Elevated hepatic iron: A confounding factor in chronic hepatitis C

Historically, iron overload in the liver has been associated with the genetic disorders hereditary hemochromatosis and thalassemia and with unusual dietary habits. More recently, elevated hepatic iron levels also have been observed in chronic hepatitis C virus (HCV) infection. Iron overload in the liver causes many changes including induction of oxidative stress, damage to lysosomes and mitochondria, altered oxidant defense systems and stimulation of hepatocyte proliferation. Chronic HCV infection causes numerous pathogenic changes in the liver including induction of endoplasmic reticulum stress, the unfolded protein response, oxidative stress, mitochondrial dysfunction and altered growth control. Understanding the molecular and cellular changes that could occur in a liver which has elevated hepatic iron levels and in which HCV replication and gene expression are ongoing has clinical relevance and represents an area of research in need of further investigation.     

Accelerated CCl4-induced liver fibrosis in Hjv-/- mice, associated with an oxidative burst and precocious profibrogenic gene expression

Hereditary hemochromatosis is commonly associated with liver fibrosis. Likewise, hepatic iron overload secondary to chronic liver diseases aggravates liver injury. To uncover underlying molecular mechanisms, hemochromatotic hemojuvelin knockout (Hjv-/-) mice and wild type (wt) controls were intoxicated with CCl₄. Hjv-/- mice developed earlier (by 2-4 weeks) and more acute liver damage, reflected in dramatic levels of serum transaminases and ferritin and the development of severe coagulative necrosis and fibrosis. These responses were associated with an oxidative burst and early upregulation of mRNAs encoding α1-(I)-collagen, the profibrogenic cytokines TGF-β1, endothelin-1 and PDGF and, notably, the iron-regulatory hormone hepcidin. Hence, CCl4-induced liver fibrogenesis was exacerbated and progressed precociously in Hjv−/− animals. Even though livers of naïve Hjv−/− mice were devoid of apparent pathology, they exhibited oxidative stress and immunoreactivity towards α-SMA antibodies, a marker of hepatic stellate cells activation. Furthermore, they expressed significantly higher (2–3 fold vs. wt, p<0.05) levels of α1-(I)-collagen, TGF-β1, endothelin-1 and PDGF mRNAs, indicative of early fibrogenesis. Our data suggest that hepatic iron overload in parenchymal cells promotes oxidative stress and triggers premature profibrogenic gene expression, contributing to accelerated onset and precipitous progression of liver fibrogenesis.     

Portal myofibroblasts are sensitive to CCN-mediated endoplasmic reticulum stress-related apoptosis with potential to attenuate biliary fibrogenesis

Portal fibroblasts are mesenchyme-derived fibroblasts surrounding the bile ducts, and activated into portal myofibroblasts (pMF) during cholestatic liver injury. pMF express α-smooth muscle actin (α-SMA) and produce the fibrogenic extracellular matrix (ECM) collagen type I and fibronectin, playing important roles in portal fibrosis. A cholestatic bile duct-ligated (BDL) model is characterized by impaired hepatobiliary excretion of bile, leading to increased bile acid accumulation. Accumulation of bile acids is known to induce endoplasmic reticulum (ER) stress leading to liver damage and cell death. Additionally, a BDL fibrotic model is also associated with upregulation of CCN (CYR61, CTGF and NOV) matricellular proteins and reported to induce ER stress both in vitro and in vivo. To explore the effects of CCN proteins, we used adenovirus-mediated CCN1-4 (Ad-CCN1-4) gene transfers into cultured pMF. Overexpression of CCN proteins leads to protein accumulation in the ER lumen, causing ER stress and unfolded protein response (UPR). We further found ER stress and UPR to mitigate fibrogenesis in pMF by decreased cellular production of fibronectin, collagen type 1 and α-SMA. In this scenario, Tauroursodeoxycholic acid, a pharmaceutical chaperone and ER stress inhibitor, attenuated Ad-CCN1-4 induced pMF apoptosis and restored collagen and fibronectin levels. Since hepatic fibrogenesis is accompanied by ER stress and upregulation of CCN proteins in a BDL, we further evaluated ER stress responses after Ad-CCN1 gene transfer in such a model and found overexpressed CCN1 to enhance the ER stress-associated proteins BiP and CHOP with positive cleaved caspase 3 and 9 staining in periportal nonparenchymal cells. This indicates that these nonparenchymal cells, most likely pMF, have the tendency to undergo apoptosis during later stages of BDL. Ad-CCN1 transduction furthermore sensitized pMF for ER stress and apoptosis. We suggest that CCN proteins are key factors in the fibrotic microenvironment impacting pMF survival during fibrogenesis and pMF apoptosis during fibrosis resolution.     

Spike‐in SILAC proteomic approach reveals the vitronectin as an early molecular signature of liver fibrosis in hepatitis C infections with hepatic iron overload

Hepatitis C virus (HCV)‐induced iron overload has been shown to promote liver fibrosis, steatosis, and hepatocellular carcinoma. The zonal‐restricted histological distribution of pathological iron deposits has hampered the attempt to perform large‐scale in vivo molecular investigations on the comorbidity between iron and HCV. Diagnostic and prognostic markers are not yet available to assess iron overload‐induced liver fibrogenesis and progression in HCV infections. Here, by means of Spike‐in SILAC proteomic approach, we first unveiled a specific membrane protein expression signature of HCV cell cultures in the presence of iron overload. Computational analysis of proteomic dataset highlighted the hepatocytic vitronectin expression as the most promising specific biomarker for iron‐associated fibrogenesis in HCV infections. Next, the robustness of our in vitro findings was challenged in human liver biopsies by immunohistochemistry and yielded two major results: (i) hepatocytic vitronectin expression is associated to liver fibrogenesis in HCV‐infected patients with iron overload; (ii) hepatic vitronectin expression was found to discriminate also the transition between mild to moderate fibrosis in HCV‐infected patients without iron overload.     

Mitochondrial dysfunction in friedreich's ataxia

Friedreich ataxia (FRDA) is an autosomal recessive degenerative disorder caused in the vast majority of cases by a GAA triplet expansion in the FRDA gene on chromosome 9q13. The FRDA gene product, frataxin, is a widely expressed mitochondrial protein which is severely reduced in FRDA patients. Loss of the homologue of frataxin in yeast is associated with mitochondrial iron overload, increased sensitivity to oxidative stress and profound deficit of oxidative phosphorylation. The demonstration that the human pathology of FRDA is also characterised by mitochondrial iron accumulation, deficit of respiratory chain complex activities and in vivo deficit of tissue energy metabolism establishes FRDA as a 'new' nuclear encoded mitochondrial disease.     

Pathogenesis of liver fibrosis: role of oxidative stress

In the liver, the progressive accumulation of connective tissue, a complex and dynamic process termed fibrosis, represents a very frequent event following a repeated or chronic insult of sufficient intensity to trigger a "wound healing"-like reaction. The fibrotic process recognises the involvement of various cells and different factors in bringing about an excessive fibrogenesis with disruption of intercellular contacts and interactions and of extracellular matrix composition. However, Kupffer cells, together with recruited mononuclear cells, and hepatic stellate cells are by far the key-players in liver fibrosis. Their cross-talk is triggered and favoured by a series of chemical mediators, with a prominent role played by the transforming growth factor beta. Both expression and synthesis of this inflammatory and pro-fibrogenic cytokine are mainly modulated through redox-sensitive reactions. Further, involvement of reactive oxygen species and lipid peroxidation products can be clearly demonstrated in other fundamental events of hepatic fibrogenesis, like activation and effects of stellate cells, expression of metalloproteinases and of their specific inhibitors. The important outcome of such findings as regards the pathogenesis of liver fibrosis derives from the observation of a consistent and marked oxidative stress condition in many if not all chronic disease processes affecting hepatic tissue. Hence, reactive oxidant species likely contribute to both onset and progression of fibrosis as induced by alcohol, viruses, iron or copper overload, cholestasis, hepatic blood congestion.     

A mutation in a mitochondrial ABC transporter results in mitochondrial dysfunction through oxidative damage of mitochondrial DNA

We have isolated a Saccharomyces cerevisiae mutant that shows an increased tendency to form cytoplasmic petites (respiration-deficient ρ⁻ or ρ⁰ mutants) in response to treatment of cells growing on a solid medium with the DNA-damaging agent methyl methanesulfonate or ultraviolet light. The mutation in this strain, atm1-1, was found to cause a single amino acid substitution in ATM1, a nuclear gene that encodes the mitochondrial ATP-binding cassette (ABC) transporter. When the mutant cells were grown in liquid glucose medium, they accumulated free iron within the mitochondria and at the same time gave rise to spontaneous cytoplasmic petite mutants, as seen previously in cells carrying a mutation in a gene homologous to the human gene responsible for Friedreich's ataxia. Analysis of the effects of free iron and malonic acid (an inhibitor of oxidative respiration in mitochondria) on the incidence of petites among the mutant cells indicated that spontaneous induction of petites was a consequence of oxidative stress rather than a direct effect of either a defect in the ATM1 gene or the accumulation of free iron. We observed an increase in the incidence of strand breaks in the mitochondrial DNA of the atm1-1 mutant cells. Furthermore, we found that rates of induction of petites and accumulation of strand breaks in mitochondrial DNA were enhanced in the atm1-1 mutant by the introduction of another mutation, mhr1-1, which results in a deficiency in mitochondrial DNA repair. These observations indicate that spontaneous induction of petites in the atm1-1 mutant is a consequence of oxidative damage to mitochondrial DNA mediated by enhanced accumulation of mitochondrial iron. Received: 26 March 1999 / Accepted: 29 June 1999     

In vitro and in vivo effects of iron on the expression and activity of glucose 6‐phosphate dehydrogenase, 6‐phosphogluconate dehydrogenase,and glutathione reductase in rat spleen

Iron is an indispensable element for vital activities in almost all living organisms. It is also a cofactor for many proteins, enzymes, and other essential complex biochemical processes. Therefore, iron trafficking is firmly regulated by Hepcidin (Hamp), which is regarded as the marker for iron accumulation. The disruption of iron homeostasis leads to oxidative stress that causes various human diseases, but this mechanism is still unclear. The aim of this study is to provide a better in vivo and in vitro understanding of how long‐term iron overload affects the gene expression and activities of some antioxidant enzymes, such as glucose 6‐phosphate dehydrogenase (G6PD), 6‐phosphogluconate dehydrogenase (6PGD), and glutathione reductase (GR) in the spleen. The findings of this study show that iron overload reduces the gene expression of G6pd, 6pgd, and Gr, but its actual effect was on the protein level.     

Proteomic analysis of iron overload in human hepatoma cells

Iron-mediated organ damage is common in patients with iron overload diseases, namely, hereditary hemochromatosis. Massive iron deposition in parenchymal organs, particularly in the liver, causes organ dysfunction, fibrosis, cirrhosis, and also hepatocellular carcinoma. To obtain deeper insight into the poorly understood and complex cellular response to iron overload and consequent oxidative stress, we studied iron overload in liver-derived HepG2 cells. Human hepatoma HepG2 cells were exposed to a high concentration of iron for 3 days, and protein expression changes initiated by the iron overload were studied by two-dimensional electrophoresis and mass spectrometry. From a total of 1,060 spots observed, 21 spots were differentially expressed by iron overload. We identified 19 of them; 11 identified proteins were upregulated, whereas 8 identified proteins showed a decline in response to iron overload. The differentially expressed proteins are involved in iron storage, stress response and protection against oxidative stress, protein folding, energy metabolism, gene expression, cell cycle regulation, and other processes. Many of these molecules have not been previously suggested to be involved in the response to iron overload and the consequent oxidative stress.     

A retinoic acid receptor agonist tamibarotene suppresses iron accumulation in the liver

Objective:

Hepatic iron overload (HIO) and iron‐induced oxidative stress have recently emerged as an important factor for the development and progression of insulin resistance. The aim of this study was to evaluate the effect of tamibarotene, a selective retinoic acid receptor α/β agonist, on hepatic iron metabolism, based on our previous findings that retinoids suppress hepatic iron accumulation by increasing hepatic iron efflux through the regulation of hemojuvelin and ferroportin expression.

Design and Methods:

We quantitated the non‐heme iron content and iron metabolism‐related gene expression in the liver, and serum lipid and blood glucose levels in KK‐A^y mice after dietary administration of tamibarotene.

Results:

It was demonstrated that tamibarotene significantly reduced blood glucose and hepatic iron, but not serum lipids, and that hemojuvelin expression significantly decreased while ferroportin increased, as observed previously.

Conclusions:

These results suggest that tamibarotene is a promising alternative for the treatment of insulin resistance associated with HIO.     

Efficacy of sauchinone as a novel AMPK-activating lignan for preventing iron-induced oxidative stress and liver injury

Iron-overload disorders cause hepatocyte injury and inflammation by oxidative stress, possibly leading to liver fibrosis and hepatocellular carcinoma. This study investigated the efficacy of sauchinone, a bioactive lignan, in preventing iron-induced liver injury and explored the mechanism of sauchinone's activity. To create iron overload, mice were injected with phenylhydrazine, and the effects on hepatic iron and histopathology were assessed. Phenylhydrazine treatment promoted liver iron accumulation and ferritin expression, causing hepatocyte death and increased plasma arachidonic acid (AA). Sauchinone attenuated liver injury (EC₅₀ = 10 mg/kg) and activated AMPK in mice. Treatment of hepatocytes with iron and AA simulated iron overload conditions: iron + AA synergistically amplified cytotoxicity, increasing H₂O₂ and the mitochondrial permeability transition. Sauchinone protected hepatocytes from iron + AA-induced cytotoxicity, preventing the induction of mitochondrial dysfunction and apoptosis (EC₅₀ = 1 μM), similar to the result using metformin. Sauchinone treatment activated LKB1, which led to AMPK activation: these events contributed to cell survival. Evidence of cytoprotection by LKB1 and AMPK activation was revealed in the reversal of sauchinone's restoration of the mitochondrial membrane potential by either dominant negative mutant AMPKα or chemical inhibitor. In conclusion, sauchinone protects the liver from toxicity induced by iron accumulation, and sauchinone's effects may be mediated by LKB1-dependent AMPK activation.     

Effects of excess dietary iron and fat on glucose and lipid metabolism

PurposeDiets rich in fat and energy are associated with metabolic syndrome (MS). Increased body iron stores have been recognized as a feature of MS. High-fat diets (HFs), excess iron loading and MS are closely associated, but the mechanism linking them has not been clearly defined. We investigated the interaction between dietary fat and dietary Fe in the context of glucose and lipid metabolism in the body.MethodsC57BL6/J mice were divided into four groups and fed the modified AIN-93G low-fat diet (LF) and HF with adequate or excess Fe for 7 weeks. The Fe contents were increased by adding carbonyl iron (2% of diet weight) (LF+Fe and HF+Fe).ResultsHigh iron levels increased blood glucose levels but decreased high-density lipoprotein cholesterol levels. The HF group showed increases in plasma levels of glucose and insulin and insulin resistance. HF+Fe mice showed greater changes. Representative indices of iron status, such hepatic and plasma Fe levels, were not altered further by the HF. However, both the HF and excess iron loading changed the hepatic expression of hepcidin and ferroportin. The LF+Fe, HF and HF+Fe groups showed greater hepatic fat accumulation compared with the LF group. These changes were paralleled by alterations in the levels of enzymes related to hepatic gluconeogenesis and lipid synthesis, which could be due to increases in mitochondrial dysfunction and oxidative stress.ConclusionsHigh-fat diets and iron overload are associated with insulin resistance, modified hepatic lipid and iron metabolism and increased mitochondrial dysfunction and oxidative stress.     

Studies on the rat liver following iron overload: An analysis of iron and lysosomal enzymes in isolated parenchymal and non-parenchymal cells

Iron overload is known to affect the liver. In order to study the effect of iron on various liver cellular and subcellular compartments and the alterations due to mobilization of iron, an experimental model has been developed previously. In this study iron stores in parenchymal and non-parenchymal cells have been investigated during iron loading and unloading. Following completion of the experimental procedures, liver cells were isolated by means of collagenase perfusion (parenchymal cells) and pronase treatment (nonparenchymal cells). It was found that iron overload did not result in significantly increased levels of three lysosomal enzymes, and that the enzyme activities were not altered as iron was mobilized. Iron stores were localized largely in parenchymal cells, and these stores decreased after cessation of iron loading. The iron content was further lowered if the animals were bled. The non-parenchymal cells of the liver initially stored a relatively small part of the administered iron but this increased in the two months following iron loading. On the other hand if the animals were bled there was a pronounced decrease in iron content of these cells as well as in parenchymal cells. It is concluded that iron overload does not affect lysosomal enzymes and that iron stores in both parenchymal and non-parenchymal cells can be mobilized in response to increased demand.     

Fatty acid-mediated intracellular iron translocation: a synergistic mechanism of oxidative injury

Fatty acid has been reported to be associated with cardiovascular diseases and cancer, but the possible mechanism remains unclear. Here, we reported a novel mechanism for the permissive role of fatty acid on iron intracellular translocation and subsequent oxidative injury. In vitro study from endothelial cells showed that iron alone had little effect, whereas in combination with PA (palmitic acid), iron-mediated toxicity was markedly potentiated, as reflected in mitochondrial dysfunction, cell death, apoptosis, and DNA mutation. We also showed that PA not only facilitated iron translocation into cells through a transferrin-receptor (TfR)-independent mechanism, but also translocated iron into mitochondria; the subsequent intracellular iron overload resulted in reactive oxygen species (ROS) overgeneration and lipid oxidation. Further investigation revealed that PA-facilitated iron translocation is due to Fe/PA-mediated extracellular oxidative stress and the subsequent membrane damage with increased membrane permeability. Fe/PA-mediated toxic effects were reduced in rho0 cells lacking mitochondrial DNA or by antioxidant enzyme SOD, especially mitochondrially localized MnSOD, suggesting a permissive role of PA for iron deposition on the vascular wall and its subsequent toxicity via mitochondrial oxidative stress. This observation was confirmed in vivo in mice, wherein higher vascular iron deposition and accompanying superoxide release were observed in the presence of a high-fat diet with iron administration.     

Iron-Induced Oxidant Stress Leads to Irreversible Mitochondrial Dysfunctions and Fibrosis in the Liver of Chronic Iron-Dosed Gerbils. The Effect of Silybin

Hepatic iron toxicity because of iron overload seems to be mediated by lipid peroxidation ofbiological membranes and the associated organelle dysfunctions. However, the basicmechanisms underlying this process in vivo are still little understood. Gerbils were dosed with weeklyinjections of iron—dextran alone or in combination with sylibin, a well—known antioxidant,by gavage for 8 weeks. A strict correlation was found between lipid peroxidation and the levelof desferrioxamine chelatable iron pool. A consequent derangement in the mitochondrialenergy-transducing capability, resulting from a reduction in the respiratory chain enzymeactivities, occurred. These irreversible oxidative anomalies brought about a dramatic drop intissue ATP level. The mitochondrial oxidative derangement was associated with thedevelopment of fibrosis in the hepatic tissue. Silybin administration significantly reduced bothfunctional anomalies and the fibrotic process by chelating desferrioxamine chelatable iron.     

Lipid peroxidation and associated hepatic organelle dysfunction in iron overload

Iron overload can have serious health consequences. Since humans lack an effective means to excrete excess iron, overload can result from an increased absorption of dietary iron or from parenteral administration of iron. When the iron burden exceeds the body's capacity for safe storage, the result is widespread damage to the liver, heart and joints, and the pancreas and other endocrine organs. Clear evidence is now available that iron overload leads to lipid peroxidation in experimental animals, if sufficiently high levels of iron are achieved. In contrast, there is a paucity of data regarding lipid peroxidation in patients with iron overload. Data from experiments using an animal model of dietary iron overload support the concept that iron overload results in an increase in an hepatic cytosolic pool of low molecular weight iron which is catalytically active in stimulating lipid peroxidation. Lipid peroxidation is associated with hepatic mitochondrial and microsomal dysfunction in experimental iron overload, and lipid peroxidation may underlie the increased lysosomal fragility that has been detected in homogenates of liver samples from both iron-loaded human subjects and experimental animals. Some current hypotheses focus on the possibility that the demonstrated functional abnormalities in organelles of the iron-loaded liver may play a pathogenic role in hepatocellular injury and eventual fibrosis. The recent demonstration that hepatic fibrosis is produced in animals with long-term dietary iron overload will allow this model to be used to further investigate the relationship between lipid peroxidation and hepatic injury in iron overload.     

Hepatic oxidative DNA damage correlates with iron overload in chronic hepatitis C patients

Hepatic oxidative stress occurs in chronic hepatitis C (CH-C), but little is known about its producing mechanisms and precise role in the pathogenesis of the disease. To determine the relevance of hepatic oxidatively generated DNA damage in CH-C, 8-hydroxy-2'-deoxyguanosine (8-OHdG) adducts were quantified in liver biopsy specimens by immunohistochemical staining, and its relationship with clinical, biochemical, and histological parameters, and treatment response was assessed in 40 CH-C patients. Hepatic 8-OHdG counts were significantly correlated with serum transaminase levels (r=0.560, p=0.0005) and histological grading activity (p=0.0013). Remarkably, 8-OHdG levels were also significantly related to body and hepatic iron storage markers (vs serum ferritin, r=0.565, p=0.0004; vs hepatic total iron score, r=0.403, p=0.0119; vs hepatic hepcidin messenger RNA, r=0.516, p=0.0013). Baseline hepatic oxidative stress was more prominent in nonsustained virological responder (non-SVR) than in SVR to interferon (IFN)/ribavirin treatment (50.8 vs 32.7 cells/10(5) microm2, p=0.0086). After phlebotomy, hepatic 8-OHdG levels were significantly reduced from 53.4 to 21.1 cells/10(5) microm2 (p=0.0125) with concomitant reductions of serum transaminase and iron-related markers in CH-C patients. In conclusion, this study showed that hepatic oxidatively generated DNA damage frequently occurs and is strongly associated with increased iron deposition and hepatic inflammation in CH-C patients, suggesting that iron overload is an important mediator of hepatic oxidative stress and disease progression in chronic HCV infection.