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Alterations in the redox state and liver damage: Hints from the EASL Basic School of Hepatology

  • Gianluca Tell
    Correspondence
    Corresponding authors. Address: Department of Medical and Biological Sciences, University of Udine, Piazzale Massimiliano Kolbe 4, 33100 Udine, Italy (G. Tell). Centro Studi Fegato, Fondazione Italiana Fegato, AREA Science Park, Campus Basovizza, Bldg Q, ss 12km 163, 5 34149 Trieste, Italy (C. Tiribelli).
    Affiliations
    Department of Medical and Biological Sciences, University of Udine, 33100 Udine, Italy
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  • Carlo Vascotto
    Affiliations
    Department of Medical and Biological Sciences, University of Udine, 33100 Udine, Italy
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  • Claudio Tiribelli
    Correspondence
    Corresponding authors. Address: Department of Medical and Biological Sciences, University of Udine, Piazzale Massimiliano Kolbe 4, 33100 Udine, Italy (G. Tell). Centro Studi Fegato, Fondazione Italiana Fegato, AREA Science Park, Campus Basovizza, Bldg Q, ss 12km 163, 5 34149 Trieste, Italy (C. Tiribelli).
    Affiliations
    Centro Studi Fegato, AREA Science Park, and Department of Medical Sciences, University of Trieste, 34149 Trieste, Italy
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Open AccessPublished:October 01, 2012DOI:https://doi.org/10.1016/j.jhep.2012.09.018

      Summary

      The importance of a correct balance between oxidative and reductive events has been shown to have a paramount effect on cell function for quite a long time. However, in spite of this body of rapidly growing evidence, the implication of the alteration of the redox state in human disease has been so far much less appreciated. Liver diseases make no exception. Although not fully comprehensive, this article reports what discussed during an EASL Basic School held in 2012 in Trieste, Italy, where the effect of the alteration of the redox state was addressed in different experimental and human models. This translational approach resulted in further stressing the concept that this topic should be expanded in the future not only to better understand how oxidative stress may be linked to a liver damage but also, perhaps more important, how this may be the target for better, more focused treatments. In parallel, understanding how alteration of the redox balance may be associated with liver damage may help define sensitive and ideally early biomarkers of the disorder.

      Abbreviations:

      ROS (reactive oxygen species), RNS (reactive nitrogen species), SOD (superoxide dismutase), ALI (acute liver injuries), CLD (chronic liver diseases), HCC (hepatocarcinoma), HBV (hepatitis B virus), HCV (hepatitis C virus), NAFLD (non-alcoholic fatty liver disease), MDA (malondialdehyde), 4-HNE (4-hydroxynonenal), APE1/Ref-1 (apurinic apyrimidinic endonuclease/redox effector factor 1), TNF (tumor necrosis factor), NOS2 (nitric oxide synthase 2), ETC (electron transport chain), I/R (ischemia/reperfusion), FA (fatty acids), NASH (non-alcoholic steatohepatitis), ASH (alcoholic steatohepatitis), 8OHdG (8-hydrossyguanosine), HIF-1 α (hypoxia-inducible factor 1α), PHD (prolyl-hydroxylases), NF-κB (nuclear factor-κB), GSH (reduced glutathione), GSSG (oxidized glutathione), CVD (cardiovascular disease), ER (endoplasmic reticulum), ERS (endoplasmic reticulum stress), HH (hereditary hemochromatosis), MPO (myeloperoxidase), HLPP (Human Liver Proteome Project), 2-DE (two-dimensional electrophoresis), MS (mass spectrometry), LC (liquid chromatography)

      Keywords

      Introduction

      All living organisms have to cope with harmful by-products of oxygen such as O2, H2O2, and OH (collectively called free radical or reactive oxygen species, here referred to as ROS), which appeared on Earth approximately 2 billion years ago and provoked the development of cell defense mechanisms [
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      ]. However, the history of free radicals is much younger since their formation in cells was first documented about 60 years ago by a study demonstrating that harmful oxygen species may be produced in animals and plants as mediators of the damaging effects of radiation [
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      ]. It was only after the discovery of the existence of superoxide dismutase enzyme (erythrocuprein or SOD), which catalyzes O2 dismutation to H2O2 [
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      Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein).
      ], that the biological interest in ROS became apparent. It soon became clear that living organisms are capable of inducing their antioxidant defense systems by relatively rapid mechanisms to cope with the oxidative stress due to an imbalance between the generation of ROS and the antioxidant defense capacity of the cell. Both animals and certain plants can induce SOD upon increased exposure to oxygen, paraquat (a known producer of O2), and X irradiation [
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      • St Clair D.K.
      • Autor A.P.
      • Oberley T.D.
      Increase in manganese superoxide dismutase activity in the mouse heart after X-irradiation.
      ]. Within the body, tissues with a higher oxygen consumption rate, such as liver, heart, and brain, constitutively express greater antioxidant enzymes than those with lower oxygen consumption [
      • Jenkins R.R.
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      ].
      Oxidative stress is a major pathogenetic event occurring in several liver diseases, ranging from metabolic to proliferative disorders. Main sources of ROS are represented by mitochondria and cytochrome P450 enzymes in the hepatocyte, Kupffer cells, and neutrophils [
      • Marí M.
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      ]. Through modulation of protein structure/function, ROS can influence gene expression profile by affecting intracellular signal transduction pathways [
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      ]. While several enzymatic and non-enzymatic markers of chronic oxidative stress are well known in the liver, early protein targets of oxidative injury are yet poorly defined [
      • Cesaratto L.
      • Vascotto C.
      • Calligaris S.
      • Tell G.
      The importance of redox state in liver damage.
      ]. Identification of these biomarkers will enable early detection of liver diseases and allow monitoring the degree of liver damage, the response to pharmacological therapies and the development of new therapeutic approaches. In the era of molecular medicine, new proteomic methodologies promise to establish a relationship between pathological hallmarks of the disease and protein structural/functional modifications, allowing a better understanding and a more rational therapy of several liver disorders.
      Liver diseases are frequent pathologies worldwide [
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      ] and may be divided into acute and chronic on the basis of the persistence of liver injury. Acute liver injuries (ALI) are characterized by a rapid resolution and a complete restitution of normal organ architecture/function after the elimination of the cause, while chronic liver diseases (CLD) are characterized by persistent liver damage with progressive alteration of organ function caused by increased cellular damage [
      • Kaplowitz N.
      Mechanisms of liver cell injury.
      ]. The most common causes at the basis of CLD are viral infections sustained by hepatitis C and B viruses (HCV and HBV), alcohol abuse and alterations of lipid/carbohydrate metabolism, also known as non-alcoholic fatty liver disease (NAFLD). All these clinical features are major risk factors for HCC development [
      • Bosch F.X.
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      ]. However, regardless of the different etiology and natural course, a common landmark of all types of liver injury is an increased production of ROS (Fig. 1) [
      • Cesaratto L.
      • Vascotto C.
      • Calligaris S.
      • Tell G.
      The importance of redox state in liver damage.
      ].
      Figure thumbnail gr1
      Fig. 1Etiologic factors and central role of oxidative stress in the pathogenetic development of hepatic diseases. Oxidative stress is the major pathogenic event occurring in several liver disorders. Chronic liver injury due to HBV and HCV infection, inadequate alcohol consumption and metabolic disorders determine a pro-oxidative state causing protein and DNA damage and lipid peroxidation. Instauration of hepatocyte oxidative stress condition results in liver fibrosis and cirrhosis, which may lead to hepatocellular carcinoma.

      Oxidative stress and liver damage

      ROS and reactive nitrogen species (RNS), such as NO, NO+, NO, and ONOO, are critical intermediates in the normal physiology and pathophysiology of the hepatocyte. When the equilibrium between ROS generation and the antioxidant defense of the cell is disrupted, a “net” oxidative stress results. In the liver, free radicals triggered by ROS and RNS are created by neutrophils, Kupffer cells, mitochondria, and cytochromes P450 (for more details about ROS and NOS generation see [
      • Diesen D.L.
      • Kuo P.C.
      Nitric oxide and redox regulation in the liver: Part I. General considerations and redox biology in hepatitis.
      ]). The damage created by oxidative stress affects all major cellular components, including lipids, proteins and DNA [
      • Sies H.
      Oxidative stress: oxidants and antioxidants.
      ]. The relevance of cellular redox imbalance in liver pathologies is outlined by a number of studies in patients with viral, alcoholic or non-alcoholic fatty liver diseases [
      • Loguercio C.
      • Federico A.
      Oxidative stress in viral and alcoholic hepatitis.
      ,
      • Kang K.J.
      Mechanism of hepatic ischemia/reperfusion injury and protection against reperfusion injury.
      ,
      • Kurose I.
      • Higuchi H.
      • Miura S.
      • Saito H.
      • Watanabe N.
      • Hokari R.
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      Oxidative stress-mediated apoptosis of hepatocytes exposed to acute ethanol intoxication.
      ,
      • Poli G.
      • Parola M.
      Oxidative damage and fibrogenesis.
      ], pointing to a correlation between organ damage and increase in pro-oxidant cellular markers, such as malondialdehyde (MDA), 4-hydroxynonenal (4-HNE) and their protein adducts, associated with a concomitant decrease of antioxidants [
      • Loguercio C.
      • Federico A.
      Oxidative stress in viral and alcoholic hepatitis.
      ,
      • Poli G.
      • Parola M.
      Oxidative damage and fibrogenesis.
      ,
      • Cardin R.
      • Saccoccio G.
      • Masutti F.
      • Bellentani S.
      • Farinati F.
      • Tiribelli C.
      DNA oxidative damage in leukocytes correlates with the severity of HCV-related liver disease: validation in an open population study.
      ]. These markers may contribute to monitor the extent of liver damage; although their alteration may represent an epiphenomenon due to the particular cellular stress [
      • Cesaratto L.
      • Vascotto C.
      • Calligaris S.
      • Tell G.
      The importance of redox state in liver damage.
      ]. Chemical modification of essential biomolecules by ROS may cause their functional inactivation and lead to either cell death or an adaptive cellular response. In particular, a complex functional modulation of the cellular protein repertoire occurs upon oxidative insult [
      • Staal F.J.
      • Anderson M.T.
      • Staal G.E.
      • Herzenberg L.A.
      • Gitler C.
      • Herzenberg L.A.
      Redox regulation of signal transduction: tyrosine phosphorylation and calcium influx.
      ,
      • Sun X.
      • Majumder P.
      • Shioya H.
      • Wu F.
      • Kumar S.
      • Weichselbaum R.
      • et al.
      Activation of the cytoplasmic c-Abl tyrosine kinase by reactive oxygen species.
      ] through the ROS-dependent modification of specific amino acids, such as Cys, Trp, Tyr, His, Arg, and Lys [
      • Scaloni A.
      Mass Spectrometry approaches for the molecular characterization of oxidatively/nitrosatively modified proteins.
      ].
      ROS also behave as molecular second messengers within the cell, as they can be generated during triggering of particular cellular responses by cytokines, hormones, growth factors, and other soluble mediators, such as extracellular ATP [
      • Lander H.M.
      An essential role for free radicals and derived species in signal transduction.
      ]. Through the activation of protein kinases and phosphatases, intracellular ROS may directly or indirectly control the function of transcription factors, such as Nrf1 and NF-κB, thus leading to profound changes in cellular gene expression profile [
      • Schwabe R.F.
      • Brenner D.A.
      Nuclear factor-kappaB in the liver: friend or foe?.
      ,
      • Xu Z.
      • Chen L.
      • Leung L.
      • Yen T.S.
      • Lee C.
      • Chan J.Y.
      Liver-specific inactivation of the Nrf1 gene in adult mouse leads to nonalcoholic steatohepatitis and hepatic neoplasia.
      ]. Moreover, ROS perpetuate and potentiate their own effects by influencing transcription and activation of cytokines and growth factors, responsible for further ROS production leading to the onset of the so called ‘vicious cycle’ [
      • Cardin R.
      • Saccoccio G.
      • Masutti F.
      • Bellentani S.
      • Farinati F.
      • Tiribelli C.
      DNA oxidative damage in leukocytes correlates with the severity of HCV-related liver disease: validation in an open population study.
      ]. Depending on the cell type and intensity/duration of the oxidative stress affecting cells, ROS may therefore act either as pro-apoptotic molecules or stimulators of cell proliferation.
      Cellular defenses coping with ROS generation are multiple and include enzymatic (superoxide anion dismutase, catalase, GSH peroxidases, peroriredoxins, glutaredoxins, thuoredoxins, sulfiredoxins) and non-enzymatic antioxidants (vitamins A, C, and E, GSH, urate, bilirubin) (for more details about antioxidant mechanisms see [
      • Marí M.
      • Colell A.
      • Morales A.
      • von Montfort C.
      • Garcia-Ruiz C.
      • Fernández-Checa J.C.
      Redox control of liver function in health and disease.
      ]). The coordinated action of antioxidant enzymes ensures efficient ROS removal. Therefore, oxidative stress may be defined as an imbalance between the generation of ROS and the antioxidant defense capacity of the cell. In spite of the fact that the innovative concept that oxidative stress requires a fine balance within antioxidant systems is gaining relevance, large-scale interventional studies in humans with antioxidants have been inconsistent in demonstrating medical benefits, particularly in cancer patients [
      • Lippman S.M.
      • Klein E.A.
      • Goodman P.J.
      • Lucia M.S.
      • Thompson I.M.
      • Ford L.G.
      • et al.
      Effect of selenium and vitamin E on risk of prostate cancer and other cancers: the Selenium and Vitamin E Cancer Prevention Trial (SELECT).
      ,
      • Tsavachidou D.
      • McDonnell T.J.
      • Wen S.
      • Wang X.
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      Selenium and vitamin E: cell type- and intervention-specific tissue effects in prostate cancer.
      ]. The idea that redox signaling may specifically involve discrete pathways within cells suggests the possibility that oxidative stress can actually occur without an overall imbalance of pro-oxidants and antioxidants, and that the disruption of redox-sensitive signaling pathways can lead to metabolic and organ specificity in oxidative stress [
      • Marí M.
      • Colell A.
      • Morales A.
      • von Montfort C.
      • Garcia-Ruiz C.
      • Fernández-Checa J.C.
      Redox control of liver function in health and disease.
      ].
      Some enzymes have a fundamental importance in maintaining cell functions during oxidative stress conditions. The nuclear protein Apurinic Apyrimidinic Endonuclease/Redox Effector Factor 1 (APE1/Ref-1) is a paradigmatic example [
      • Tell G.
      • Damante G.
      • Caldwell D.
      • Kelley M.R.
      The intracellular localization of APE1/Ref-1: more than a passive phenomenon?.
      ]. This protein is involved in both transcriptional regulation of gene expression during adaptive cellular response to oxidative stress and the base excision repair pathway of DNA lesions generated as a consequence of ROS-induced base damages [
      • Tell G.
      • Damante G.
      • Caldwell D.
      • Kelley M.R.
      The intracellular localization of APE1/Ref-1: more than a passive phenomenon?.
      ]. A significant upregulation and relocalization of this protein has been described during HCC progression, accounting for a causative role of oxidative stress in the pathogenesis of HCC and suggesting APE1/Ref-1 as a new biomarker of the transformation process [
      • Di Maso V.
      • Avellini C.
      • Crocè L.S.
      • Rosso N.
      • Quadrifoglio F.
      • Cesaratto L.
      • et al.
      Subcellular localization of APE1/Ref-1 in human hepatocellular carcinoma: possible prognostic significance.
      ].
      In most of the liver diseases, of both metabolic and viral origin and associated with transformation processes, a chronic oxidative stress condition represents the main common determinant. During acute and chronic damage, hepatocytes are exposed to increased levels of oxidants, cytokines and bile acids. In spite of their powerful antioxidant resources, hepatocytes suffer from the cytotoxic effect of oxidative stress, leading to cell death. It is still a matter of debate whether cell death induced by ROS occurs either by necrosis or apoptosis, and which are the pathways involved in dead processes [
      • Conde de la Rosa L.
      • Schoemaker M.H.
      • Vrenken T.E.
      • Buist-Homan M.
      • Havinga R.
      • Jansen P.L.
      • et al.
      Superoxide anions and hydrogen peroxide induce hepatocyte death by different mechanisms: involvement of JNK and ERK MAP kinases.
      ,
      • Hong J.Y.
      • Lebofsky M.
      • Farhood A.
      • Jaeschke H.
      Oxidant stress-induced liver injury in vivo: role of apoptosis, oncotic necrosis, and c-Jun NH2-terminal kinase activation.
      ].
      In the liver, inflammatory cells, cholangiocytes, and Kupffer cells are the main sources of tumor necrosis factor α (TNFα). TNFα, together with other inflammatory cytokines, contribute to mitochondrial dysfunction by interfering with the mitochondrial respiratory chain and by forming O2 [
      • Sánchez-Alcázar J.A.
      • Schneider E.
      • Martínez M.A.
      • Carmona P.
      • Hernández-Muñoz I.
      • Siles E.
      • et al.
      Tumor necrosis factor-alpha increases the steady-state reduction of cytochrome b of the mitochondrial respiratory chain in metabolically inhibited L929 cells.
      ]. An indirect effect of TNFα in promoting mitochondrial dysfunction is the increased production of RNS as a consequence of the induction of nitric oxide synthase 2 (NOS2) [
      • Palmer R.M.
      • Rees D.D.
      • Ashton D.S.
      • Moncada S.
      L-arginine is the physiological precursor for the formation of nitric oxide in endothelium-dependent relaxation.
      ]. RNS, such as NO, NO+, NO, and ONOO, play an important role in controlling the cellular redox state (Fig. 2) [
      • Stamler J.S.
      • Singel D.J.
      • Loscalzo J.
      Biochemistry of nitric oxide and its redox-activated forms.
      ]. Interestingly, some of the physiological effects of RNS are mediated through the formation of S-nitroso-Cys or S-nitroso-GSH intermediates [
      • Gow A.J.
      • Stamler J.S.
      Reactions between nitric oxide and haemoglobin under physiological conditions.
      ]. RNS may functionally inactivate proteins of the mitochondrial respiratory chain through nitration of their Tyr residues or intermediate formation of S-nitrosated protein adducts at Cys residues [
      • Gow A.J.
      • Stamler J.S.
      Reactions between nitric oxide and haemoglobin under physiological conditions.
      ].
      Figure thumbnail gr2
      Fig. 2Main pathways for the formation of ROS and NOS. Mitochondria are the major source of cellular ROS during respiratory processes. Electron flow leads to the formation of superoxide anion (O2) that is generated by the univalent reduction of molecular oxygen (O2). This process may also be mediated by enzymes such as NADPH oxidase and xanthine oxidase. Superoxide dismutase (SOD) catalyzes the dismutation of two superoxide anions into hydrogen peroxide (H2O2) and oxygen. H2O2 can react with reduced transition metals, via Fenton’s reaction, to produce the highly reactive hydroxyl radical (OH). Alternatively, H2O2 could be converted into water by enzymes catalase (CAT) and glutathione peroxidase (GPX). Due to its relative long half-life (10−5 s), H2O2 could damage lipids, DNA and proteins leading to cell death. Inflammatory cells are the main source of TNFα. This cytokine contribute to mitochondrial dysfunction indirectly leading to the formation of RNS as a consequence of the induction of nitric oxide synthase 2 (NOS2) and formation of nitric oxide (NO), which reacting with O2 generates peroxynitrate (ONOO).
      Mitochondria play a crucial role in controlling apoptotic cell death, particularly in the hepatocyte. Activation of death receptors induces amplified apoptotic pathways involving caspase 8 and mitochondrial membrane proteins, which abolish the flow of electrons in the electron transport chain (ETC), increase mitochondrial ROS production and finally trigger the apoptosome (Fig. 2) [
      • Feldmann G.
      • Haouzi D.
      • Moreau A.
      • Durand-Schneider A.M.
      • Bringuier A.
      • Berson A.
      • et al.
      Opening of the mitochondrial permeability transition pore causes matrix expansion and outer membrane rupture in Fas-mediated hepatic apoptosis in mice.
      ]. ROS can also damage the mitochondria directly, by oxidizing various mitochondrial biomolecules, or by further increasing lipid peroxidation. Mitochondria are involved in both fatty acids (FA) β-oxidation and ROS generation and increasing evidence indicates that respiratory-chain defects are a key determinant of mitochondrial dysfunction, which in turn occurs as a result of ischemia/reperfusion (I/R) and excess of FA (lipotoxicity) [
      • Begriche K.
      • Igoudjil A.
      • Pessayre D.
      • Fromenty B.
      Mitochondrial dysfunction in NASH: causes, consequences and possible means to prevent it.
      ]. Mitochondrial impairment causes enhanced ROS production, which initiates a self-sustaining loop through the generation of MDA and 4-HNE, resulting from cellular lipid peroxidation, which are able to inhibit cytochrome c oxidase of mitochondrial complex IV. On the other hand, ROS per se damage both mtDNA and Fe–S cluster enzymes of the respiratory chain, leading to chronic organelle damage [
      • Paradies G.
      • Ruggiero F.M.
      • Gadaleta M.N.
      • Quagliariello E.
      The effect of aging and acetyl-l-carnitine on the activity of the phosphate carrier and on the phospholipid composition in rat heart mitochondria.
      ].
      The increased availability of FA in hepatic pathologies characterized by fat accumulation (NASH or ASH) determines the activation of microsomal cytochrome P-450 isoforms CYP2E1 and CYP4A10/4A14, involved in FA ϖ-oxidation, leading to an increased ROS production and uncoupling mitochondrial respiration [
      • Begriche K.
      • Igoudjil A.
      • Pessayre D.
      • Fromenty B.
      Mitochondrial dysfunction in NASH: causes, consequences and possible means to prevent it.
      ,
      • Leclercq I.A.
      • Farrell G.C.
      • Field J.
      • Bell D.R.
      • Gonzalez F.J.
      • Robertson G.R.
      CYP2E1 and CYP4A as microsomal catalysts of lipid peroxides in murine nonalcoholic steatohepatitis.
      ]. Of notice is the finding that a non-mitochondrial source of ROS, such as the NADPH-oxidase system of Kupffer cells, is also activated in NASH models, probably as a consequence of lipoperoxide or endotoxin phagocytosis [
      • De Minicis S.
      • Bataller R.
      • Brenner D.A.
      NADPH oxidase in the liver: defensive, offensive, or fibrogenic?.
      ]. This pathway is also present in activated hepatic stellate cells, possibly contributing to the general oxidative stress condition. Moreover, some viral proteins, such as the HCV protein NS5A, may affect intracellular Ca2+ concentration and signaling, thus triggering the elevation of ROS concentration in mitochondria and the translocation of NF-κB and STAT-3 into the nucleus with the consequent activation of target genes [
      • Berridge M.J.
      • Bootman M.D.
      • Lipp P.
      Calcium – a life and death signal.
      ,
      • Gong G.
      • Waris G.
      • Tanveer R.
      • Siddiqui A.
      Human hepatitis C virus NS5A protein alters intracellular calcium levels, induces oxidative stress, and activates STAT-3 and NF-kappa B.
      ,
      • Liu P.
      • Kimmoun E.
      • Legrand A.
      • Sauvanet A.
      • Degott C.
      • Lardeux B.
      • et al.
      Activation of NF-kappa B, AP-1 and STAT transcription factors is a frequent and early event in human hepatocellular carcinomas.
      ].
      Besides the involvement in development of CLD, oxidative stress is also generated under physiological conditions, such as during I/R upon liver transplantation. The I/R damage of the transplanted liver is one of the main determinants of primary non-function or initial poor function of the graft, which may contribute to a poor transplant outcome [
      • Cesaratto L.
      • Vascotto C.
      • Calligaris S.
      • Tell G.
      The importance of redox state in liver damage.
      ]. Hepatic I/R injury can be categorized into warm I/R and cold-storage reperfusion injury [
      • Teoh N.C.
      Hepatic ischemia reperfusion injury: contemporary perspectives on pathogenic mechanisms and basis for hepatoprotection – the good, bad and deadly.
      ]. ROS are generated very early after warm ischemia (within the first 4 h of reperfusion) and revascularization of ischemic tissue, in which the initial cell death triggers an inflammatory response with activation of tissue macrophages and recruitment of neutrophils [
      • McCord J.M.
      Oxygen-derived free radicals in postischemic tissue injury.
      ]. Primary sources of ROS during hepatic I/R are impairment of mitochondrial ETC, activation of NADPH oxidase in neutrophils and Kupffer cells and conversion of xanthine dehydrogenase into the ROS-producing form xanthine oxidase [
      • Jaeschke H.
      • Woolbright B.L.
      Current strategies to minimize hepatic ischemia-reperfusion injury by targeting reactive oxygen species.
      ] (Fig. 2). Although the latter mechanism was often considered as a critical source of ROS during I/R injury, recent evidence tends to limit the contribution of this pathway in ROS formation [
      • Jaeschke H.
      • Woolbright B.L.
      Current strategies to minimize hepatic ischemia-reperfusion injury by targeting reactive oxygen species.
      ].

      Role of oxidative stress during HCV infection

      A close relationship exists between HCV infection and oxidative stress, and hepatitis C virus infection is associated with severe alteration of the host redox status [
      • Choi J.
      • Ou J.H.
      Mechanisms of liver injury. III. Oxidative stress in the pathogenesis of hepatitis C virus.
      ]. Lipid peroxidation products are increased in serum, peripheral blood mononuclear cells (PBMC), and liver tissue, and 4-HNE and 8-hydroxyguanosine (8OHdG) are also elevated [
      • Mahmood S.
      • Kawanaka M.
      • Kamei A.
      • Izumi A.
      • Nakata K.
      • Niiyama G.
      • et al.
      Immunohistochemical evaluation of oxidative stress markers in chronic hepatitis C.
      ]. In addition, there is a significant reduction of hepatic, plasmatic, and lymphocytic GSH levels in patients chronically infected with HCV associated with an increased percentage of oxidized GSH (GSSG), suggesting an increased GSH turnover [
      • Mahmood S.
      • Kawanaka M.
      • Kamei A.
      • Izumi A.
      • Nakata K.
      • Niiyama G.
      • et al.
      Immunohistochemical evaluation of oxidative stress markers in chronic hepatitis C.
      ].
      This increased oxidative stress in hepatitis C may be explained by chronic inflammation, and the continued generation of ROS and NOS may be due to an increased activity of NADPH oxidase (Nox 2 protein) of Kupffer and polymorphonuclear cells in the liver [
      • Forman H.J.
      • Fukuto J.M.
      • Torres M.
      Redox signaling: thiol chemistry defines which reactive oxygen and nitrogen species can act as second messengers.
      ]. NS3 protein of HCV has been shown to activate Nox 2 protein of phagocytes and trigger apoptosis and dysfunction of T cells, natural killer cells, and natural killer T cells. Nox 2 protein is located on phagosomal and plasma membranes, leading to increased generation of ROS and other reactive species that can exert oxidative stress to the nearby cells.
      The excess iron deposits found in the liver tissue of some HCV patients may promote the generation of free radicals in these individuals [
      • Choi J.
      • Lee K.J.
      • Zheng Y.
      • Yamaga A.K.
      • Lai M.M.
      • Ou J.H.
      Reactive oxygen species suppress hepatitis C virus RNA replication in human hepatoma cells.
      ]. The mRNAs of TNF-α and cytochrome P-450 (CYP2E1), both of which can increase ROS production, might also be elevated in hepatitis C patients [
      • Gochee P.A.
      • Jonsson J.R.
      • Clouston A.D.
      • Pandeya N.
      • Purdie D.M.
      • Powell E.E.
      Steatosis in chronic hepatitis C: association with increased messenger RNA expression of collagen I, tumor necrosis factor-alpha and cytochrome P450 2E1.
      ]. Furthermore, there is some indication that HCV can directly induce oxidative stress in the hepatocyte [
      • Abdalla M.Y.
      • Ahmad I.M.
      • Spitz D.R.
      • Schmidt W.N.
      • Britigan B.E.
      Hepatitis C virus-core and non structural proteins lead to different effects on cellular antioxidant defenses.
      ]. Gene expression in HCV core has been associated with increased ROS, decreased intracellular and/or mitochondrial GSH content, and increased levels of oxidized thioredoxin and lipid peroxidation products [
      • Korenaga M.
      • Wang T.
      • Li Y.
      • Showalter L.A.
      • Chan T.
      • Sun J.
      • et al.
      Hepatitis C virus core protein inhibits mitochondrial electron transport and increases reactive oxygen species (ROS) production.
      ,
      • Okuda M.
      • Li K.
      • Beard M.R.
      • Showalter L.A.
      • Scholle F.
      • Lemon S.M.
      • et al.
      Mitochondrial injury, oxidative stress, and antioxidant gene expression are induced by hepatitis C virus core protein.
      ]. Therefore, it may be suggested that HCV produces oxidative stress through multiple mechanisms that include chronic inflammation, iron overload, and liver injury. Some of the HCV proteins may contribute to this process.
      Sensitive oxidative stress biomarkers may be important in the diagnostic approach to HCV infection and help monitor disease progression and the efficacy of therapies. However, in spite of the amount of information available on the pathogenic role of oxidative stress in HCV infection and the significant advances made in the last few years, the translation to clinical practice is still far [
      • González-Gallego J.
      • García-Mediavilla M.V.
      • Sánchez-Campos S.
      Hepatitis C virus, oxidative stress and steatosis: current status and perspectives.
      ].

      ROS and hypoxia in HCC

      Human HCC is the fifth most frequent neoplasm worldwide and the third cause of cancer-estimated deaths. There are multiple etiological agents that are associated with the development of HCC, the most frequent being HBV and HCV infections and metabolic disease as NASH [
      • Ha H.L.
      • Shin H.J.
      • Feitelson M.A.
      • Yu D.Y.
      Oxidative stress and antioxidants in hepatic pathogenesis.
      ]. Globally, up to 80% of HCC is attributable to HBV or HCV [
      • Perz J.F.
      • Armstrong G.L.
      • Farrington L.A.
      • Hutin Y.J.
      • Bell B.P.
      The contributions of hepatitis B virus and hepatitis C virus infections to cirrhosis and primary liver cancer worldwide.
      ]. The risk of HCC is increased 5- to 15-fold in chronic HBV carriers [
      • El-Serag H.B.
      • Rudolph K.L.
      Hepatocellular carcinoma: epidemiology and molecular carcinogenesis.
      ] and 11.5- to 17-fold in HCV-infected patients [
      • Donato F.
      • Tagger A.
      • Gelatti U.
      • Parrinello G.
      • Boffetta P.
      • Albertini A.
      • et al.
      Alcohol and hepatocellular carcinoma: the effect of lifetime intake and hepatitis virus infections in men and women.
      ]. Antiviral therapy is effective in preventing HCC in only a proportion of patients [
      • Tai A.W.
      • Chung R.T.
      Treatment failure in hepatitis C: mechanisms of non-response.
      ]. Moreover, sustained clearance of HBV or HCV may be difficult to accomplish, particularly among cirrhotic patients.
      It has been shown that oxidative DNA damage in cirrhotic HCV-infected patients is associated with increased risk of developing HCC [
      • Tanaka H.
      • Fujita N.
      • Sugimoto R.
      • Urawa N.
      • Horiike S.
      • et al.
      Hepatic oxidative DNA damage is associated with increased risk for hepatocellular carcinoma in chronic hepatitis C.
      ]. In patients with chronic hepatitis C, an increased 8OHdG in DNA extracted from liver tissue was reported [
      • Mahmood S.
      • Kawanaka M.
      • Kamei A.
      • Izumi A.
      • Nakata K.
      • Niiyama G.
      • et al.
      Immunohistochemical evaluation of oxidative stress markers in chronic hepatitis C.
      ,
      • Shimoda R.
      • Nagashima M.
      • Sakamoto M.
      • Yamaguchi N.
      • Hirohashi S.
      • Yokota
      • et al.
      Increased formation of oxidative DNA damage, 8-hydroxydeoxyguanosine, in human livers with chronic hepatitis.
      ,
      • Fujita N.
      • Horiike S.
      • Sugimoto R.
      • Tanaka H.
      • Iwasa M.
      • Kobayashi Y.
      • et al.
      Hepatic oxidative DNA damage correlates with iron overload in chronic hepatitis C patients.
      ]. Although these reports suggest that oxidative stress may be involved in the progression of liver disease, they did not show a direct participation of oxidative stress in hepatocarcinogenesis.
      Intracellular ROS generation may also represent a critical event linking hypoxia to angiogenesis and HCC development. Indeed, hypoxic areas are very common in HCC and hypoxia-related induction of ROS formation has been reported to stabilize the hypoxia-inducible factor 1α (HIF-1 α), through inhibition of prolyl-hydroxylases (PHD) or redox-dependent activation of protein phosphorylation cascades, and to lead to upregulation of HIF-1 target genes [
      • Fernández M.
      • Semela D.
      • Bruix J.
      • Colle I.
      • Pinzani M.
      • Bosch J.
      Angiogenesis in liver disease.
      ]. Mitochondria are the major source of ROS generated during hypoxia [
      • Brunelle J.K.
      • Bell E.L.
      • Quesada N.M.
      • Vercauteren K.
      • Tiranti V.
      • Zeviani M.
      • et al.
      Oxygen sensing requires mitochondrial ROS but not oxidative phosphorylation.
      ]. Hypoxia increases mitochondrial ROS via the transfer of electrons from ubisemiquinone to molecular oxygen at the Qo site of complex III of the mitochondrial ETC [
      • Bell E.L.
      • Klimova T.A.
      • Eisenbart J.
      • Moraes C.T.
      • Murphy M.P.
      • Budinger G.R.
      • et al.
      The Qo site of the mitochondrial complex III is required for the transduction of hypoxic signaling via reactive oxygen species production.
      ]. Release of reactive oxygen species from the inner mitochondrial membrane to the intermembrane space leads to the activation of transcription factors, including HIF-1 α [
      • Lluis J.M.
      • Buricchi F.
      • Chiarugi P.
      • Morales A.
      • Fernandez-Checa J.C.
      Dual role of mitochondrial reactive oxygen species in hypoxia signaling: activation of nuclear factor-{kappa}B via c-SRC and oxidant-dependent cell death.
      ]. Furthermore, hypoxia-induced ROS generation has been shown to enhance the DNA binding of NF-κB through a redox dependent mechanism, leading to transcriptional activation of target genes [
      • Chandel N.S.
      • Trzyna W.C.
      • McClintock D.S.
      • Schumacker P.T.
      Role of oxidants in NF-kappa B activation and TNF-alpha gene transcription induced by hypoxia and endotoxin.
      ].
      Hypoxia represents a strong selective pressure reported to favor cancer progression through the activation of adaptive transcriptional programs that promote angiogenesis, cell survival, motility, and invasiveness of malignant cells, including the ability to induce epithelial mesenchymal transition (EMT) and increased invasiveness in human epithelial cancer cells [
      • Cannito S.
      • Novo E.
      • di Bonzo L.V.
      • Busletta C.
      • Colombatto S.
      • Parola M.
      Epithelial-mesenchymal transition: from molecular mechanisms, redox regulation to implications in human health and disease.
      ,
      • Pani G.
      • Giannoni E.
      • Galeotti T.
      • Chiarugi P.
      Redox-based escape mechanism from death: the cancer lesson.
      ,
      • Semela D.
      • Dufour J.F.
      Angiogenesis and hepatocellular carcinoma.
      ]. This novel, ROS- and hypoxia-related perspective is particularly relevant to HCC that usually develops on the background of a chronically damaged liver. Indeed, hypoxia, through redox signaling, has been described to induce EMT and increased invasiveness in human liver cancer cells [
      • Pani G.
      • Giannoni E.
      • Galeotti T.
      • Chiarugi P.
      Redox-based escape mechanism from death: the cancer lesson.
      ], which is relevant for a tumor like HCC that is more invasive than metastatic [
      • Cannito S.
      • Novo E.
      • di Bonzo L.V.
      • Busletta C.
      • Colombatto S.
      • Parola M.
      Epithelial-mesenchymal transition: from molecular mechanisms, redox regulation to implications in human health and disease.
      ,
      • Semela D.
      • Dufour J.F.
      Angiogenesis and hepatocellular carcinoma.
      ]. Moreover, aberrant angiogenesis and vascular remodeling are critical events for HCC growth [
      • Cannito S.
      • Novo E.
      • di Bonzo L.V.
      • Busletta C.
      • Colombatto S.
      • Parola M.
      Epithelial-mesenchymal transition: from molecular mechanisms, redox regulation to implications in human health and disease.
      ,
      • Semela D.
      • Dufour J.F.
      Angiogenesis and hepatocellular carcinoma.
      ]. The angiogenic switch is sustained by hypoxia, cytokines and GFs as well as by mutation in suppressor genes and oncogenes, with hypoxia being the most relevant one.

      The metabolic syndrome: role of oxidative stress in the progression of NAFLD

      The definition of metabolic syndrome involves a group of metabolic and cardiovascular risk factors that help identify subjects at high risk of type 2 diabetes and cardiovascular disease (CVD). Even if the term “syndrome” implies a specific causative aetiology, there is not a clear, unifying pathophysiological cause for the metabolic syndrome. Nevertheless, abdominal adiposity, physical inactivity, and insulin resistance appear to be at the core of this condition [
      • Mazzucco S.
      • Agostini F.
      • Biolo G.
      Inactivity-mediated insulin resistance is associated with upregulated pro-inflammatory fatty acids in human cell membranes.
      ]. The latest definition of the metabolic syndrome, supplied by the International Diabetes Federation, shows as typical marks: abdominal obesity, defined by increased waist circumference (⩾94 cm in men and ⩾80 cm in women), and two or more of the following factors including elevated blood pressure, raised fasting plasma glucose, increase serum triglyceride levels and/or low HDL cholesterol [
      • Alberti K.G.
      • Zimmet P.
      • Shaw J.
      Metabolic syndrome – a new world-wide definition. A consensus statement from the International Diabetes Federation.
      ]. An increase in triglyceride levels is one of the most prevalent metabolic syndrome components. Recent epidemiological data indicate that NAFLD is the most frequent hepatic lesion in Western countries. This condition is often benign, but in about 20–30% of NAFLD patients the disease can progress to NASH and cirrhosis.
      Increased levels of oxidative stress have been proposed to play a relevant pathogenic role in NAFLD progression according to the original ‘two-hit’ theory of Day and James [
      • Day C.P.
      • James O.F.
      Steatohepatitis: a tale of two “hits”?.
      ] and to the more recent insights coming from reliable animal models [
      • Tipoe G.L.
      • Ho C.T.
      • Liong E.C.
      • Leung T.M.
      • Lau T.Y.
      • Fung M.L.
      • et al.
      Voluntary oral feeding of rats not requiring a very high fat diet is a clinically relevant animal model of non-alcoholic fatty liver disease (NAFLD).
      ]. Along these lines, several mechanisms favoring increased generation of oxidative stress mediators in NAFLD have been proposed, including mitochondrial, increased fatty acid oxidation in either endoplasmic reticulum (ω-oxidation), by CYP-2E1 and CYP4A isoforms, or in peroxisomes (β-oxidation) by acyl-CoA oxidase [
      • Browning J.D.
      • Horton J.D.
      Molecular mediators of hepatic steatosis and liver injury.
      ]. Moreover, ROS and other oxidative stress-mediators, such as aldehydic end-products of lipid peroxidation are believed to sustain fibrotic progression of chronic liver diseases of different aetiology towards the end point of cirrhosis, being able to elicit cell injury and death as well as fibrosis and inflammatory response [
      • Browning J.D.
      • Horton J.D.
      Molecular mediators of hepatic steatosis and liver injury.
      ,
      • Parola M.
      • Robino G.
      Oxidative stress-related molecules and liver fibrosis.
      ].

      Molecular events in NASH

      The accumulation of triglycerides in hepatocytes is the hallmark of NAFLD, a spectrum of hepatic abnormalities usually associated with altered metabolism [
      • Alberti K.G.
      • Zimmet P.
      • Shaw J.
      Metabolic syndrome – a new world-wide definition. A consensus statement from the International Diabetes Federation.
      ]. In approximately 30% of the cases, steatosis is associated with hepatocellular damage, evident as ballooning, inflammation and fibrosis. This more aggressive form of the disease, known as NASH, may be associated with fibrosis and has the ability to progress to cirrhosis and its complications, including hepatocellular carcinoma [
      • Marchesini G.
      • Bugianesi E.
      • Forlani G.
      • Cerrelli F.
      • Lenzi M.
      • Manini R.
      • et al.
      Nonalcoholic fatty liver, steatohepatitis, and the metabolic syndrome.
      ]. The mechanisms leading to the appearance of NASH and its progression to fibrosis are still uncertain and focus of active investigation. Genetic factors certainly play a role, as shown for the polymorphisms of adiponutrin (PNPLA3), which are associated with steatosis and predisposition to injury and fibrosis [
      • Daly A.K.
      • Ballestri S.
      • Carulli L.
      • Loria P.
      • Day C.P.
      Genetic determinants of susceptibility and severity in nonalcoholic fatty liver disease.
      ]. Studies in animal models have increased our understanding of the molecular mechanisms that lead to the appearance and progression of NASH. A central point in the emergence of NASH is the toxic effects of lipids accumulating in the liver, a process known as lipotoxicity [
      • Neuschwander-Tetri B.A.
      Hepatic lipotoxicity and the pathogenesis of nonalcoholic steatohepatitis: the central role of nontriglyceride fatty acid metabolites.
      ]. This event is mediated by fatty acids and some of their metabolites, whereas triglycerides represent a relatively safe form of fat storage. Of interest was the observation that cholesterol per se may play a role in the progression of fatty liver to more steatohepatitis. Both nutritional and genetic models of hepatic steatosis showed that the accumulation of cholesterol in mitochondria was associated with a selective glutathione depletion which in turn was associated with a higher sensitivity to TNF and Fas [
      • Marí M.
      • Caballero F.
      • Colell A.
      • Morales A.
      • Caballeria J.
      • Fernandez A.
      • et al.
      Mitochondrial free cholesterol loading sensitizes to TNF- and Fas-mediated steatohepatitis.
      ]. This observation pointed to the conclusion that not only the amount but also the type of lipids accumulated in the liver may be important, a concept confirmed in different experimental models [
      • Chavez-Tapia N.C.
      • Rosso N.
      • Tiribelli C.
      In vitro models for the study of non-alcoholic fatty liver disease.
      ]. One of the consequences of lipotoxicity is the appearance of endoplasmic reticulum stress (ERS), which results from improperly folded proteins accumulating in the ER [
      • Wierzbicki A.S.
      • Oben J.
      Nonalcoholic fatty liver disease and lipids.
      ]. ERS is linked to the activation of NF-κB, c-Jun N-terminal kinase, and oxidative stress pathways. More recently, autophagy has also been implicated in the pathogenesis of insulin resistance and fat-mediated damage [
      • Rautou P.E.
      • Mansouri A.
      • Lebrec D.
      • Durand F.
      • Valla D.
      • Moreau R.
      Autophagy in liver diseases.
      ].
      Another pathway strictly linked to lipotoxicity is the generation of oxidative stress-related products, which contribute to hepatocellular damage, inflammation and fibrosis [
      • Tilg H.
      • Moschen A.R.
      Evolution of inflammation in nonalcoholic fatty liver disease: the multiple parallel hits hypothesis.
      ]. Inflammation is part of the wound healing response and is regulated by a complex network of soluble mediators, including cytokines such as TNF-α or IL-6. In this context, chemokines such as CCL2 or CCL5 also play a relevant role [
      • Haukeland J.W.
      • Damås J.K.
      • Konopski Z.
      • Løberg E.M.
      • Haaland T.
      • Goverud I.
      • et al.
      Systemic inflammation in nonalcoholic fatty liver disease is characterized by elevated levels of CCL2.
      ]. The chemokine system is not only linked to inflammation but also contributes to the development of fibrosis via direct actions on hepatic stellate cells. In this respect, activation of the receptors CCR5 and CCR2 play a major role [
      • Wasmuth H.E.
      • Tacke F.
      • Trautwein C.
      Chemokines in liver inflammation and fibrosis.
      ].
      The development of steatohepatitis and fibrosis is also influenced by extrahepatic factors. Differences in the microbiota have been recently suggested to participate in the pathogenesis of NASH [
      • Machado M.V.
      • Cortez-Pinto H.
      Gut microbiota and nonalcoholic fatty liver disease.
      ]. These actions are mediated, at least in part, by activation of the toll-like receptors, which recognize microbial patterns providing danger signals. Adipokines, cytokines secreted at the level of the adipose tissue, represent another group of signals relevant to the development of steatohepatitis and fibrosis. Leptin and adiponectin exert many opposing actions on inflammation and fibrosis. In addition, adiponectin limits the development of insulin resistance. Modulation of adipokine imbalance can explain some of the favorable effects of weight loss [
      • Dumas M.E.
      • Barton R.H.
      • Toye A.
      • Cloarec O.
      • Blancher C.
      • Rothwell A.
      • et al.
      Metabolic profiling reveals a contribution of gut microbiota to fatty liver phenotype in insulin-resistant mice.
      ].

      Iron induced liver damage

      Accumulating evidence indicates that deregulation of iron metabolism is involved in the pathogenesis of ischemic/reperfusion injury, neurodegeneration, insulin resistance and diabetes mellitus, atherosclerosis, and liver diseases [
      • Fargion S.
      • Valenti L.
      • Fracanzani A.L.
      Beyond hereditary hemochromatosis: new insights into the relationship between iron overload and chronic liver diseases.
      ]. The most studied disease model of iron toxicity is represented by hereditary hemochromatosis (HH), where defective regulation of systemic iron metabolism due to defective release or activity of the hormone hepcidin leads to hepatic and later parenchymal systemic iron overload [
      • Pietrangelo A.
      Hemochromatosis: an endocrine liver disease.
      ]. In HH, iron accumulation within hepatocytes has been associated with oxidative damage to DNA and activation of fibrogenesis [
      • Gualdi R.
      • Casalgrandi G.
      • Montosi G.
      • Ventura E.
      • Pietrangelo A.
      Excess iron into hepatocytes is required for activation of collagen type I gene during experimental siderosis.
      ], and often results in progressive liver disease. In association with obesity and metabolic syndrome, it represents the leading cause of hepatic iron overload in Western countries [
      • Dongiovanni P.
      • Fracanzani A.L.
      • Fargion S.
      • Valenti L.
      Iron in fatty liver and in the metabolic syndrome: a promising therapeutic target.
      ], with frequent progression to advanced fibrosis and HCC [
      • Fargion S.
      • Valenti L.
      • Fracanzani A.L.
      Hemochromatosis gene (HFE) mutations and cancer risk: expanding the clinical manifestations of hereditary iron overload.
      ].
      Although a long-lasting hypothesis is that the damaging effect of iron is related to its ability to catalyze the generation of ROS via the Fenton reaction, the molecular pathways of cellular and tissue dysfunction are less clear. Recent data have implicated an upregulation of the p53 pathway, which would lead to an initial induction of antioxidant enzymes, and later to cell senescence and death [
      • Dongiovanni P.
      • Fracanzani A.L.
      • Cairo G.
      • Megazzini C.P.
      • Gatti S.
      • Rametta R.
      • et al.
      Iron-dependent regulation of MDM2 influences p53 activity and hepatic carcinogenesis.
      ]. Furthermore, fatty acids would further increase ROS production when iron accumulation is associated with fatty liver, and, when present, excess alcohol.
      A recent study by Dixon et al. describes a non-apoptotic form of cell death dependent on the oncogenic RAS-selective lethal small molecule erastin. This molecule triggers a unique iron-dependent form of non-apoptotic cell death that the authors termed “ferroptosis”. Ferroptosis is dependent upon intracellular iron and is morphologically, biochemically, and genetically distinct from apoptosis, necrosis, and autophagy. Erastin, like glutamate, inhibits cystine uptake by the cystine/glutamate antiporter (system x(c)(−)), creating a void in the antioxidant defenses of the cell and ultimately leading to iron-dependent, oxidative death [
      • Dixon S.J.
      • Lemberg K.M.
      • Lamprecht M.R.
      • Skouta R.
      • Zaitsev E.M.
      • Gleason C.E.
      • et al.
      Ferroptosis: an iron-dependent form of nonapoptotic cell death.
      ].
      The role of ROS in the pathogenesis of iron-related liver damage was supported by the demonstration that a functional promoter polymorphism of myeloperoxidase (MPO), a major site of ROS production in phagocytes, is associated with cirrhosis and HCC in HH patients [
      • Osterreicher C.H.
      • Datz C.
      • Stickel F.
      • Hellerbrand C.
      • Penz M.
      • Hofer H.
      • et al.
      Association of myeloperoxidase promotor polymorphism with cirrhosis in patients with hereditary hemochromatosis.
      ]. Similarly, in NAFLD it was recently demonstrated that the C47T polymorphism, resulting in a decreased import and activity of the mitochondrial SOD2 involved in the detoxification of ROS, was strongly associated with the susceptibility to advanced fibrotic disease even when I148M PNPLA3 genotype was controlled [
      • Al-Serri A.
      • Anstee Q.M.
      • Valenti L.
      • Nobili V.
      • Leathart J.B.
      • Dongiovanni P.
      • et al.
      The SOD2 C47T polymorphism influences NAFLD fibrosis severity: evidence from case-control and intra-familial allele association studies.
      ]. The same genetic variant predisposes to cardiomyopathy and HCC in HH [
      • Valenti L.
      • Conte D.
      • Piperno A.
      • Dongiovanni P.
      • Fracanzani A.L.
      • Fraquelli M.
      • et al.
      The mitochondrial superoxide dismutase A16V polymorphism in the cardiomyopathy associated with hereditary haemochromatosis.
      ].
      Recent observations suggest that as in NASH, ERS plays an important role in iron-induced liver injury and that ROS are mediators of the ERS response. ERS is an adaptive response to cellular stress induced by the accumulation of unfolded proteins, caused by unbalanced oxidative stress overcoming the protein folding capacity of ER. An excessive and protracted ERS results in cellular dysfunction by the alteration of glucose and lipid metabolism and by favoring cell death. Finally, the complex interplay between iron, ROS, and ERS response was emphasized by the evidence that accumulation of unfolded proteins in ER induces hepcidin expression via the ERS response [
      • Vecchi C.
      • Montosi G.
      • Zhang K.
      • Lamberti I.
      • Duncan S.A.
      • Kaufman R.J.
      • et al.
      ER stress controls iron metabolism through induction of hepcidin.
      ], thus linking ERS to iron homeostasis. On the other hand, oxidative stress has also been shown to decrease hepcidin release by altering the chromatin structure of its promoter region during HCV infection and alcohol abuse [
      • Harrison-Findik D.D.
      • Klein E.
      • Crist C.
      • Evans J.
      • Timchenko N.
      • Gollan J.
      Iron-mediated regulation of liver hepcidin expression in rats and mice is abolished by alcohol.
      ,
      • Fujita N.
      • Sugimoto R.
      • Takeo M.
      • Urawa N.
      • Mifuji R.
      • Tanaka H.
      • et al.
      Hepcidin expression in the liver: relatively low level in patients with chronic hepatitis C.
      ], thus contributing to the increased iron absorption characteristic of these diseases.
      Increased ROS production leading to unbalanced oxidative stress plays a key role in the pathogenesis of liver disease associated with hepatic iron accumulation, due to both genetic and acquired factors, and excess iron which may synergize with steatosis. The mechanisms include direct stimulation of fibrogenesis, mitochondrial damage and activation of the p53 pathway, activation of the ERS response, and DNA damage, collectively favoring HCC occurrence. Conversely, the oxidative stress and ERS response affect iron metabolism by influencing hepcidin expression. Therefore, the modulation of oxidative stress represents an attractive therapeutic target for the prevention of clinical complications of iron overload and steatosis.

      Modern proteomics will help in new biomarker discovery for early diagnosis of oxidative stress-based liver pathologies

      Antioxidant supplements for liver diseases did not provide convincing evidence to support or refute antioxidants in treating liver diseases [
      • Bjelakovic G.
      • Nikolova D.
      • Gluud L.L.
      • Simonetti R.G.
      • Gluud C.
      Antioxidant supplements for prevention of mortality in healthy participants and patients with various diseases.
      ]. This conclusion may be due to the lack of biomarkers predictive of severity and progression of the disease. Accordingly, the understanding of the sources of ROS formation during liver injury process is critical for the interpretation of experimental results and for designing an effective therapeutic intervention.
      The liver has been the subject of dedicated investigations since the first introduction of basic proteomic technologies in the early ‘90s [
      • Hochstrasser D.F.
      • Frutiger S.
      • Paquet N.
      • Bairoch A.
      • Ravier F.
      • Pasquali C.
      • et al.
      Human liver protein map: a reference database established by microsequencing and gel comparison.
      ]. After the launch of the Human Liver Proteome Project (HLPP) in 2002, a number of holistic studies have been undertaken on human and mouse/rat models in an attempt to reach a functional map of the organ, expanding the proteomic description to its “physic-ome” and “path-ome”, with the aim to accelerate the development of liver-specific diagnostics and therapeutics [
      • Zheng J.
      • Gao X.
      • Beretta L.
      • He F.
      The Human Liver Proteome Project (HLPP) workshop during the 4th HUPO World Congress.
      ]. Thus, various dozens of investigations, based on the use of 2D chromatography/electrophoresis combined with different MS approaches, have been published to describe the quantitative level of liver proteins under basal conditions [
      • Scaloni A.
      • Codarin E.
      • Di Maso V.
      • Arena S.
      • Renzone G.
      • Tiribelli C.
      • et al.
      Modern strategies to identify new molecular targets for the treatment of liver diseases: the promising role of proteomics and redox proteomics investigations.
      ]. These researches have been performed directly on liver tissues or, in other cases, on hepatocyte, hepatic stellate, Chang and Kupffer cells. Moreover, a large number of differential studies have been undertaken to describe the quantitative proteomic variations in hepatic cells during embryo organ development [
      • Ying W.
      • Jiang Y.
      • Guo L.
      • Hao Y.
      • Zhang Y.
      • Wu S.
      • et al.
      A dataset of human fetal liver proteome identified by subcellular fractionation and multiple protein separation and identification technology.
      ,
      • Lee N.P.
      • Leung K.W.
      • Cheung N.
      • Lam B.Y.
      • Xu M.Z.
      • Sham P.C.
      • et al.
      Comparative proteomic analysis of mouse livers from embryo to adult reveals an association with progression of hepatocellular carcinoma.
      ] or senescence [
      • Cho Y.M.
      • Bae S.H.
      • Choi B.K.
      • Cho S.Y.
      • Song C.W.
      • Yoo J.K.
      • et al.
      Differential expression of the liver proteome in senescence accelerated mice.
      ], after organ transplantation/resection [
      • Vascotto C.
      • Cesaratto L.
      • D’Ambrosio C.
      • Scaloni A.
      • Avellini C.
      • Paron I.
      • et al.
      Proteomic analysis of liver tissues subjected to early ischemia/reperfusion injury during human orthotopic liver transplantation.
      ,
      • Hirsch J.
      • Hansen K.C.
      • Choi S.
      • Noh J.
      • Hirose R.
      • Roberts J.P.
      • et al.
      Warm ischemia-induced alterations in oxidative and inflammatory proteins in hepatic Kupffer cells in rats.
      ], following treatment with various toxic agents [
      • Glückmann M.
      • Fella K.
      • Waidelich D.
      • Merkel D.
      • Kruft V.
      • Kramer P.J.
      • et al.
      Prevalidation of potential protein biomarkers in toxicology using iTRAQ reagent technology.
      ,
      • Low T.Y.
      • Leow C.K.
      • Salto-Tellez M.
      • Chung M.C.
      A proteomic analysis of thioacetamide-induced hepatotoxicity and cirrhosis in rat livers.
      ], or different liver diseases. In particular, a great effort has been spent in the proteomic analysis of in vitro models or liver tissue of HCV [
      • Ichibangase T.
      • Moriya K.
      • Koike K.
      • Imai K.
      A proteomics method revealing disease-related proteins in livers of hepatitis-infected mouse model.
      ,
      • Diamond D.L.
      • Jacobs J.M.
      • Paeper B.
      • Proll S.C.
      • Gritsenko M.A.
      • Carithers Jr, R.L.
      • et al.
      Proteomic profiling of human liver biopsies: hepatitis C virus-induced fibrosis and mitochondrial dysfunction.
      ] and HBV infection [
      • Yang F.
      • Yan S.
      • He Y.
      • Wang F.
      • Song S.
      • Guo Y.
      • et al.
      Expression of hepatitis B virus proteins in transgenic mice alters lipid metabolism and induces oxidative stress in the liver.
      ,
      • Tong A.
      • Wu L.
      • Lin Q.
      • Lau Q.C.
      • Zhao X.
      • Li J.
      • et al.
      Proteomic analysis of cellular protein alterations using a hepatitis B virus-producing cellular model.
      ], fibrosis and cirrhosis [
      • Tong A.
      • Wu L.
      • Lin Q.
      • Lau Q.C.
      • Zhao X.
      • Li J.
      • et al.
      Proteomic analysis of cellular protein alterations using a hepatitis B virus-producing cellular model.
      ,
      • Liu E.H.
      • Chen M.F.
      • Yeh T.S.
      • Ho Y.P.
      • Wu R.C.
      • Chen T.C.
      • et al.
      A useful model to audit liver resolution from cirrhosis in rats using functional proteomics.
      ], HCC [
      • Feng J.T.
      • Shang S.
      • Beretta L.
      Proteomics for the early detection and treatment of hepatocellular carcinoma.
      ,
      • Vivekanandan P.
      • Singh O.V.
      High-dimensional biology to comprehend hepatocellular carcinoma.
      ,
      • Sun W.
      • Xing B.
      • Sun Y.
      • Du X.
      • Lu M.
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      ]. Sensitive and specific biomarkers of some liver diseases have also been searched in the serum of patients by 2-DE/MS or SELDI-MS approaches [
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      Enrichment of low molecular weight fraction of serum for MS analysis of peptides associated with hepatocellular carcinoma.
      ], proposing a number of disease-associated biomarkers. Developments in immunoaffinity depletion and various fractionation approaches in combination with substantial improvements in LC–MS platforms have enabled the plasma/tissue proteome to be profiled with a considerably greater dynamic range of coverage, allowing the identification of several proteins at low ng/ml levels. Despite these significant advances, major challenges associated with the dynamic range of measurements and extent of proteome coverage, confidence of peptide/protein identifications, quantitation accuracy, analysis throughput, and robustness of present instrumentation must be addressed before a proteomics profiling platform suitable for efficient clinical applications may be routinely used in the accurate diagnosis of early liver damage, as well as to monitor disease progression and assess treatment efficacy [
      • Beretta L.
      Proteomics from the clinical perspective: many hopes and much debate.
      ].
      Based on previous investigations describing the main targets of ROS and RNS-dependent protein oxidation/nitrosation, redox proteomics approaches [
      • Lin T.K.
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      • Muratovska A.
      • Blaikie F.H.
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      • et al.
      Specific modification of mitochondrial protein thiols in response to oxidative stress: a proteomics approach.
      ] were performed in vitro experiments by treating liver tissue or hepatocytes with specific oxidants, such hydrogen peroxide, diamide, menadione, t-butyl-hydroperoxide and S-nitroso-l-cysteine [
      • Lin T.K.
      • Hughes G.
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      • Brookes P.S.
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      Specific modification of mitochondrial protein thiols in response to oxidative stress: a proteomics approach.
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      ,
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      Detection and proteomic identification of S-nitrosated proteins in human hepatocytes.
      ,
      • Cesaratto L.
      • Vascotto C.
      • D’Ambrosio C.
      • Scaloni A.
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      • et al.
      ]. These studies identified early protein markers of thiol modification, which are subjected to glutathionylation, sulphenation or nitrosation reactions. Although still preliminary and inconclusive, this scenario emphasizes the potential role of proteomics and redox proteomics technologies in the development of liver-specific diagnostics and therapeutics. These holistic hold promise for the identification of biomarker proteins implicated in the development of CLD and of protein targets of the ROS/RNS insult during pathophysiological phenomena. It has to be mentioned, however, that while various proteomic studies have described the quantitative changes in the protein profile of hepatic cells or tissues during the pathological income, a very limited number of investigations have described their redox-associated modifications.

      Financial support

      This work was supported by Grants from MIUR (FIRB RBRN07BMCT) and AIRC (to G.T.), and by the European Union Seventh Framework Programme (FP7/2007-2013) under Grant agreement No. Health-F2-2009-241762, for the project FLIP and an in house grant from the Fondazione Italiana Fegato (to C.T.).

      Conflict of interest

      The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

      Acknowledgements

      We want to deeply acknowledge the entire faculty and students for their active participation in the inspiring presentations and discussion.

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