODE), major lipid peroxidation products produced from linoleic acid, were significantly elevated in patients with NASH compared with patients with steatosis and that a strong correlation was observed between these oxidation products and liver histopathology such as inflammation, fibrosis, and steatosis [24]. It is noted that these HODEs were racemic, suggesting that these HODEs were produced by free radical oxidation. Furthermore, it was reported that a decrease in HODE and HETE correlated with improved histological disease in the setting of a therapeutic trial in NASH [17]. Other studies also observed higher levels of lipid oxidation products in NAFLD/NASH patients than healthy control subjects [15], but often the responsible oxidants are not clear. The elucidation of responsible oxidants is important for development of drugs against NAFLD/NASH. Many, if not all, studies showed the ameliorating effects of radicalscavenging antioxidants against NAFLD/NASH [25,26], suggesting the involvement of free radicals in the pathogenesis. Notably, recent studies suggesting the beneficial effect of vitamin E against NAFLD/NASH have attracted much attention [27–29]. It is noteworthy that vitamin E acts as an efficient peroxyl radical-scavenging antioxidant in vivo to prevent lipid peroxidation, but not against the oxidation mediated by enzymes and non-radical oxidants such as hypochlorite [30,31]. The fatty acid residues of phospholipids and cholesteryl esters are oxidized similarly as free fatty acids. Interestingly, lipoxygenase oxidizes cholesteryl esters in

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Free radical and oxidative stress in NAFLD/NASH

intact form [32]. It was reported that the levels of cholesterylester hydroperoxide in the liver of patients with NASH was increased when compared with isolated steatosis and normal liver [33], although it is not clear whether this hydroperoxide was produced by free radicals or by lipoxygenase. Lipids are oxidized by non-radical mechanisms as well. As noted above lipoxygenase and cyclooxygenase oxidize PUFA selectively and specifically. Hypochlorite produced by myeloperoxidase oxidizes unsaturated fatty acids and cholesterol by non-radical mechanisms, although it may produce free radicals in the reactions with amines and thiols [34]. Singlet oxygen produced by the reaction of hypochlorite and hydrogen peroxide oxidizes unsaturated lipids non-selectively by non-radical mechanism. For example, singlet oxygen oxidizes linoleic acid to yield 9-, 10-, 12-, and 13-hydroperoxyoctadecadienoic acid (HPODE), 10- and 12-HPODE being specific products of singlet oxygen oxidation. Various cytochrome P450 isoforms have been identified and characterized [35]. They act principally as monooxygenase and oxidizes PUFA to give hydroxides and epoxides. They also play major role in detoxification of xenobiotics in liver. The oxidation of lipids by CYP proceeds by non-radical mechanisms which cannot be inhibited by vitamin E. CYP oxidizes arachidonic acid to give all the regio-isomers of HETE produced by lipid peroxidation, that is 5-, 8-, 9-, 11-, 12-, and 15-HETE, although the enzymatic oxidation gives only cis,trans-HETEs, whereas free radical oxidation gives both cis,trans- and trans,trans-HETEs. CYP induces ω-hydroxylation of arachidonic acid to give 19and 20-HETE and epoxidation to give 5,6- 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acid [36]. Cholesterol is also oxidized by multiple oxidants [20,37–39]. Various cytochrome P450 isoforms oxidize cholesterol specifically to give 4α-, .7α-, 24-, and 27hydroxycholesterol, while 25-hydroxycholesterol is formed by cholesterol 25-hydroxylase which is not a CYP enzyme [38,40,41]. The levels of side chain hydroxycholesterols have been observed to be higher in patients with NAFLD than in control healthy subjects [42]. It is noted that hydroxycholesterols are ligand for liver X receptor (LXR) which is involved in the control of various aspects of lipid metabolism in liver [43]. Fatty acid metabolism is transcriptionally regulated by two reciprocal systems: peroxisome proliferator-activated receptor α controls fatty acid degradation, whereas sterol regulatory element-binding protein-1c activated by LXR regulates fatty acid synthesis. Hypochlorous acid oxidizes cholesterol to produce cholesterol chlorohydrins and epoxide [39]. Free radical oxidation of cholesterol gives both 7α- and 7βhydroperoxycholesterol as the primary product which undergoes secondary reaction to give 7-hydroxy- and 7-ketocholesterol. Singlet oxygen oxidizes cholesterol to give 5α-hydroperoxycholesterol. 7β-Hydroxycholesterol and 7-ketocholesterol may serve as a biomarker for free radical-mediated oxidation of cholesterol in vivo.

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CYP2E1 has been shown to play a significant role in alcohol-induced liver injury [44,45]. Increased levels of CYP2E1 have been observed in patients with NAFLD/ NASH [46–48]. CYP2E1 oxidizes PUFA as hydroxylase and epoxydase without production of free radicals [36], while it reacts with various heteroatom-containing compounds such as chlorinated compounds to yield ROS and possibly free radicals [35]. The increased evidence showing the involvement of oxidative stress in the pathogenesis of NAFLD/NASH has stimulated studies on the effects of antioxidants. Various antioxidants have been tested in animal experiments and human studies. Notably, as described later, vitamin E and its derivatives [49] have been studied most extensively. Metal chelators and peroxidases as well as radical-scavenging antioxidants may be useful for inhibition of free radical-mediated oxidation. It should be noted that the effects of antioxidants are dependent on the active species. In the following sections, the role of oxidative stress and effects of antioxidants in the pathogenesis of NAFLD/ NASH observed in animal and human studies will be reviewed focusing specifically on free radicals. As described above, oxidative stress is mediated by multiple oxidants by different mechanisms and it is important to elucidate the role of free radicals using appropriate biomarkes for understanding the underlying mechanisms and developing drugs. Animal studies Carbon tetrachloride intoxication It has been known for many years that carbon tetrachloride induces fat accumulation and damage in the liver [50]. Slater and his colleagues conducted a series of pioneering studies on the experimental liver injury induced by carbon tetrachloride and showed that the hepatotoxicity was mediated by free radicals [51–54]. This was a monumental achievement showing that free radicals play a causative role in the induction of liver diseases and that free radical-scavenging antioxidants are effective against free radical-mediated diseases. It was shown that carbon tetrachloride was metabolized in liver by cytochrome P-450, CYP2E1, to yield trichloromethyl radical [46,55], which after reaction with oxygen-induced lipid peroxidation, leading eventually to liver damage [54]. The formation of trichloromethyl radical from carbon tetrachloride was confirmed by spin trapping technique [56]. It was found that the administration of carbon tetrachloride gave both cis,trans- and trans,trans-conjugate diene products [57], the latter being the specific products of free radical-mediated lipid oxidation [19,20,23]. It has widely been confirmed that the administration of carbon tetrachloride induced an increase in lipid peroxidation products such as trans,trans-HODE and F2-isoprostanes in liver and plasma, which was followed by an increase in plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) [58].

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872 Y. Sumida et al. Fatty liver induced by free radical initiator

Lipid oxidation

Alcohol, high fat diet, methionine–chloine deficient diet, D-galactosamine, and lipopolysaccharide as well as carbon tetrachloride have been known to induce fatty liver and liver damage in experimental animals. Free radicals may be produced in vivo by several methods. High energy radiation and radical-generating compounds are useful for generating free radicals at controlled rate and, more importantly, without bio-transformation and affecting other biological responses. The azo compounds, R-N ⫽ N-R, are considered to be a convenient source of free radicals, since they are stable and safe, but decompose thermally without biotransformation. Azo compound is not biologically relevant, but the free radicals produced from azo compounds are assumed to behave similarly as those produced in vivo and that this is a useful method for studying the effects of free radicals and antioxidants in vivo [59]. By choosing appropriate R group, free radicals may be produced at convenient and controlled rate. 2,2′-Azobis(2-amidinopropane) dihydrochloride (AAPH) used in this study generates free radicals at a rate of approximately 10 nM/sec under the reaction conditions employed here. On the other hand, stable azo compound such as azomethane (R ⫽ CH3), the half-life being 100 years at 37°C, does not exert any deleterious effects in vivo and in cultured cells. Interestingly, it was found that intraperitoneal administration of AAPH, an azo compound which produces free radicals by thermal decomposition without biotransformation, induces fatty liver in mice, which was inhibited by several radical-scavenging antioxidants in a dosedependent manner [60]. Significant intoxication was observed in liver, kidney, and thymus. Small fat droplets appeared in the cytoplasm of the hepatocytes by 3 h after administration of AAPH and these fat droplets increased in number and size with time, 0.8–4 μm in diameter as measured by electron micrograph. The lipid droplets were diffusely distributed throughout the lobules of the liver. Since this first observation, several other studies confirmed that the administration of AAPH induced fatty liver in mouse and rat [61–64]. Two major outcomes of AAPH administration are the fat accumulation and lipid peroxidation. The administration of azo initiator dissolved in drinking water also induced fat increase and lipid peroxidation in liver of mice [64,65].

Lipid oxidation has been implicated in the pathogenesis of various diseases including NAFLD/NASH, although it has not been proven unequivocally whether the lipid oxidation plays a causative role or it is simply a consequence of diseases. However, many studies observed a link between an increase in lipid oxidation products and disease progression and also between the decrease in lipid oxidation by antioxidant and improvement of disease state [14]. As described above, free radical-mediated lipid peroxidation and enzymatic oxidation by lipoxygenase, cyclooxygenase, and CYP are the major pathways of lipid oxidation in vivo [20]. More than hundred kinds of products are formed by lipid oxidation. The detailed analyses of lipid oxidation products may elucidate responsible active oxidants and mechanisms and may shed light on the development of effective drugs. It was found that administration of azo compound to mice and rats increased lipid peroxidation products such as HODE, HETE, and 8-isoprostaglandin F2α (8-isoP) [64,65]. The lipid extracts from the liver were treated with triphenylphosphine and then hydrolyzed to convert hydroperoxides and hydroxides of linoleic and arachidonic acid moieties of triglycerides, phospholipids and other esters to the corresponding free fatty acid (FFA) hydroxides, that is, HODE and HETE, respectively. The esters of isoprostanes were also measured as free acid form. Linoleates, arachidonates and docosahexaenoates are the major polyunsaturated fatty acid in the liver. The levels of HODE, HETE and isoprostanes fluctuated considerably between the mice, but as a whole AAPH increased the lipid peroxidation products. It is noteworthy that high fat diet also increased trans,trans–HODE, a specific marker for free radical lipid peroxidation. It is noteworthy that a good correlation was observed between the levels of trans,trans-HODE and triglycerides [64], implying that free radical-mediated lipid peroxidation plays an important role. Cholesterol is another important substrate of lipid oxidation in vivo. Cholesterol is oxidized by similar mechanisms as PUFA to give diverse products collectively termed oxysterols [37,66]. Oxysterols are not only a biomarker for lipid oxidation in vivo but also biological mediator. It has been known that chronic ethanol feeding increases 7-hydroperoxycholesterol and 7-ketocholesterol in rat liver [67]. It was reported that oxysterols induced proinflammatory and profibrogenic mechanisms in mouse liver cells [68]. The role of iron has been suggested in the initiation of lipid peroxidation. Iron may be potential therapeutic target [69] and, in fact, phlebotomy is known as one of the therapeutic treatments in the liver diseases. The increased induction of CYP 2E1 and resulting formation of RONS have been suggested [46–48,70]. It may be added that lipid peroxidation proceeds by the same chain mechanisms and produces the same products independent of the nature of attacking free radicals, that is, chain propagation proceeds similarly independent of the type of chain initiation [20].

Accumulation of fats Electron micrograph analysis clearly showed that administration of AAPH induced the formation of fat droplets in the cytoplasm of hepatocytes which were surrounded by mitochondria [60]. As in the case of carbon tetrachloride, AAPH induced a marked increase in triglyceride and concomitant decrease in phospholipids. The most striking observation was that the plot of increase in triglyceride against decrease in phospholipids induced by either azo compound administration or high fat diet gave the same line [64].

Free radical and oxidative stress in NAFLD/NASH

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Role of antioxidants and Nrf2 The accumulating evidence showing the involvement of oxidative stress and lipid peroxidation in liver injury of experimental animals has attracted much attention to the protective effects of antioxidants. The protective effects of vitamin E against liver injury have been confirmed in many animal studies [49,57,71–78]. Interestingly, triglyceride accumulation was found in the liver of rats fed vitamin E deficient diet for 4 months [79] and chronic ethanol feeding caused a marked increase in hepatic α-tocopheryl quinone [80], a metabolite of free radical scavenging reaction by α-tocopherol [81], suggesting the role of vitamin E as antioxidant in preventing fatty liver in experimental animals. Several radical-scavenging antioxidants such as 2-carboxy-2,5,7,8-tetramethyl-6chromanol (Trolox), a water soluble analog of vitamin E, glutathione, cysteine, uric acid [60] and diarylamines [61] were found to reduce the accumulation of fats in the mouse induced by administration of AAPH. Further, catechins were found to ameliorate the increase in enzyme markers of liver injury such as ALT and AST and also in the formation TBARS, a marker of lipid peroxidation [63]. The aerobic organisms are protected against oxidative stress by an efficient defense network composed of various antioxidant compounds and enzymes with diverse functions [82]. One of such functions is the adaptive response, in which various antioxidant or drug metabolizing proteins and enzymes are induced by oxidative stress and lipid oxidation products. Kelch-like ECH-associated protein 1-nuclear erythroid 2-related factor 2 (Keap 1-Nrf2) system plays a significant physiological role in the response to endogenous, environmental, and pharmacological electrophiles including lipid peroxidation products produced by oxidative stress [83,84]. The transcription factor Nrf2 regulates hundreds of genes that are involved in the cytoprotective response against oxidative stress. The role of Nrf2 in the progression of NASH has been studied [85]. It was found that high fat diet induced an increase in lipid peroxidation and Nrf2 expression which strongly correlated with the degree of hepatic steatosis and inflammation [86]. The activation of Nrf2 was observed also in NAFLD patients [87]. It was reported that Nrf2 was increased by CYP2E1 in rodent liver and HepG2 cells and protected against oxidative stress caused by CYP2E1 [88]. The development of NASH was greatly accelerated in mice lacking Nrf2 when they were challenged with a methionine and choline deficient diet [89] and further deletion of Nrf2 in mice resulted in the higher hepatic triglycerides, MDA, and iron [90].

Human studies Oxidative stress, free radicals, and lipid oxidation in patients with NAFLD/NASH Free radicals and oxidative stress are considered to be one of the key mediators of hepatocellular injury and

873

implicated in the progression from benign NAFL to NASH [91], although the responsible oxidants have not been identified. Several studies have measured systemic markers of oxidative stress status in NAFLD and an increase in markers such as HODE, HETE, 8-isoP, TBARS, MDA, oxidized LDL, hydroxydeoxyguanosine (8-OHdG), and thioredoxin (TRX) has been observed in NASH patients (Table I). Several groups found higher levels of lipid peroxidation products in NASH patients compared with NAFL or healthy people [11,14,17,33]. Hepatic level of 8-OHdG, a reliable marker of oxidative DNA damage, was increased in NAFLD patients, especially in NASH patients [92]. Hepatic iron overload and insulin resistance are likely to be independently associated with elevated 8-OHdG. TRX, an oxidative stress -inducible thiol-containing protein, has important roles in the redox regulation. Our data have demonstrated that serum TRX level is a parameter for discriminating NASH from simple NAFL as well as a predictor of the severity of NASH [93]. Our previous data have showed that visceral fat may have the most important roles in not only hepatic steatosis but also NASH fibrosis [94]. A decrease in antioxidant defenses is also a major factor promoting oxidative stress in NASH patients. Decreases in antioxidant factors including coenzyme Q10, Cu-Zn SOD, catalase activity, glutathione (GSH), and GSH S-transferase correlate with the severity of liver disease [8]. The expression of hemeoxygenase-1 (HO-1), which may protect the cells against oxidative stress, is induced in NASH patients as an adapting response and its increase reflected the severity of the disease [95]. CYP2E1 has recently emerged as a potentially important cause of ROS overproduction. Increased hepatic expression of CYP2E1 was first shown in a mouse model of NASH and later also reported for human NASH [47,48]. Malaguarnera et al. reported that human Kupffer cells in NASH patients overproduce chitotriosidase (Chit) which is selectively expressed in chronically activated tissue macrophages, like the lipid laden storage cells that accumulate in large quantities in the spleen, liver, and other tissues of Gaucher patients [96]. The increased levels of Chit expression was well related to superoxide production and to lipid peroxidation levels. Iron as a potent source of free radicals/oxidative stress in NAFLD/NASH Iron is a potent agent which generates free radicals via Fenton or Haber-Weiss reaction. Iron deposition seems to be found in about 30% of NAFLD patients. NASH patients are likely to have higher degrees of hepatic iron deposits than NAFL. Iron deposition may have important roles in the fibrosis progression and hepatocarcinogensis in NASH patients. Although genetic factors, insulin resistance, dysregulation of iron-regulatory molecules, and erythrophagocytosis by Kupffer cells may be responsible for hepatic iron accumulation in NASH, the mechanisms of iron accumulation in NASH are not precisely understood [97]. Phlebotomy targeting hepatic iron overload with resultant increased oxidative stress is

874 Y. Sumida et al. Table I. Indicators of free radicals/oxidative stress in NAFLD/NASH.

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Author

References

Population

Loguercio

[121]

NASH ⬎ NAFL ⬎ Control

[122]

Serum RBC Liver

HNE, MDA

Seki

NAFLD (n ⫽ 81) NASH/NAFL ⫽ 34:14 NASH (n ⫽ 17) NAFL (n ⫽ 23) Control (n ⫽ 7) NASH (n ⫽ 25) NAFL (n ⫽ 15) Control (n ⫽ 17) NASH (n ⫽ 18) Control (n ⫽ 16)

8-OHdG HNE

NASH ⬎ Control NASH ⫽ or ⬎ NAFL

Serum

TRX

NASH ⬎ NAFL, Control

Serum

NASH (n ⫽ 21) Control (n ⫽ 19) NASH (n ⫽ 22) Control (n ⫽ 22) NAFLD (n ⫽ 51) Control (n ⫽ 30) Viral hepatitis (n ⫽ 30) NASH (n ⫽ 43) Control (n ⫽ 33)

Serum

GSH, MDA, NO SOD GSH-Px, GR Oxidized LDL, TBARS

NASH ⬎ Control NASH ⬍ Control NASH ⫽ Control NASH ⬎ Control

TAR Total plasma peroxide MDA Cu,Zn- SOD, catalase, coenzyme Q

NASH ⬍ Control NASH ⬎ Control NASH ⬎ Control NASH ⬍ Control

8-OHdG Reduced GSH, Reduced GSH/ oxidized GSH ratio TAS, Vitamin E GSH-Px, GR 8-OHdG

NASH ⫽ Control NASH ⬍ Control NASH ⬎ Control NASH ⫽ Control NASH ⬎ Control NAFL⬎ NASH, control NASH, NAFL⬍ control NASH ⬍⬍ NAFL ⬍ control NASH ⫽ NAFL ⫽ control NASH ⬎ NAFL, control NASH ⬍ NAFL ⬍ control NASH ⬎ NAFL, control NASH ⬎ NAFL, control

Sumida

[93]

Koruk

[123]

Chalassani

[124]

Horoz

[125]

Yesilova

[126]

Machado

[127]

Fujita

[92]

Videla

[8]

Malaguarnera

[95]

Oliveira

[33]

Sample

Serum Serum

Serum RBC

Parameter

NASH (n ⫽ 38) NAFL (n ⫽ 24) Control (n ⫽ 10) NASH (n ⫽ 16) NAFL (n ⫽ 15) Control (n ⫽ 12)

Liver

NASH (n ⫽ 35) NAFL (n ⫽ 15) Control (n ⫽ 10) NASH (n ⫽ 17) NAFL (n ⫽ 19) Control (n ⫽ 3)

Liver

Protein carbonyl SOD, GSH CAT GSH-Px, CYP2E1 FRAP HO-1

Liver

Hydroperdoxides

Liver Plasma

Results

NAFL, non-alcoholic fatty liver; HNE, 4-hydroxy-2-nonenal; MDA, malondialdehyde; 8-OHdG, 8-hydroxy deoxyguanosine; TRX, thioredoxin; GSH, glutathione; NO, nitric oxide; TBARS, thiobarbituric acid-reactive substances; SOD, superoxide dismutase; GSH-Px, glutathione peroxidase; TAR, total antioxidant response; TAS, total antioxidant status; GR, glutathione reductase; FRAP, ferric reducing ability of plasma.

another potential therapeutic modality that has been investigated in the treatment of NASH [69,98]. In a few pilot studies in NASH patients [99], quantitative phlebotomies were performed with induction of iron depletion and reduction in serum ferritin levels. Results of these studies suggest that iron reduction therapy by phlebotomy can lead to improvement in aminotransferase levels and insulin resistance [100]. According to a Phase II clinical trial of phlebotomy for NAFLD, paired liver biopsies demonstrated the improvement of NAFLD activity scores [101]. Phase III trial has been registered with the US National Institute of Health (clinicaltrials.gov, Identifier NCT 00641524). Anti-oxidative therapies for NASH There has been no established pharmacologic treatment for NAFLD/NASH. The depletion of antioxidants within

hepatocytes resulting in impaired ROS inactivation is the basis for antioxidant supplementation as a potential treatment for NASH. Several encouraging pilot studies of various agents indicate potential beneficial effects which may be related to their antioxidant effects. These include vitamin E, betaine, ursodeoxycholic acid (UDCA), probucol, L-carnitine, pentoxifylline, metformin, and N-acetylcysteine (NAC) (Table II). Among those, vitamin E seems to be one of the most promising agents for the treatment of NASH. Vitamin E, a lipid-soluble antioxidant, has been investigated to treat NASH [reviewed in 29]. A recent study revealed that NASH patients had much lower levels of serum vitamin E than healthy controls [9]. Vitamin E has been shown to inhibit lipid peroxidation and suppress inflammatory cytokines such as TNF-α, and its use in the treatment of NAFLD/NASH has been studied. In Japan, a study with vitamin E treatment (300 mg/d

Free radical and oxidative stress in NAFLD/NASH

875

Table II. Anti-oxidative therapies for NAFLD/NASH based on randomized controlled trials. Drug

RCT

Biochemical effects

Histological effects

Comments

↓steatosis, inflammation, and NAS

No significant change in body weight and insulin sensitivity

Vitamin E

↓oxidative stress

[27,104,128]

↓LFTs

Betaine UDCA

↓oxidative stress Hepatoprotective

[109] [110–113]

No effects over placebo Marginal benefit

?effect on fibrosis No effects over placebo No effects over placebo

[114]

↓LFTs

Not assessed

A larger RCT is essential

[116]

↓LFTs

↓steatosis and NAS

A larger RCT is essential

[17,117,118]

↓LFTs

↓steatosis, inflammation, and NAS

No significant effect on IL-6 and TNFα

[119]

↓LFTs

Not assessed

A larger RCT is essential

Pentoxifylline

↓oxidative stress Lipid-lowering agent with strong antioxidant properties Modulator of mitochondria FFA transport and oxidation Anti-TNF agent

NAC

Hepatoprotective A precursor of glutathione

Probucol L-carnitine

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Mechanisms

No beneficial effect in large RCT

RCT, randomized controlled trial; LFT, liver function test; UDCA, ursodeoxycholic acid; NAS, NAFLD activity score; FFA, free fatty acid.

during 1 year) confirmed improvement in liver tests with reduction of plasma levels of TGF-β1, but revealed no change in degree of steatosis, fibrosis and inflammation on post-treatment liver biopsy [102]. Kawanaka et al. revealed that serum transaminase activities, TRX, and TBARS were significantly decreased after the 6-month course of vitamin E therapy [103]. A group of biopsyproven NASH patients were randomized to vitamin E (1,000 IU/day) plus vitamin C (1,000 mg/day) or placebo for 6 months [104]. The patients showed no improvement in serum aminotransferase levels but repeat liver biopsies at the end of the trial demonstrated decreased fibrosis within the vitamin group (especially in patients with diabetes). Recently, Non-alcoholic Steatohepatitis Clinical Research Network (NASH CRN) conducted the Pioglitazone versus Vitamin E versus Placebo for the Treatment of Non-diabetic Patients with Non-alcoholic Steatohepatitis (PIVENS) trial, a phase 3, multicenter, randomized, placebo-controlled, doubleblind clinical trial of pioglitazone or vitamin E for the treatment of adults without diabetes who had biopsyconfirmed NASH. By histological analysis, vitamin E was superior to placebo in regard to the resolution of NASH and there was no benefit of pioglitazone over placebo [27]. In the PIVES trial, however, vitamin E exerted no significant effect on hepatic fibrosis. The Treatment of NAFLD in Children (TONIC) trial compared the efficacy of vitamin E or metformin to placebo in 8–17-year olds with NAFLD. Although the primary outcome of sustained reduction of ALT was not different among the three groups, there were statistically significant improvements in NAFLD activity score and resolution of NASH with vitamin E treatment compared to placebo over 96 weeks [105]. Despite these limitations and, in summary, in the recent AASLD guidelines, vitamin E is the first line pharmacotherapy in non-diabetic adult patients with

biopsy-proven NASH. However, vitamin E is not recommended to treat NASH in diabetic patients, NAFLD without liver biopsy, NASH cirrhosis, or cryptogenic cirrhosis, because available data supporting the efficacy of this drug do not exist [6]. One concern with vitamin E is the controversial issue of whether it increases all-cause mortality. Some meta-analyses have reported an increase in all-cause mortality with high dose vitamin E, but others failed to confirm such an association. A recently published RCT revealed that vitamin E administrated at a dose of 400 IU/day increased the risk of prostate cancer in relatively healthy men [106]. In the future, we should examine whether vitamin E treatment may prevent hepatic carcinogenesis or hepatic failure, leading to improved overall survival in NAFLD/NASH patients. Betaine, a methyl donor for the remethylation of homocysteine, raises S-adenosylmethionine levels, which may reduce hepatic steatosis. A placebo-controlled study including 191 patients with NAFLD treated for 8 weeks with combination of betaine glucuronate (300 mg/d), diethanolamine and nicotinamide ascorbate showed significant improvement in liver tests and degree of steatosis in the treated group [107]. A 1-year trial of betaine supplementation in NASH patients also reduced transaminase activities with the histological improvement of steatosis, inflammation, and fibrosis [108]. However, a randomized placebo-controlled trial did not prove the efficacy of betaine for NASH patients [109]. UDCA, a widely-used hepatoprotective agent which has antioxidant activity, has been expected to be a first-line therapy for NAFLD/NASH. However, a standard dose of UDCA exerted no beneficial effects over placebo [110]. Conflicting results exist regarding efficacy of higher dose UDCA for the treatment of NASH [111,112]. Therefore, in AASLD guidelines, UDCA is not recommended for the treatment of NASH/NAFLD [2]. In a small randomized controlled trial, the treatment with UDCA in combination

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876 Y. Sumida et al. with vitamin E improved laboratory values and hepatic steatosis of patients with NASH [113]. Probucol, which is a lipid-lowering agent with strong antioxidant properties, appears to be effective in decreasing ALT levels in NASH patients, according to a doubleblind RCT by Merat et al. [114]. This group also revealed that hepatic inflammation was reduced in NASH patients after use of probucol for 1 year [115]. Supplementation to diet of L-carnitine, a modulator of mitochondrial free fatty acid transport and oxidation, reduced TNF-α and C-reactive protein and improved liver function, glucose plasma level, lipid profile, and histological manifestations of NASH [116]. Pentoxifylline has a variety of functions such as inhibition of inflammatory cytokines including TNF-α, hepatoprotective effects, antioxidant effects, and anti-fibrotic effects. According to the results of three RCT [17,117,118], pentoxifylline may have beneficial biochemical and histological effects on NASH without remarkable adverse effects. It was found that pentoxifylline decreased the plasma levels of lipid peroxidation products such as HODEs and HETEs [14]. NAC, a precursor of GSH, ameliorates NASH histology in animal models of NASH. According to a recently published RCT in patients with NAFLD, NAC resulted in a significant decrease of serum ALT after 3 months, compared to vitamin C [119]. Overall, antioxidants represent a novel class of medications that have shown some promising initial results in the treatment of NASH but further well-designed large RCTs are required (Table II) and it is likely that these therapies will be used in combination regimens with other agents that affect insulin sensitivity, such as pioglitazone or metformin [120]. The research agenda for the future includes establishing the role of free radicals/ oxidative stress in NAFLD/NASH, identifying better predictors of the disease, and defining effective treatment. While waiting for these trials and based on the recent AASLD guidelines as mentioned above, we recommend using vitamin E (800 IU/day) for non-diabetic adults with biopsy proven NASH in addition to lifestyle changes and risk factors control.

suggesting the involvement of multiple active species in the pathogenesis of these diseases. The detailed analysis of the products is necessary for understanding the underlying mechanisms and elucidation of responsible oxidants. HODEs and HETEs with trans,trans-form and isoprostanes may be appropriate biomarker for the free radical-mediated oxidative stress. Practically, 9- and 13-trans,trans-HODE may be the most suitable products, since they are produced by straightforward mechanism and their physiological levels are high. The trans,trans-HETEs are also specific products of free radical-mediated lipid peroxidation and their levels in human plasma are similar to those of HODEs, but HETEs are mixtures of six regio-isomers and their quantitative analysis is not easy. It is noteworthy that lipid oxidation products may exert multiple effects: they are cytotoxic and modify proteins and DNA, but may also induce adaptive response to increase antioxidant compounds and enzymes such as glutathione and HO-1, respectively [20,95]. They may also act as ligands and antagonists for various receptors and further act in the activation and resolution of inflammation. Lipid oxidation products may also affect the expression and activation of various signaling messengers such as TNF-α, nuclear factor-κB (NF-κB) and activating protein-1 (AP-1). These multiple functions make the effects of lipid oxidation and oxidative stress more complex. Collectively, there is no doubt that oxidative stress is elevated in NAFLD/NASH patients and that free radicals act as one of the responsible species. Apparently, more studies are required to understand the roles of free radicals in the pathogenesis of these diseases and to explore the effective prevention and treatment. The detailed analyses of the alterations in the experimental animals and patients are essential. NAFLD/NASH are considered as a multifactorial disease in relation to the pathogenic mechanisms and concurrence of multiple pathogenic mechanisms should be confronted with multiple drugs with different functions, an issue that requires future studies. Future studies need to characterize which biomarkers of oxidative stress are better suited for NASH diagnosis or follow-up. This may also shed light on the responsible active species and mechanisms.

Perspectives and concluding remarks This review shows accumulating results suggesting the role of free radicals in the pathogenesis of NAFLD/NASH and also potential beneficial effects of radical-scavenging antioxidants for prevention and/or treatment of diseases. It was emphasized that oxidative stress is mediated by multiple oxidants which produce the same lipid oxidation products and that the efficacy of antioxidants is determined by the type of oxidants. Vitamin E may be effective when free radicals play a causative role, but may not be so against the oxidation mediated by non-radical oxidants. The results described above show that various antioxidants, some are free radical scavengers while other are not, may be effective for treatment of NAFLD/NASH,

Declaration of interest The authors have declared that no competing interests exist. YS received scholarship funds from MSD Co., Ltd. YN received scholarship funds from Otsuka Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Ltd., and Mitsubishi Tanabe Pharma Co., Ltd. References [1] Wierzbicki AS, Oben J. Nonalcoholic fatty liver disease and lipids. Curr Opin Lipidol 2012;23:345–352.

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