Journal of Hepatology
Volume 35, Issue 2 , Pages 297-306, August 2001

Oxidative stress-related molecules and liver fibrosis

Dipartimento di Medicina e Oncologia Sperimentale, Sezione di Patologia Generale, Università degli Studi di Torino, Corso Raffaello 30, 10125 Turin, Italy

Received 25 January 2001; received in revised form 21 May 2001; accepted 28 May 2001.

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1. Introduction 

Liver fibrosis can be considered as a dynamic and highly integrated cellular response to chronic liver injury [1]. Whatever the etiology, the evolution of chronic liver disease (CLD) is characterized by perpetuation of parenchymal necrosis, chronic hepatitis and qualitative as well as quantitative alterations in extracellular matrix (ECM) composition, whereas activation of hepatic stellate cells (HSC) and involvement of macrophages and Kupffer cells predominate at cellular level [1], [2], [3]. At the molecular level, growth factors, cytokines and chemokines, changes in ECM organization and composition as well as reactive molecules originated by oxidative stress have been suggested to play a pathogenetic role [1], [2], [3]. Evidence of oxidative stress has been detected in almost all the clinical and experimental conditions of CLD with different etiology and fibrosis progression rate (Table 1 and Refs. [4], [5], [6]), often in association with decreased antioxidant defenses. As already proposed for atherosclerosis [7] and chronic degenerative diseases of CNS [8], oxidative stress-related molecules may act as mediators able to modulate tissue and cellular events responsible for the progression of liver fibrosis [1], [3], [4], [5], [6]. This review will highlight major concepts and recent insights in the field, and the definition ‘oxidative stress-related molecules’ will be used to indicate reactive oxygen intermediates (ROI, i.e. oxygen-centered free radicals or intermediates) as well as aldehydes from lipid peroxidation (i.e. a major feature of hepatic oxidative stress). Major concepts and findings related to the role of nitric oxide (NO) and reactive nitrogen oxide intermediates (RNOI) in chronic liver injury, with special reference to NO interactions with ROI and potential antifibrogenic action of NO, will be also presented.

Table 1. Major clinical and experimental conditions of chronic liver injury in which involvement of oxidative stress (mainly lipid peroxidation) has been detected in vivoa
Clinical conditionsExperimental animal models
Chronic HCV infectionBile duct ligation
Alcoholic liver diseaseChronic CCl4 administration
Genetic hemochromatosisChronic ethanol administration
Wilson's diseaseModels of iron overload
Primary biliary cirrhosisModels for copper overload
Various cholestatic diseasesMixed models of hepatotoxins (ethanol+iron, CCl4+ethanol)

a In most of these conditions also a decrease in either hepatic or plasma antioxidant defenses has been reported to occur (see Refs. [4], [5], [6], [19], [20], [29] and references therein).

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2. Oxidative stress in CLD: basic and clinically relevant concepts 

The term oxidative stress has been often employed to indicate the outcome of oxidative damage to biologically relevant macromolecules such as nucleic acids, proteins, lipids and carbohydrates [9]. This occurs when oxidative stress-related molecules, generated in the extracellular environment or within the cell, exceed cellular antioxidant defenses and, in the past, this aspect has been mainly related to the potential cytotoxic consequences of oxidative stress. At present this definition has been implemented by recent data indicating that major ROI, such as hydrogen peroxide (H2O2) and superoxide anion (O2·−), as well as 4-hydroxy-2,3-nonenal (HNE) and related 4-hydroxy-2,3-alkenals (HAKs), major aldehydic end-products of lipid peroxidation, can act as potential mediators able to affect signal transduction pathways as well as the proliferative and functional response of target cells [6], [8], [10]. H2O2 and O2·− may be also generated as molecular messengers within the cell as part of the cellular response to defined growth factors, cytokines and other mediators [8], [10]. As it will be emphasized later in this review, the final consequence at tissue, cellular and molecular level (overt cytotoxicity, apoptotic cell death or modulation of cell response and fibrosis progression) is primarily affected by the steady state concentration of oxidative stress-related molecules. This parameter, in turn, may be modulated by conditions that can have a relevant impact on both basic mechanisms and clinical manifestations [5], [6], [8], [9], [10], [11], [12], [13]. These include the following.

2.1. Antioxidant defenses 

The normal liver is a well equipped organ in terms of either enzymic or non enzymic antioxidants (see Fig. 1) although most of the hepatic antioxidant defenses are essentially confined to parenchymal cells [14]. Kupffer cells, HSC or endothelial cells are potentially more exposed or sensitive to oxidative stress-related molecules (see later in this review). Published experimental evidence clearly indicates that hepatic as well as plasma antioxidant defenses (in particular, GSH and α-tocopherol) are often significantly decreased in several CLD [4], [5], [14].

  • View full-size image.
  • Fig. 1. 

    ROI, HAKs and NO in the pathogenesis of liver fibrosis. ROI, and then HAKs, may be generated by activated inflammatory cells (NADH/NADPH oxidase systems in neutrophils, macrophages and Kupffer cells), by ‘leaking’ or damaged mitochondria (shift of electrons through respiratory chain to molecular oxygen to give superoxide anion in a univalent reduction) as well as following hepatocellular injury or induction of CYP2E1 isoforms. Extent of oxidative stress and the nature of reactive species is modulated by a number of factors of potential clinical impact as well as by available intracellular and plasma antioxidant defenses. Both ROI and HAKs may elicit and/or sustain crucial pathogenetic features in CLDs whereas emerging evidence suggest that NO, generated by eNOS and iNOS, may act also or mainly as ‘defensive’ intermediate by down-modulating amplification and perpetuation of inflammatory response as well as progression of fibrosis by affecting HSC activation and/or reducing oxidative stress. GSH, reduced glutathione; GSH-PXs, GSH peroxidases; SODs, superoxide dismutases; Ald-DH, aldehyde dehydrogenase; GSSG-red, glutathione disulfide reductase; GSTs, GSH-S-transferases; ·NO, nitric oxide.

2.2. The nature, the specificity of the reactions and the site of production of the different reactive molecules 

Origin of ROI and other oxidative stress-related molecules as well as their reactions relevant to this review are summarized in Fig. 1 and Table 2. Different ROI have highly variable half life and reactivity: as a general rule, the more unstable intermediate has the highest reactivity and the lowest half life time and vice versa. Hydroxyl radical (·OH), probably the most reactive and cytotoxic oxygen centered radical, has a mean half life of 10−9 seconds, it can diffuse less than 2 nm from the site in which is generated and, as a main consequence, it will essentially react non-specifically with any biological molecules available at the site of production. H2O2 and O2·−, that exert manifold effects, are less reactive, and may have a longer half life; however, only H2O2 can easily diffuse across plasma membrane and throughout the cell, whereas O2·− diffuses poorly across cell membranes [9]. NO and the highly lipophilic aldehyde HNE, that have a relatively high diffusion range and long half-life, may also react in a less unspecific way with biological macromolecules [12], [15]. However, this is just a theoretic scenario: for example, NO can react at extremely fast rate constants when produced in the same microenvironment in which superoxide anion (i.e. by activated inflammatory cells) or carbon dioxide are generated. Moreover, any ROI may rapidly change into other radicals or intermediates depending on local chemical and biochemical conditions, including pH value and the presence of metals [9], [15].

Table 2. Major consequences of reaction of ROI, HAKs and NO with biologically relevant macromolecules that may mediate pathophysiological effects of these compoundsa
ROIDNA: oxidation, strand breaks, genotoxicity
Proteins: oxidation, fragmentation, formation of carbonyls
Lipids: lipid peroxidation and degradation
HAKsDNA: adducts (low doses), strand breaks, genotoxicity (high doses)
Proteins: adducts (Michael type reactions on Lys, Cys and His residues)
NODNA: oxidation, strand breaks
Proteins: oxidation, nitrosation, nitration (nytrosylation of tyrosine)
Lipids: lipid peroxidation and degradation

a Note the following: (a) ROI may act primarily as oxidising agents or by eliciting lipid peroxidation; (b) HAKs do not act as oxidising entities but rather as nucleophilic agents; (c) NO exerts reported effects mainly under the form of N2O3 or interacting with superoxide anion to form ONOO−. More details on chemistry and biochemistry of ROI, HAKs as well as NO and RNOI may be found in specialized reviews (see Refs. [9], [12], [15], [89]).

2.3. Hepatic levels of metal catalysts of oxidative stress 

Hepatic iron overload is associated with hepatocellular injury, inflammation, fibrosis, cirrhosis and hepatocellular cancer [4]. Iron accumulates in the liver as a consequence of genetic defects in the absorption, as in genetic hemochromatosis associated with either HFE and non-HFE mutations, or following repeated parenteral administration (i.e. transfusions). Excess free iron represents a potent deleterious hepatotoxic as well as pro-fibrogenic cofactor in the presence of chronic alcohol abuse, viral hepatitis or hepatotoxic xenobiotics [4], [16]. Iron is the ideal metal catalyst for generation of ROI and other free radical intermediates as well as for the induction of lipid peroxidation, and oxidative stress is a common finding in all conditions of iron overload [4], [5]. Antioxidant supplementation prevents liver injury and fibrosis progression in animal models of iron overload [17], [18] although iron may be also fibrogenic per se [4]. Since copper is another excellent catalyst of oxidative stress [9], similar considerations should apply also to Wilson's disease and related animal models in which the involvement of oxidative stress has been detected [5].

2.4. Alcohol consumption 

Detection of oxidative stress and reduced antioxidant defenses has been found in either human alcoholics and in experimental animal models [19], [20]. Relationships between alcohol consumption and CLD are crucial for alcoholics and for patients with iron overload; moreover alcohol consumption is one of the few relevant host-related factor able to accelerate the progression of fibrosis and the development of cirrhosis in patients affected by chronic hepatitis C [21]. A major determinant of oxidative stress during chronic alcohol consumption is represented by the induction of cytochrome P450 isoform CYP2E1 in hepatocytes and in Kupffer cells [19], [20], but not in hepatic stellate cells (HSC) [22]. Induction of CYP2E1 is responsible for most of ethanol metabolism to acetaldehyde but also for the increased vulnerability of alcoholics to the toxicity of various drugs, industrial solvents and anesthetics [20]. CYP2E1 has a very high NADPH oxidase activity and extensively produce O2·− and H2O2 as well as hydroxyethyl radicals that are likely to be responsible for ethanol induction of oxidative stress and lipid peroxidation [19], [20]. Hydroxyethyl radicals have been shown to alkylate proteins and to induce, particularly in human alcoholics, the production of related specific antibodies, in addition to antibodies directed against adducts generated by the interaction of proteins with acetaldehyde [19], [20]. These antibodies recognize hydroxyethyl radical-derived antigens on the plasma membrane of hepatocytes (one identified as CYP2E1 itself) exposed to ethanol, and are able to elicit antibody-dependent cell-mediated immunological reactions towards parenchymal cells [19], [23]. As a consequence, oxidative stress-related intermediates may contribute to liver injury during alcohol abuse also by means of immunological mechanisms.

2.5. Age, obesity and non-alcoholic steatohepatitis (NASH) 

Aging is usually associated with an increased susceptibility to oxidative stress and a significant reduction of antioxidant defenses [24], and this reduction may sustain, at least in part, the increased rate of fibrosis progression observed in older patients. Age at infection is considered a relevant individual factor able to accelerate fibrosis progression in chronic HCV patients [21] and a significant association between age (>50 years) and septal fibrosis has also been described in overweight patients [25]. NASH is a liver disease characterized by histopathological features similar to those observed in alcoholic liver disease, in the absence of significant alcohol consumption [26]. Oxidative stress and lipid peroxidation have been implicated in the pathogenesis of NASH, possibly as the results of two conditions [26]: (a) it has been proposed that non-insulin dependent diabetes mellitus (NIDDM) and rapid weight loss in obese patients, two recognized risk factors for NASH, may lead to increased concentration of free fatty acids in mitochondria, saturation of mitochondrial β-oxidation and excess H2O2 generation during peroxisomal β-oxidation; (b) CYP2E1 is induced in either human NASH patients as well as in a related experimental model of NASH [27], [28]; recently, also the CYP4A isoform has been suggested as a catalyst of lipid peroxide generation in experimental NASH [28]. Moreover, CYP2E1 is also induced by free fatty acids and ketones and, indeed, diabetes (NIDDM) and obesity are considered risk factors for NASH [26].

2.6. Bile acids and cholestasis 

In the last decade, several reports have proposed the involvement of oxidative stress and decreased antioxidant defenses in experimental and clinical cholestatic liver injury, including primary biliary cirrhosis [5], [6]. Whether oxidative stress, mainly lipid peroxidation, may represent a component of bile acids cytotoxicity, the consequence of activation of inflammatory cells or of decreased antioxidant defenses [5], [29] is still unclear. Moreover, the scenario is even more complicated by the fact that bilirubin may act as an antioxidant [5], [7], [9].

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3. ROI and HAKs as cytotoxic agents: necrosis versus apoptosis, a matter of concentration? 

Parenchymal liver necrosis and its perpetuation is a common condition in the natural history and progression of CLD [1], [2], [3]. Severe oxidative stress, considered a major cause of liver necrosis (i.e. acute liver injury), can be elicited by several pro-oxidants and hepatotoxic agents or drugs, leading to steady state concentrations of approx. 0.15 μM for H2O2 and 0.25 μM for total ROI [13] as well as 10−5 M for HNE and HAKs [12]. In addition, oxidative stress constitutes a key feature of hepatitis induced by various conditions, including anoxic/reoxygenation injury, autoimmune hepatitis, viral hepatitis and alcoholic hepatitis [11]. Activated neutrophils, macrophage and Kupffer cells are a major source of ROI during inflammation, whereas individual differences (i.e. induction of cytochrome P450 isoforms, genetic polymorphisms, antioxidant status, etc.) may also play a role. If oxidative stress is severe, all the major cellular structures (particularly mitochondria and cytoskeletal proteins), macromolecules (DNA, lipids, enzymatic proteins) and metabolic pathways can be directly or indirectly oxidized, damaged and then blocked or inactivated, leading eventually to necrotic cell death [11]. Less severe oxidative stress, not sufficient to impair irreversibly mitochondrial functions or to inactivate caspases, has been suggested to cause liver cells apoptosis [11]. Similarly, high levels (25–50 μM or more) of HAKs, particularly HNE, can inactivate several enzymes, cellular functions and metabolisms leading to cell death, as shown for hepatocytes and HSC [6], [12], [30]. HNE may be also able to elicit apoptosis in liver cell populations but this specific aspect is still unexplored. Whether very high levels of ROI and HAKs (i.e. lethal oxidative stress) may occur in vivo during CLD is still matter of debate and a precise and definitive in vivo answer has not yet been provided. It is possible to hypothesize that in defined conditions known to occur in CLD (elevated catalytic free iron, induction of CYP2E1, decrease of antioxidant defenses at tissue or cellular level, strict contact between activated inflammatory cells and target cells, etc.) local high levels of oxidative stress may be achieved. However, it is reasonable to think that only mild to moderate levels of ROI and HAKs may be generated in vivo during CLD; these relatively low levels of oxidative stress, as discussed in the next paragraph, may sustain fibrosis progression.

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4. ROI and HAKs as pro-fibrogenic biological signals in the liver: the present and the future 

The progression of fibrosis in CLD of different etiology is, at least in part, sustained by the activation and phenotypical modulation of HSC towards the so-called myofibroblast-like phenotype [1], [2], [3]. This process follows a relatively well defined and programmed temporal sequence that recognizes early (initiation or pre-inflammatory stage) and late events (perpetuation of HSC activation) in which the activated phenotype is fully expressed [1]. Phenotypic responses of activated HSC include proliferative attitude, synthesis and degradation/remodeling of extracellular matrix, chemotaxis, contractility, pro-inflammatory activity (i.e. release of cytokines and chemokines) and retinoid loss [1]. As a consequence several laboratories have focused their efforts to analyze whether ROI and HAKs may act as profibrogenic mediators or signals able to modulate phenotypic responses of HSC.

4.1. Antioxidant defenses in HSC 

Activated rat HSC can remove H2O2 more efficiently than quiescent cells: induction of significant amount of catalase has been detected in HSC differentiated in culture into myofibroblasts as well as in rat HSC isolated from fibrotic livers [31], [32]. Efficient removal of H2O2, presumably by catalase, has been confirmed also by others [32]. Manganese superoxide dismutase (Mn-SOD), absent in freshly isolated HSC, is transiently expressed during early steps of activation in culture but, interestingly, is not expressed in HSC isolated from fibrotic livers [31]. Data concerning levels of reduced glutathione (GSH, the main water soluble cellular antioxidant) in HSC are less straightforward. GSH levels in rat HSC rapidly increase in the first few days of culture, reaching an apparent plateau after 6–8 days as a consequence of induction of γ-glutamylcysteine synthetase. However, no significant change in HSC levels of GSH was found in cells isolated from fibrotic rat livers [33] and manipulation of intracellular GSH levels by l-buthionine sulfoximine or diethylmaleate apparently do not modulate collagen synthesis in rat HSC [34]. On the other hand, it has been proposed that intracellular GSH levels may be relevant to modulate the synthesis of TGFβ1 by rat HSC [31].

The ability to metabolize reactive aldehydes by HSC represents an additional interesting issue. Data obtained on either human [35] or rat [36] HSC indicate that activated HSC are potentially much more sensitive to HNE than quiescent HSC or hepatocytes, a feature depending on a lower expression of GSH-S-transferase and aldehyde dehydrogenase isoforms active on HNE [35], [36]. Therefore, an emerging feature is that activated HSC may be relatively well equipped in terms of antioxidant defenses against H2O2 and ROI but more vulnerable to aldehydic end products of lipid peroxidation.

4.2. ROI, HAKs and collagen synthesis by HSC 

Oxidative stress may represent a direct or indirect relevant pro-fibrogenic stimulus for HSC, as suggested by in vivo experimental studies in which administration of antioxidants prevents oxidative stress, lipid peroxidation and liver fibrosis [17], [18], [37]. Moreover, signs of oxidative stress and lipid peroxidation are concomitant or precede HSC activation and collagen deposition [16], [38], [39], [40], [41]. Although it has been questioned that HSC may be not directly subjected to oxidative stress in experimental fibrosis [41], exposure of cultured human or rat HSC to pro-oxidant systems or to medium containing products released from hepatocytes undergoing oxidative stress (i.e. to mimic a possible paracrine effect by damaged parenchymal cells) is followed by increased procollagen type I gene expression and synthesis. This effect is prevented by antioxidants [42], [43], [44] or by NO donors [43]. Procollagen type I gene expression is also strongly elicited by very low levels (10−6 M) of extracellular HNE and other HAKs in human HSC [42], [45] and by H2O2 and malondialdehyde (MDA) in rat HSC [46], [47]. A role for either intracellular or extracellular ROI as profibrogenic mediators of oxidative stress has been confirmed in rat HSC transfected with human CYP2E1 cDNA, a model in which procollagen type I expression was found to be proportional to CYP2E1 levels [32]. Moreover, CYP2E1 transfected HSC overexpress procollagen type I after exposure to ethanol or arachidonic acid [48], a result prevented by antioxidants or by the specific CYP2E1 inhibitor diallylsulfide [32], [48].

Few data are available on the mechanisms involved in oxidative stress-mediated upregulation of procollagen type I. In activated human HSC, HNE, at very low and pro-fibrogenic doses (10−6 M), elicits the activation and nuclear translocation of c-Jun NH2-terminal kinases (JNKs), upregulation of c-jun and increased AP-1 binding [35]. Activation of JNKs and involvement of AP-1 is major determinant in the upregulation of procollagen type I gene expression as induced by exposure of HSC to UV irradiation [49], a process known to involve oxidative stress, lipid peroxidation and HNE generation. Results obtained in a series of studies [44], [50] have suggested that oxidative stress may modulate collagen synthesis also through the activation of Na+/H+ exchanger and the increase in intracellular pH. The use of antioxidants (resveratrol) or specific inhibitors of the Na+/H+ exchanger, amiloride and 5-N-ethyl-N-isopropyl-amiloride (EIPA), inhibits collagen synthesis by activated HSC in vitro and in vivo [44], [45]. H2O2 and O2·− may induce the activation of collagen type I gene also by upregulating cyclo-oxygenase 2 (COX-2) and then, possibly, through the action of metabolites of arachidonic acid [48]. In addition, in HSC H2O2 may act as intracellular signal mediator of the pro-fibrogenic action of TGFβ [31], [47] or of acetaldehyde [51] and may upregulate collagen type I gene by activation and binding of p35 C/EBPβ protein to a specific region of the promoter of the collagen α1(I) gene.

4.3. Effects of ROI, HAKs on HSC proliferation and α-smooth muscle actin (α-SMA) expression 

A series of in vivo and in vitro studies ([52] and references therein) has outlined a close relationship between oxidative stress, redox sensitive transcription factor NF-κB, increased expression of c-myb proto-oncogene and activation of HSC in terms of α-SMA expression and proliferation. In cultured rat HSC, this pathway is stimulated by the pro-oxidant system ascorbate-FeSO4 and relatively high concentrations of MDA (200 μM) but also by the contact of quiescent HSC with collagen type I. α-Tocopherol, used as antioxidant, prevented all the features of HSC activation. The oxidative stress-dependent role of c-myb is based on the redox state of a specific cysteine residue [52]. Other groups have confirmed that the use of phenolic antioxidants such as resveratrol and quercetin or the thiol derivative 2-mercapto-ethanol, can inhibit α-SMA expression in HSC [53], [54]. HSC proliferation induced by ROI can be also inhibited by resveratrol as well as amiloride and its derivative EIPA [44], [50], again indicating an involvement of the Na+/H+ exchanger.

Data concerning the action of HNE and other HAKs on HSC proliferation differ from those just reported for ROI, suggesting that the overall final response of a target HSC may depend on several factors, including local concentration of ROI and HAKs, presence of activated inflammatory cells, growth factors and other mediators in the micro-environment, as well as the actual state of HSC antioxidant defenses. In this connection, HAKs do not elicit proliferation in human HSC at any of the concentrations used but, rather, they inhibit DNA synthesis at high and cytotoxic doses [35], [30]. HNE, a non-oxidant that induce formation of adducts by means of nucleophilic Michael type reactions (see Table 2), does not elicit NF-κB [35] and specifically inhibit c-myb expression [6]. In addition, HAKs, at very low and non cytotoxic doses identical to those found to elicit procollagen type I synthesis, abolish PDGF-BB mitogenic signaling and proliferation of human HSC [30]. This occurs through specific inhibition of intrinsic tyrosine kinase activity associated with the PDGF-β receptor subunit; autophosphorylation of other receptors, such as PDGF-α receptor or EGF receptor, is not affected. The inhibitory effect of HAKs is transient and HSC recover the ability to proliferate as a response to PDGF within 48 h; such a recovery is associated with HAKs induced upregulation of PDGF-β receptors [30].

4.4. ROI, HAKs and the perpetuation of inflammatory response 

Activation of inflammatory cells represents a major source of oxidative stress-related molecules that may mediate either cytotoxic as well profibrogenic effects (see Fig. 1) but ROI and HAKs and other related molecules may also serve as pro-inflammatory mediators. Early work by Tsukamoto and coworkers demonstrated that Kupffer cells treated with α-tocopherol decreased significantly their release of tumor necrosis factor alpha (TNFα) and interleukin (IL)-6, confirming previous results obtained in human peripheral blood mononuclear cells ([55] and references therein). In addition, these authors identified the existence of in vitro and in vivo (BDL model) relationships between MDA levels and TNFα and IL-6 mRNA levels. Similarly, α-tocopherol administration prevented not only oxidative stress, lipid peroxidation and collagen deposition in the chronic CCl4 model [37], but also inhibited in vivo expression of TGFβ1 [56]. Along these lines, HNE elicits increased transcription of TGFβ1 in human monocyte/macrophage cell lines and in rat Kupffer cells isolated from fibrotic livers [57]. Moreover, ROI and, to a less extent, HNE upregulate MCP-1 expression in HSC: MCP-1 expression depends on NF-κB redox-sensitive transcription factor and is highly expressed during active fibrogenesis to recruit and activate monocytes and lymphocytes [58], [59]. Once again, α-tocopherol has been found to inhibit in vivo MCP-1 expression, and then monocyte recruitment and the extent of parenchymal liver injury, in an experimental model [59]. It should be noted that since HAKs are known to elicit leukocyte chemotaxis at extremely low concentrations (10−8–10−6 M, see Ref. [6] and references therein), α-tocopherol may also reduce the recruitment of leukocytes by inhibiting HAKs-stimulated chemotaxis.

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5. NO and RNOI: cytoprotective, cytotoxic and signal intermediates in liver injury 

Nitric oxide (NO) is a short-lived gaseous free radical known to exert many actions in the liver as well as in other tissues and organs [15]. In this review only major concepts, potentially relevant in conditions of CLD, with special reference to interactions with ROI at molecular level and effects at cellular level (particularly those on activated HSC), will be summarized [60], [61].

5.1. NO and RNOI as cytoprotective and cytotoxic intermediates 

In normal liver low fluxes of NO are produced by constitutive endothelial NO synthase (eNOS, mainly in endothelial cells) and are considered sufficient to maintain perfusion of liver sinusoids by acting on vascular tone (i.e. vasodilation) and on vascular permeability. Moreover, endothelial cells control sinusoid perfusion by increasing their production of NO in response to flow [60], [61], [62]. NO is supposed also to regulate leukocyte adhesion to sinusoidal endothelium and to inhibit platelet adhesion and aggregation. In pathological conditions, including endotoxemia and chronic inflammation, inducible NO synthase (iNOS) is upregulated in almost all liver cell populations, including HSC [63], by several mediators and, consequently, NO generation is increased [60], [61]. Under these conditions NO has been documented to act either as a (cyto)protective as well as (cyto)toxic and deleterious agent. NO can elicit its effects either by reacting directly with several macromolecules, metal complexes and free radicals or as a consequence of interaction with O2 or O2·− to generate reactive nitrogen oxide intermediates (RNOI) such as dinitrogen trioxide (N2O3) and peroxynitrite (ONOO−). The final effect, cytoprotection or cytotoxicity, mostly depends by the local ratio of ROI and NO generation, by the nature of mediators that lead to iNOS induction, by the cells that produce either ROI or NO and by other chemical and biochemical considerations (pH, presence of transition metals, etc.) [15]. Protective effects seem to predominate essentially in the presence of low levels of ROI generation: in this condition, the use of NOS inhibitors usually exacerbates liver injury, whereas NO donors usually reduce liver necrosis [60], [61], [64]. NO can decrease liver injury by inhibiting generation of OH· in a superoxide anion-driven, redox active Fe3+ catalyzed, Fenton reaction. Moreover, NO can either inhibit initiation or propagation of lipid peroxidation by rapidly reacting with a number of ROI, including lipid alkoxyl (LO·) and lipid hydroperoxyl (LOO·) radicals which are generated as intermediates during lipid peroxidation [15]. Cytotoxic action of NO seems to occur at relatively high levels of ROI and at the right ratio NO/O2·− as a consequence of interaction of NO with either O2 or O2·− to generate N2O3 and ONOO− that, as is well known, can elicit cytotoxicity [15]. However, this concept should be considered with caution. Both N2O3 and ONOO− can be deleterious, by inducing oxidation, nitrosation and nitration of biological macromolecules, including formation of DNA strand breaks, induction of lipid peroxidation, inactivation of enzymes or structural proteins, etc. (see Table 2). N2O3, for example, is a potent nitrosating agent for many biological targets, leading to generation of potentially carcinogenic nitrosamines amines and S-nitrosothiol derivatives (RNSO). RNSO, however, may also act as signaling mediators for vasodilating effects of NO donors and S-nitrosation of critical SH groups on endothelial cells and PMNL may represent the basis for the well known NO donor-mediated inhibition of leukocyte adhesion in vivo [15]. Similarly, high levels of NO, in the presence of high levels of ROI, can induce apoptosis in several cell types through the generation of ONOO− (see Ref. [61] and references therein). However, one of the most interesting protective effects of NO is represented by the NO-dependent block of hepatocyte apoptosis induced either by removal of growth factors or by exposure to TNFα or anti-Fas antibody: this antiapoptotic effect has been ascribed to S-nitrosylation of several members of caspase family, including caspase 3 and 8, and consequent inhibition of caspase's activity [65], [66].

Moreover, in spite of the fact that simultaneous generation of superoxide anion and NO is known to generate the potent pro-oxidant and cytotoxic intermediate ONOO·, the same reaction can serve as a relevant detoxification and anti-inflammatory pathway by removing superoxide anion and related generation of hydrogen peroxide. NO-mediated inhibition of oxidative damage and of lipid peroxidation has been described in experimental model of liver injury induced by CCl4 and ethanol [67], [68].

5.2. NO: a mediator able to counteract activation of HSC? 

Recent data seem to indicate that NO and then NO donors may also block or reduce proliferative responses of activated HSC. NO donors can efficiently inhibit PDGF-dependent proliferation and chemotaxis in activated human HSC by activating a peculiar ibuprofen-sensitive, PGE2 and cAMP-dependent pathway which interferes negatively with PDGF signaling [69]. Similar results (i.e. inhibition of stimulated proliferation of HSC by NO donors) have been found with angiotensin II as proliferative stimulus [70].

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6. Antioxidants as potential therapeutics? 

Several experimental reports have stated that antioxidant treatment in vivo is effective in preventing or reducing liver fibrosis. Liver fibrosis induced by chronic ethanol consumption is prevented by polyenylphosphatidylcholine (PPC), CYP2E1 inhibitors such as diallylsulfide (DAS) or phenylethylisothiocyanate (PIC) as well as by S-adenosyl-methionine (SAMe, used to replenish GSH levels) (reviewed in Refs. [19], [20]). SAMe is effective also in animal models [71] and positive results have been obtained also using α-tocopherol (inhibits lipid peroxidation and the synthesis of HNE and HAKs) [18], [38], the flavonoids sylimarin [72], [73] and quercetin [74] as well as the Japanese herbal medicine sho-saiko-to, possibly through the action of flavonoid bacalein (similar to sylimarin) contained in this herbal mixture [75], [76]. Also, the antifibrogenic action displayed by estrogens on dimethylnitrosamine-induced fibrosis [77] may be mediated, at least in part, by an antioxidant mechanism.

Data concerning the use of antioxidants in clinical trial are relatively few. They have been performed using very different doses or therapeutic combinations for quite limited period of time and on patients with long-standing CLD or cirrhosis. Having in mind these limitations, results provided by these studies are sometimes conflicting and definitively less impressive than experimental studies, particularly if significant changes in laboratory data and histological analysis are concerned [78], [79], [80], [81], [82], [83], [84], [85], [86], [87], but nevertheless some positive aspects are emerged. α-Tocopherol (vitamin E) has been employed in trials of different length (from 8 weeks to 1 year in different patients) as single agent [78], [79], [80] or in combination with other antioxidants in patients treated with α-interferon [81] or alone in patients refractory to α-interferon [82]: positive results include a decrease in oxidative stress markers [78], [79], [80], and HSC activation [78], replenishment of α-tocopherol plasma levels [79] and, particularly, a reduction of clotting activation in cirrhotics [80] a higher response rate to α-IFN for chronic HCV patients [81], and decrease of aminotransferase levels [82]. Positive clinical results were reported for silymarin in one study (2 years of treatment) in cirrhotic alcoholics or in Child A patients but not in more advanced stages of the disease [83]. However, a more recent multicenter trial failed to confirm positive effects of silymarin on the course of disease in cirrhotic alcoholics [84]. Encouraging results derive from the use of the sho-saiko-to herbal medicine [85], [86] and of GSH pro-drugs [87], particularly in terms on inhibition of cytokine production in cirrhotic patients such as TNFα, IL-6, IL-8 and IL-12.

If these data are compared with those obtained in experimental models and with the concepts provided in this review the following comments may be offered: (1) classic natural occurring antioxidants or drugs with established antioxidant activity should be administered at rather high doses to humans to match effective doses reached in experimental animals; (2) experimental protocols on animals are designed to make the antioxidant available during the development of fibrosis (i.e. to directly affect fibrosis progression) whereas most of human studies have employed them in cirrhotics or in patients with advanced stages of the diseases. Since these limitations are difficult to overcome in clinical practice, antioxidants should be administered as early as possible during the natural history of the disease and, hopefully, more potent and diffusible compounds will be available in the next future. In addition, antioxidants may be more effective if used in combination with other antifibrotic drugs, possibly designed to target essentially liver cells and, particularly, HSC, as outlined recently [2]. Finally, an ideal antifibrotic agent should act either as an antioxidant or by means of other actions: in this connection, data obtained with flavonoids such as bacalein [75], quercetin [53] and the polyphenol trans-resveratrol [44], [88], that appear to inhibit aspects of HSC activation in vitro by different mechanisms, are of increasing interest.

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7. Conclusions 

Current literature indicate that ROI, HAKs and NO (including RNOI) are likely to be involved in the progression of liver fibrosis during CLD of different etiology. They may contribute to such progression by eliciting cytotoxicity or, more likely, by modulating tissue events and the functional response of potential target cells, particularly HSC phenotypic responses, but several possible aspects of their intervention, known to operate in other diseases [7], [8], still remain to be explored. Future studies should outline a more complete hepatic spectrum of action for these molecules as well as to provide more details on molecular mechanisms of action of these reactive intermediates. Hepatologists also need more powerful antioxidants from natural sources or synthetic and specifically designed and liver targeted, for a possible use as therapeutics.

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Acknowledgements 

This work has been supported by Ministero dell'Università e della Ricerca Scientifica e Tecnologica (MURST, Rome, Italy), National Project on Molecular and Cellular Biology of Hepatic Fibrosis. The authors are sincerely indebted to Professor Mario Umberto Dianzani for his continuous support and encouragement throughout the years.

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PII: S0168-8278(01)00142-8

Journal of Hepatology
Volume 35, Issue 2 , Pages 297-306, August 2001

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