Role of transferrin receptor 2 in hepatic accumulation of iron in patients with chronic hepatitis C

Background and Aim: Iron deposition in the liver is a common finding in patients with chronic hepatitis C (CH‐C). The mechanism of this hepatic accumulation of iron is not completely understood. This study assessed if the protein expression of transferrin receptor 2 (TfR2) is upregulated in the liver of patients with CH‐C and if TfR2 protein mediates iron accumulation during hepatitis C virus (HCV) infection. Method: Liver specimens from patients with CH‐C that underwent interferon (IFN) therapy (n = 23) and from patients with CH‐B (n = 18) were evaluated. Hepatic expression of TfR2 protein was analyzed by immunohistochemistry. Total hepatic iron score (THIS) was evaluated by Prussian blue staining. Results: TfR2 protein was expressed in the cell membrane and cytosol of hepatocytes. Cytosol TfR2 protein was found to co‐localize with Tf. THIS (P = 0.0198) and hepatic TfR2 (P = 0.0047) expression were significantly higher in CH‐C than in CH‐B. The change in THIS values (rho = 0.580, P = 0.0079) and the grade of histological activity (rho = 0.444, P = 0.0373) were significantly correlated with changes in TfR2 expression after IFN therapy. Conclusions: The protein expression of TfR2 is significantly associated with iron deposition in the liver in patients with CH‐C. HCV infection may affect the hepatic expression of TfR2, leading to iron accumulation in the liver.     

Successful interferon therapy reverses enhanced hepatic iron accumulation and lipid peroxidation in chronic hepatitis C

OBJECTIVES: Hepatic iron deposition has been reported in chronic hepatitis C (CH-C), and iron-induced lipid peroxidation may be involved in the pathogenesis of CH-C. The aims of the present study were: 1) to determine whether patients with CH-C have evidence of enhanced hepatic lipid peroxidation and to evaluate its relation to iron status, compared with that in patients with chronic hepatitis B (CH-B); and 2) to assess the effect of interferon (IFN) therapy on hepatic iron and lipid peroxidation. METHODS: In the liver biopsies of 40 patients with CH-C and 26 patients with CH-B, immunohistochemical detection of 4-hydroxy-2-nonenal (HNE)-protein adducts for evaluation of lipid peroxidation was performed, and hepatic iron status was biochemically and histologically assessed. In 16 CH-C patients with normal serum transaminases and undetectable serum HCV-RNA >6 months after the end of IFN treatment (responders) and in 11 nonresponders, hepatic HNE-protein adducts and siderosis were evaluated in pre- and posttreatment liver biopsies. RESULTS: Hepatocytic HNE-protein adducts and iron deposits were more abundant in the patients with CH-C than in those with CH-B. No correlation was found between the levels of hepatocytic HNE-protein adducts and hepatic iron status in either of the two groups. In the responders to IFN treatment for CH-C, hepatocytic HNE-protein adducts disappeared or attenuated with improvement of hepatic siderosis after the treatment, whereas IFN treatment did not improve hepatocytic expression of HNE-protein adducts and hepatic siderosis in the nonresponders. CONCLUSIONS: Patients with CH-C have evidence of enhanced hepatic iron accumulation and lipid peroxidation compared to those with CH-B. In CH-C, hepatic siderosis and lipid peroxidation are improved with successful IFN treatment. These results suggest that hepatic lipid peroxidation and iron may potentially play contributory roles in the pathogenesis of CH-C.     

Comparison of hepatic oxidative DNA damage in patients with chronic hepatitis B and C

Summary.  8-Hydroxydeoxyguanosine (8-OHdG) is a promutagenic DNA lesion produced by hydroxyl radicals and is recognized as a useful marker in estimating DNA damage induced by oxidative stress. The aim of this study was to clarify the clinical significance of hepatic 8-OHdG levels in patients with chronic viral hepatitis. Hepatic 8-OHdG accumulation was investigated in patients with chronic hepatitis C (CH-C) ( n  = 77) and chronic hepatitis B (CH-B) ( n  = 34) by immunohistochemical staining of liver biopsy samples. 8-OHdG positive hepatocytes were significantly higher in patients with CH-C compared to CH-B (median 55.0 vs 18.8 cells/10⁵ μm², P  < 0.0001). The number of positive hepatocytes significantly increased with the elevation of serum aminotransferase levels, especially in CH-C patients (8-OHdG vs alanine aminotransferase (ALT)/aspartate aminotrasferase (AST) were r  = 0.738/0.720 in CH-C and 0.506/0.515 in CH-B). 8-OHdG reactivity was strongly correlated with body and hepatic iron storage markers in CH-C ( vs serum ferritin, r  = 0.615; vs hepatic total iron score, r  = 0.520; vs hepatic hepcidin mRNA levels, r  = 0.571), although it was related to serum HBV-DNA titers ( r  = 0.540) and age of patients ( r  = –0.559) in CH-B. These results indicate that hepatic oxidative DNA damage is common in chronic viral hepatitis, in particular chronic HCV-infected patients, suggesting a possible link between chronic hepatic inflammation and hepatocarcinogenesis. The strong positive correlation between hepatic DNA damage and iron overload suggests that iron content is one of the most likely mediators of hepatic oxidative stress and iron reduction may be beneficial to reduce the incidence of hepatic cancer in CH-C patients.     

Up-regulation of Transferrin Receptor Expression in Hepatocytes by Habitual Alcohol Drinking Is Implicated in Hepatic Iron Overload in Alcoholic Liver Disease

Background Hepatic iron overload is often seen in alcoholic liver disease (ALD). We previously reported that the expression of 4-hydroxy-2-nonenal-protein adducts, which is a lipid peroxidative product and can be used as a marker of radical-mediated cellular damage, was increased in iron-overloaded hepatocytes with ALD. However, the mechanism of hepatic iron overload in ALD has not been clarified. In this study, to elucidate the mechanism of hepatic iron overload in ALD, we immunohistochemically investigated the expression of transferrin receptor (TfR), which mainly acts for cellular iron uptake.
Methods Hepatic tissues were obtained from 31 patients with ALD and 5 normal livers by percutaneous needle biopsy under laparoscopy or ultrasound guidance. Chemical detection of hepatic iron accumulation was performed by Perls' Prussian blue stain. Immunohistochemical detection of TfR expression was done using human monoclonal anti-TfR antibody (TR104) according to the avidin-biotin complex method with alkaline phosphatase.
Results Excess iron accumulation was found in 22 hepatic tissues with ALD but not in any normal hepatic tissues. TfR expression was increased in hepatocytes of 18 hepatic tissues with ALD but was not detected in any normal hepatic tissues. The mean duration of abstinence of patients who demonstrated positive TfR expression in hepatocytes was significantly shorter than that of patients who demonstrated negative TfR expression (positive: 14 days; negative: 30 days). However, total ethanol consumption, daily ethanol intake, and serum aspartate aminotransferase and γ-glutamyl transpeptidase values on admission were not significantly correlated with TfR expression in hepatocytes.
Conclusions The up-regulation of TfR expression in hepatocytes is implicated in hepatic iron overload in ALD, and habitual alcohol drinking is an important factor for the induction of TfR expression.     

Plasma tissue inhibitor of metalloproteinases-1 and transforming growth factor beta 1--possible non-invasive biomarkers of hepatic fibrosis in patients with chronic B and C hepatitis

BACKGROUND/AIMS: The role of transforming growth factor-beta 1 (TGF-beta 1) in liver fibrosis is in part related to impairment of extracellular matrix breakdown by stimulation of tissue inhibitor of metalloproteinases-1 (TIMP-1) gene. The aim of the study was to evaluate association between TGF-beta 1 and TIMP-1 in relation to liver injury in chronic viral hepatitis B and C. METHODOLOGY: Association between plasma TGF-beta 1 and TIMP-1 was evaluated in 28 consecutive patients undergoing liver biopsy for chronic viral hepatitis B and C (CH-B, CH-C) and these tests were correlated with hepatic fibrosis, inflammation and liver function tests. Moreover carboxyterminal cross-linked telopeptide of type 1 procollagen (ICTP) and carboxyterminal propeptide of type 1 collagen (PICP) were also measured for assessment of extracellular matrix breakdown or synthesis, respectively. RESULTS: Chronic viral hepatitis B and C resulted in a significant increase in plasma TIMP-1 levels but not TGF-beta 1. Among biochemical markers of liver injury, significant correlation with TGF-beta 1 and TIMP-1 was demonstrated in respect to aminotransferase activities in both groups. TIMP-1 showed significant correlation with ICTP levels in both CH-B (r = 0.59) and CH-C (r = 0.62), whereas TGF-beta 1 was correlated with ICTP only in CH-C patients (r = 0.75). PICP did not demonstrate any correlation with either TGF-beta 1 or TIMP-1. Hepatic fibrosis, but not inflammation, correlated significantly with TGF-beta 1 (CH-B: r = 0.73; CH-C: r = 0.79) and TIMP-1 (CH-B: r = 0.66; CH-C: r = 0.71) in both groups and there was a significant correlation between TIMP-1 and TGF-beta 1 in the CH-B group (r = 0.83) and CH-C group (r = 79). CONCLUSIONS: These results support the role of TIMP-1 in a TGF-beta 1-dependent mechanism for liver fibrosis and suggest their plasma levels can be used as a possible early non-invasive marker of liver fibrosis useful for chronic hepatitis management.     

Iron state in association with retinoid metabolism in non‐alcoholic fatty liver disease

Aim: We have recently reported that hyperdynamic state of retinoid metabolism, which may lead to the shortage of retinoid, is observed in patients with non‐alcoholic fatty liver disease (NAFLD). Hepatic iron overload, which causes production of reactive oxygen species (ROS), is also frequently seen in NAFLD patients. The aim of the study is to examine iron state and retinoid metabolic state simultaneously, and to clarify the relationship between two disorders. Methods: Thirty‐six persons, comprising 17 patients with simple steatosis (SS), 11 with NASH, and 8 normal controls (N), were examined on hepatic expression of iron metabolism‐related genes including hemojuvelin (HJV), hepcidin (HEPC), transferrin receptor 1 and 2 (TfR1, TfR2), ferroportin (FPN), neogenin (NEO) and ferritin heavy chain (FtH) and hepatic iron contents in addition to expression 51 genes which is involved in retinoid metabolism and antioxidative action. Results: In patients with NAFLD, expression of HJV, TfR2, FPN, TfR1, FtH, SOD and catalase was increased, compared with that in N. In addition, hepatic iron content, which was increased in NASH, was correlated with expression level of TfR2. Expression of cellular retinoid binding protein (CRBP1), alcohol dehydrogenase 1 (ADH1) and cytochrome P450 26A1(CYP26A1) was significantly correlated with that of HJV, TfR2 and FPN, respectively. Conclusion: The results of the present study suggest that the reasons responsible for iron accumulation in NASH in the present study may partly be due to enhanced expression of TfRs, especially TfR2, and hyperdynamic state of retinoid metabolism is closely related to iron metabolism in the disease.     

Different signaling pathways in the livers of patients with chronic hepatitis B or chronic hepatitis C

The clinical manifestations of chronic hepatitis B (CH-B) and chronic hepatitis C (CH-C) are different. We previously reported differences in the gene expression profiles of liver tissue infected with CH-B or CH-C; however, the signaling pathways underlying each condition have yet to be clarified. Using a newly constructed cDNA microarray consisting of 9614 clones selected from 256,550 tags of hepatic serial analysis of gene expression (SAGE) libraries, we compared the gene expression profiles of liver tissue from 24 CH-B patients with those of 23 CH-C patients. Laser capture microdissection was used to isolate hepatocytes from liver lobules and infiltrating lymphoid cells from the portal area, from 16 patients, for gene expression analysis. Furthermore, the comprehensive gene network was analyzed using SAGE libraries of CH-B and CH-C. Supervised and nonsupervised learning methods revealed that gene expression was correlated more with the infecting virus than any other clinical parameters such as histological stage and disease activity. Pro-apoptotic and DNA repair responses were predominant in CH-B with p53 and 14-3-3 interacting genes having an important role. In contrast, inflammatory and anti-apoptotic phenotypes were predominant in CH-C. These differences would evoke different oncogenic factors in CH-B and CH-C. In conclusion, we describe the different signaling pathways induced in the livers of patients with CH-B or CH-C. The results might be useful in guiding therapeutic strategies to prevent the development of hepatocellular carcinoma in cases of CH-B and CH-C.     

Characteristic Laparoscopic Findings of Chronic Hepatitis C: A Comparison with Hepatitis B.

Abstract: We performed laparoscopy on 72 patients with chronic hepatitis C (CH-C) to elucidate its morphological features and characteristics relating to its progressive clinical course. We then compared these findings with those of 188 patients with chronic hepatitis B (CH-B). The frequency of reddish hepatic markings was almost the same in the patients with CH-B (21%) and the patients with CH-C (24%). In the patients with CH-B, reddish markings were present more frequently in the subjects with sublobular hepatic necrosis (34%) than in those without it (5%, p<0.01), but in CH-C the difference was not significant. Reddish markings were more even in the patients with CH-B with respect to form, size and distribution, whereas those in the patients with CH-C were more uneven. Prenodular patchy markings were present more frequently in the patients with CH-B (14%) than in the patients with CH-C (6%, p<0. 05). These findings suggested that the progression of hepatic necrosis was more variable in different parts of the liver, and that the regeneration of hepatocytes was less active in the patients with CH-C, compared with the patients with CH-B.     

Iron and HFE or TfR1 mutations as comorbid factors for development and progression of chronic hepatitis C

BACKGROUND/AIMS: Recent evidence implicates iron as a comorbid factor for development of non-hemochromatotic liver diseases. Mutations or polymorphisms in the HFE gene or the TfR1 gene may influence the accumulation of iron in the liver or other tissues or may influence chronic viral hepatitis apart from effects on iron homeostasis. The aim of this study was to assess the role of hepatic iron, HFE and TfR1 variations on development and progression of chronic hepatitis C infection. METHODS: We studied 119 consecutive patients with chronic hepatitis C, correlating clinical, laboratory, histopathological, and genetic data. Frequencies of genetic variations were compared with local and national controls. RESULTS: HFE mutations were more common in patients than controls (48% vs. 38%, P=0.04), and advanced degrees of fibrosis developed at younger ages in subjects with the C282Y mutation (38.6 vs. 46.5 years, P=0.03). Patients carrying C282Y had higher mean hepatic iron concentrations (P=0.02), hepatic iron indices (P<=0.0001), and hepatic fibrosis scores (P=0.01). Hepatic fibrosis was correlated with hepatic iron concentration (P=0.03). TfR1 polymorphisms bore no detectable relation to disease severity or response to therapy. CONCLUSIONS: Hepatic iron and HFE mutations are comorbid factors that increase development and progression of chronic hepatitis C.     

Serum thioredoxin elucidates the significance of serum ferritin as a marker of oxidative stress in chronic liver diseases

BACKGROUND/AIMS: Serum thioredoxin (TRX) levels have recently been established as an indicator of oxidative stress in various diseases. The aim of the present study was to clarify the clinical significance of serum ferritin in chronic liver diseases. METHODS: Levels of ferritin, transferrin saturation (TS), aspartate aminotransferase (AST), and TRX were measured in the sera of patients with chronic hepatitis C (CH-C, n=92), chronic hepatitis B (CH-B, n=28), nonalcoholic fatty liver (FL, n=31), or alcoholic liver diseases (ALD, n=17). Serum TRX levels were evaluated with a recently established sandwich enzyme-linked immunosorbent assay kit. RESULTS: Serum TRX levels were significantly higher in CH-C, FL, and ALD than in healthy volunteers. A larger proportion of patients with CH-C, FL, and ALD had elevated levels of serum ferritin than CH-B. Serum ferritin levels were positively correlated with levels of TS, AST, and TRX in CH-C, but were merely correlated with TS values in CH-B. Ferritin levels were also well correlated with AST and TRX, but not with TS in FL and ALD. CONCLUSION: Oxidative stress, which was evaluated by measuring serum TRX, in addition to storage iron and hepatocyte damage is a cause of increasing serum ferritin levels in chronic liver diseases. An elevated serum ferritin level, which was correlated with TS, indicates that iron-induced oxidative stress contributes to CH-C. Elevated ferritin levels in FL and ALD may be mostly due to iron-unrelated stresses.     

Health-related quality of life in patients with chronic hepatitis B.

Although chronic hepatitis C (CH-C) has consistently been shown to impair patients' health-related quality of life (HRQL), the impact of chronic hepatitis B (CH-B) on HRQL has not been fully explored. AIM: Compare HRQL between patients with CH-B, CH-C, primary biliary cirrhosis (PBC) and healthy controls. Design: Three HRQL questionnaires [Chronic Liver Disease Questionnaire (CLDQ), Short Form 36 (SF-36) and the Health Utility Index (HUI Mark-2 and Mark-3)] were administered prospectively. Additional clinical and laboratory data and normative data for healthy individuals, were available. ANALYSIS: Scores were compared using analysis of variance and multiple regression. RESULTS: One hundred and forty-six patients with CH-B, CH-C and PBC were included [mean age 47.1 years (+/-11.6), 41% female, 33% cirrhosis]. CH-C and PBC patients scored the lowest on all CLDQ, SF-36 and HUI domains compared with CH-B patients and healthy controls. CH-B patients had scores similar to the healthy population, measured by most CLDQ and SF-36 scales. However, the HUI scores for CH-B patients showed more impairment than population norms. Having CH-B and not having cirrhosis were predictive of utility and HRQL scores in multivariate models. CONCLUSIONS: CH-B patients have better HRQL than CH-C, PBC and population norms. CH-B patients' overall utility scores are lower than population norms.     

Hepatitis C virus protein and iron overload induce hepatic steatosis through the unfolded protein response in mice

Background/Aim: Hepatic iron overload and steatosis play critical roles in the progression of hepatitis C virus (HCV)‐associated chronic liver disease. However, how these two pathophysiological features affect each other remains unknown. The aim of this study was to investigate how hepatic iron overload contributes to the development of hepatic steatosis in the presence of HCV proteins. Methods: Male C57BL/6 transgenic mice expressing the HCV polyprotein and nontransgenic littermates were fed an excess‐iron diet or a control diet. Mice in each group were assessed for the molecules responsible for fat accumulation in the liver. Results: Hepatic iron levels were positively correlated with triglyceride concentrations in the liver for all mice. As compared with the livers of nontransgenic mice fed the control diet, the livers of transgenic mice fed the excess‐iron diet showed a lower expression of carnitine palmitoyl transferase I, a higher expression of sterol‐regulatory element‐binding protein 1 and fatty acid synthetase and an activated unfolded protein response indicated by a higher expression of unspliced and spliced X‐box DNA‐binding protein 1 (XBP‐1), phosphorylated eukaryotic initiation factor‐2α (p‐eIF2α), CCAAT/enhancer‐binding protein homology protein (CHOP) and abundant autophagosomes concomitant with increased production of reactive oxygen species. Six‐month treatment with the anti‐oxidant N‐acetyl cysteine dramatically reduced hepatic steatosis in transgenic mice fed the excess‐iron diet through decreased expression of unspliced and spliced XBP‐1, p‐eIF2α, and CHOP. Conclusions: The iron‐induced unfolded protein response appears to be one of the mechanisms responsible for fat accumulation in the liver in transgenic mice expressing the HCV polyprotein.     

Dysregulated expression of fatty acid oxidation enzymes and iron‐regulatory genes in livers of Nrf2‐null mice

Background and Aim: Hepatic excessive iron may play a role in the pathogenesis of non‐alcoholic steatohepatitis (NASH). Nrf2 is a master regulator of antioxidative responses. However, the role of Nrf2 in lipid and iron homeostasis remains unclear. Accordingly, it was examined how Nrf2 regulates lipid‐related and iron‐regulatory genes after feeding a high‐fat diet (HFD) with iron. Methods: Wild‐type and Nrf2‐null mice were fed the following diets: (i) control diet (4% soybean oil) for 12 weeks, (ii) control diet for 8 weeks followed by control diet containing 0.5% carbonyl iron for 4 weeks, (iii) HFD (4% soybean oil and 16% lard) for 12 weeks, (iv) HFD for 8 weeks followed by HFD containing 0.5% carbonyl iron for 4 weeks. Blood and livers were removed after 12 weeks. Results: Nrf2‐null control mice exhibited a tendency towards higher hepatic triglycerides compared to wild‐type control mice. Hepatic malondialdehyde was higher and hepatic iron levels tended to be higher in Nrf2‐null mice than wild‐type counterparts while on a HFD. The HFD with iron synergistically induced mRNA expression of Pparα targets, including Acox and Cpt1 in wild‐type mice, yet the induction was diminished in Nrf2‐null mice. Hepatic hepcidin and ferroportin 1 mRNA expression were increased in wild‐type mice after feeding a HFD with iron, but were unchanged in any group of Nrf2‐null mice. Conclusions: Nrf2 deletion dysregulates hepatic mRNA expression of β‐oxidation enzymes and iron‐related genes, possibly causing a trend for increased hepatic triglyceride and iron concentrations. Nrf2 may have roles in the progression of NASH.     

mRNA expression of iron regulatory genes in beta-thalassemia intermedia and beta-thalassemia major mouse models

beta-Thalassemia is an inherited anemia in which synthesis of the hemoglobin beta-chain is decreased. The excess unmatched alpha-globin chains accumulate in the growing erythroid precursors, causing their premature death (ineffective erythropoiesis). Clinical features of beta-thalassemia include variably severe anemia and iron accumulation due to increased intestinal iron absorption. The most anemic patients require regular blood transfusions, which exacerbate their iron overload and result in damage to vital organs. The hepatic peptide hepcidin, a key regulator of iron metabolism in mammals, was recently found to be low in the urine of beta-thalassemia patients, compared with healthy controls, despite their iron overload. In our work, we measured by RQ-PCR the liver mRNA expression of hepcidin and other iron regulatory genes in beta-thalassemia major mouse model (C57Bl/6 Hbb(th3/th3)), and compared it with beta-thalassemia intermedia mouse model (C57Bl/6 Hbb(th3/+)) and control mice. We found decreased expression of hepcidin and TfR2 and increased expression of TfR1 and NGAL in the beta-thalassemia mouse models, compared with the control mice. Significant down-regulation of hepcidin expression in beta-thalassemia major, despite iron overload, might explain the increased iron absorption typically observed in thalassemia.