Hepatitis C virus-induced reactive oxygen species raise hepatic iron level in mice by reducing hepcidin transcription.

BACKGROUND & AIMS: Despite abundant clinical evidence, the mechanisms by which hepatic iron overload develops in patients with hepatitis C virus (HCV)-associated chronic liver disease remain unknown. The aim of this study was to investigate how hepatic iron overload develops in the presence of HCV proteins. METHODS: Male transgenic mice expressing the HCV polyprotein and nontransgenic control mice (C57BL/6) were assessed for iron concentrations in the liver, spleen, and serum and iron regulatory molecules in vivo and ex vivo. RESULTS: Transgenic mice had increased hepatic and serum iron concentrations, decreased splenic iron concentration, and lower hepcidin expression in the liver accompanied by higher expression of ferroportin in the duodenum, spleen, and liver. In response to hepatocellular iron excess, transferrin receptor 1 expression decreased and ferritin expression increased in the transgenic liver. Transgenic mice showed no inflammation in the liver but preserved the ability to induce hepcidin in response to proinflammatory cytokines induced by lipopolysaccharide. Hepcidin promoter activity and the DNA binding activity of CCAAT/enhancer-binding protein alpha (C/EBP) were down-regulated concomitant with increased expression of C/EBP homology protein, an inhibitor of C/EBP DNA binding activity, and with increased levels of reactive oxygen species in transgenic mice at the ages of 8 and 14 months. CONCLUSIONS: HCV-induced reactive oxygen species may down-regulate hepcidin transcription through inhibition of C/EBPalpha DNA binding activity by C/EBP homology protein, which in turn leads to increased duodenal iron transport and macrophage iron release, causing hepatic iron accumulation.

Stronger Neo-Minophagen C, a glycyrrhizin-containing preparation, protects liver against carbon tetrachloride-induced oxidative stress in transgenic mice expressing the hepatitis C virus polyprotein.

BACKGROUND/AIM: Stronger Neo-Minophagen C (SNMC), a glycyrrhizin-containing preparation, has been used as a treatment for chronic hepatitis for more than 30 years in Japan, and shown to be effective in preventing the development of hepatocellular carcinoma in chronic hepatitis C patients, but its underlying mechanisms remain elusive. The aim of this study was to investigate if SNMC had an anti-oxidative effect, as oxidative stress has been proposed to be one of the mechanisms of liver injury in hepatitis C virus (HCV)-associated chronic liver diseases. METHODS: The protective effect of SNMC against carbon tetrachloride (CCl4)-induced liver injury was examined using transgenic mice expressing the HCV polyprotein. RESULTS: A small dose of CCl4 (10 microl/kg of body weight) significantly increased the serum alanine aminotransferase (ALT) level and hepatic malondialdehyde content, decreased hepatic reduced glutathione (GSH) content and induced ultrastructural alterations of hepatic mitochondria in transgenic mice, but not in nontransgenic mice. A single SNMC treatment equivalent to a clinical dose significantly restored the serum ALT level and hepatic malondialdehyde and GSH contents, attenuated the ultrastructural alterations of hepatic mitochondria, and increased mRNA expression of gamma-glutamylcysteine synthetase (gamma-GCS). CONCLUSIONS: Transgenic mice expressing the HCV polyprotein are abnormally vulnerable to oxidative stress. SNMC protects hepatocytes against CCl4-induced oxidative stress and mitochondrial injury in the presence of HCV proteins by restoring depleted cellular GSH.

Alpha-tocopherol [corrected] and ascorbic acid attenuates the ribavirin [corrected] induced decrease of eicosapentaenoic acid in erythrocyte membrane in chronic hepatitis C patients.

BACKGROUND: Oxidative damage of the erythrocyte membrane plays an important role in ribavirin-induced anemia. The purpose of the present paper was to assess whether supplementation of alpha-tocopherol and ascorbic acid (vitamins) causes changes in the erythrocyte membrane fatty acid composition during interferon and ribavirin combination therapy for chronic hepatitis C patients. METHODS: Fatty acid compositions in erythrocyte membrane phospholipids were determined by gas chromatography at 0, 2, 4, 8 weeks, and at the end of combination therapy (26 weeks) for interferon with ribavirin in 32 patients with chronic hepatitis C who were randomized to receive vitamins or not (controls). RESULTS: Good compliance with orally administered vitamins and ribavirin were confirmed by their concentrations in erythrocytes or plasma. The hemoglobin level was negatively correlated with the ribavirin concentration at 8 weeks (r = 0.59, P = 0.01) after initiation of therapy in controls, but not in the vitamin group. Among the 26 kinds of fatty acids analyzed, only eicosapentaenoic acid (EPA) significantly decreased at 8 weeks after initiation of therapy (P = 0.03) and at the end of therapy (P = 0.004) in controls. Vitamins did not inhibit ribavirin-induced anemia, but attenuated the decrease of EPA in erythrocytes. The EPA level was negatively correlated with the drop in hemoglobin levels at 8 weeks after initiation of therapy in controls (r = 0.58, P = 0.015), but not in the vitamin group. CONCLUSIONS: Supplementation of alpha-tocopherol and ascorbic acid attenuates the ribavirin-induced decrease of EPA in erythrocyte membrane phospholipids in chronic hepatitis C patients.

Hepatic iron overload induces hepatocellular carcinoma in transgenic mice expressing the hepatitis C virus polyprotein.

BACKGROUND & AIMS: Despite the evidence of hepatic iron overload in patients with chronic hepatitis C, it remains unknown if iron overload is related to hepatocarcinogenesis in this condition. The aim of this study was to determine whether iron overload contributes to development of hepatocellular carcinoma (HCC) in transgenic mice expressing the hepatitis C virus (HCV) polyprotein. METHODS: Male C57BL/6 transgenic mice expressing the HCV polyprotein and nontransgenic littermates were fed an excess-iron diet or control diet. Mice in each group were assessed for altered liver morphology and function and the development of liver tumors. RESULTS: Hepatic iron concentrations in mice fed the excess-iron diet were comparable to those of patients with chronic hepatitis C. There was no inflammation in transgenic and nontransgenic livers. Compared with mice in 3 other groups, transgenic mice fed the excess-iron diet showed marked hepatic steatosis including the centrilobular microvesicular type, ultrastructural alterations of the mitochondria and decreased degradation activity of fatty acid at 6 months, and greater hepatic content of lipid peroxidation products and 8-hydroxy-2'-deoxyguanosine at 12 months after initiation of feeding. The number of proliferating hepatocytes was significantly increased in mice fed the excess-iron diet but was not different between transgenic and nontransgenic mice. Hepatic tumors including HCC developed in 5 of 11 (45%) transgenic mice fed the excess-iron diet but not in mice in other groups at 12 months after initiation of feeding. CONCLUSIONS: Iron overload induces mitochondrial injury and increases the risk of HCC development in transgenic mice expressing the HCV polyprotein.

Hepatitis C virus core protein inhibits deoxycholic acid-mediated apoptosis despite generating mitochondrial reactive oxygen species.

BACKGROUND: Hepatitis C virus (HCV) core protein is known to cause oxidative stress and alter apoptosis pathways. However, the apoptosis results are inconsistent, and the real significance of oxidative stress is not well known. The aim of this study was twofold. First, we wanted to confirm whether core-induced oxidative stress was really significant enough to cause DNA damage, and whether it induced cellular antioxidant responses. Second, we wanted to evaluate whether this core-induced oxidative stress and the antioxidant response to it was responsible for apoptosis changes. METHODS: HCV core protein was expressed under control of the Tet-Off promoter in Huh-7 cells and HeLa cells. We chose to use deoxycholic acid (DCA) as a model because it is known to produce both reactive oxygen species (ROS) and apoptosis. RESULTS: Core expression uniformly increased ROS and 8-hydroxy-2'-deoxyguanosine (8-OHdG) under basal and DCA-stimulated conditions. Core protein expression also increased manganese superoxide dismutase levels. Core protein inhibited DCA-mediated mitochondrial membrane depolarization and DCA-mediated activation of caspase-9 and caspase-3, despite the increase in ROS by DCA. Core protein inhibited DCA-mediated apoptosis by increasing Bcl-x(L) protein and decreasing Bax protein, without affecting the proportion of Bax between mitochondria and cytosol, resulting in suppression of cytochrome c release from mitochondria into cytoplasm. CONCLUSIONS: HCV core protein induces oxidative DNA damage, whereas it inhibits apoptosis that is accompanied by enhancement of ROS production. Thus, oxidative stress and apoptosis modulation by core protein are independent of each other.

Vitamin E and C supplementation prevents decrease of eicosapentaenoic acid in mononuclear cells in chronic hepatitis C patients during combination therapy of interferon alpha-2b and ribavirin.

OBJECTIVE: We investigated the effects of vitamin E and C supplementation on the fatty acid composition of mononuclear cells and on the clinical observations in patients who had chronic hepatitis C and received interferon-alpha-2b (IFN-alpha-2b) and ribavirin combination therapy. METHODS: Patients were randomly allocated to receive daily 500 mg of vitamin E and 750 mg of vitamin C (vitamin group, n = 14) or no supplement (non-vitamin group, n = 16) in addition to IFN-alpha-2b and ribavirin therapy. The fatty acid composition of mononuclear cell phospholipids was analyzed before and at 2, 4, and 8 wk after treatment. RESULTS: After vitamin supplementation, plasma and red blood cell alpha-tocopherol and plasma ascorbic acid levels increased in the vitamin group. Serum levels of alanine aminotransferase decreased significantly after 2 wk of treatment in both groups. At the start of treatment, a lower level of eicosapentaenoic acid (EPA) and a higher level of the molar ratio of arachidonic acid to EPA in mononuclear cells were observed in the present patients compared with healthy volunteers, and a significant correlation between the molar ratio and serum alanine aminotransferase level was found. The EPA level of mononuclear cells was maintained in the vitamin group during treatment, whereas a significant decrease was observed in the non-vitamin group at 4 and 8 wk after treatment. CONCLUSIONS: Antioxidant vitamin supplementation during IFN-alpha-2b and ribavirin therapy prevented a decrease in EPA of mononuclear cell phospholipids. If a further decrease in the ratio of arachidonic acid to EPA can be achieved by using oral EPA supplementation, the efficacy of IFN-alpha-2b and ribavirin therapy may be improved.

In situ detection of oxidized n-3 polyunsaturated fatty acids in chronic hepatitis C: correlation with hepatic steatosis.

BACKGROUND: Oxidative stress contributes to the pathogenesis of chronic hepatitis C. The aim of this study was to assess the peroxidation of n-3 polyunsaturated fatty acids (PUFAs) in the liver and its relation to hepatic steatosis in chronic hepatitis C. METHODS: We immunohistochemically detected malondialdehyde (MDA)-, 4-hydroxy-2-nonenal (HNE)-, and 4-hydroxy-2-hexenal (HHE)-protein adducts in liver biopsy specimens from 55 patients with chronic hepatitis C. Cells stained positively for HHE-protein adducts were quantified using computer-based image analysis. Fatty-acid composition was determined, by gas chromatography, for the noncancerous portions of resected livers, with or without steatosis, obtained from two patients with hepatitis C virus-associated hepatocellular carcinoma. RESULTS: The detection rate of HHE-protein adducts (63.6%) was significantly higher than that of MDA-protein adducts (21.8%; P < 0.001) or HNE-protein adducts (29.1%; P < 0.001). Areas positively stained for HHE-protein adducts (HHE-positive areas) were significantly larger in 18 patients with steatosis (6.2 +/- 3.6%) than in 17 patients without steatosis (3.4 +/- 2.6%; P = 0.01). Resected liver tissue with steatosis showed a larger HHE-positive area (18.6%) and higher ratio of n-6 PUFA content to n-3 PUFA content (3 : 1) than liver tissue without steatosis (7.2%; 2 : 3). On multivariate analysis, the HHE-positive area (odds ratio, 1.55; 95% confidence interval [CI], 1.08-2.23; P = 0.019) was a factor associated with the presence of hepatic steatosis. CONCLUSIONS: HHE-protein adducts, which are a good marker for oxidative stress, are associated with steatosis in chronic hepatitis C.

Mitochondrial dysfunction in hepatitis C.

Chronic hepatitis C induces a state of hepatic oxidative stress that is more pronounced than that present in many other inflammatory liver diseases. This review summarizes recent information that the hepatitis C virus (HCV) core protein plays an important role in this phenomenon. Core protein localizes to mitochondria, particularly at the points of contact between mitochondrial outer membrane and endoplasmic reticulum. Its expression causes inhibition of electron transport at complex I, increased complex I reactive oxygen species (ROS) production, decreased mitochondrial glutathione, and increased mitochondrial permeability transition in response to exogenous oxidants and tumor necrosis factor-alpha. Possible mechanisms of the core protein effects include direct interaction with electron carriers and indirect effects mediated by changes in mitochondrial calcium. These results suggest that antioxidant approaches may prove beneficial for patients with chronic hepatitis C.

Hepatitis C virus infection upregulates expression of the type I interferon receptor in human peripheral blood mononuclear cells.

To assess whether hepatitis C virus infection (HCV) affects type I interferon (IFN) receptor expression, the expression levels of two subunits of this receptor, IFNAR1 and IFNAR2, in peripheral blood mononuclear cells (PBMCs) were determined by flow-cytometric assay in 68 anti-HCV antibody-positive patients (47 positive for HCV RNA and 21 negative for HCV RNA) and 14 healthy controls. The percentages of IFNAR1- and IFNAR2-expressing cells were significantly higher in HCV RNA-positive patients than in HCV RNA-negative patients or healthy controls. In multiple regression analysis, the presence of HCV RNA in serum was independently associated with the expression of both IFNAR1 and IFNAR2 in PBMCs (P=0.007 for IFNAR1 and P=0.003 for IFNAR2). The frequency of IFN-gamma-producing peripheral CD4(+) and CD8(+) cells was also significantly higher in HCV RNA-positive patients than in HCV RNA-negative patients or healthy controls and there was a significant correlation between IFN receptor expression and the frequency of IFN-gamma-producing cells. These results suggest that HCV infection upregulates the expression of the type I IFN receptor in PBMCs through enhanced IFN-gamma production by peripheral CD4(+) and CD8(+) cells as one of mechanisms that regulate the expression of the type I IFN receptor.

Hepatic expression of type I interferon receptor for predicting response to interferon therapy in chronic hepatitis C patients: a comparison of immunohistochemical method vs. competitive polymerase chain reaction assay.

The focus of this study is to determine both mRNA and protein expression levels of the type I interferon (IFN) receptor subunits, IFNalpha receptor (IFNAR1) and IFNalpha/beta receptor (IFNAR2), in the liver, and to assess which quantification method is most useful in predicting response to IFN in chronic hepatitis C patients. Liver biopsy specimens from 41 chronic hepatitis C patients subsequently treated with IFN were immunostained with IFNAR1 and IFNAR2 antibodies, and also analyzed for both receptor subunit mRNA levels, using competitive polymerase chain reaction. Immunostaining of IFNAR1 and IFNAR2 was observed in the cell membrane and cytoplasm of hepatocytes in all liver specimens. Hepatic expression of IFNAR1 (r=0.45, P=0.012) or IFNAR2 mRNA (r=0.46, P=0.009) was weakly correlated with their protein expression in hepatocytes. The labeling indexes of IFNAR1 (136.7+/-94.1 vs. 77.4+/-76.8, P=0.032) and IFNAR2 (153.1+/-80.2 vs. 87.2+/-75.5, P=0.011) in liver specimens were significantly higher in sustained virologic responders (n=17) than in non-sustained virologic responders (n=24), while mRNA levels of either receptor subunit did not differ between response groups. These results suggest that protein expression of the type I IFN receptor in liver is a more useful measure than its mRNA expression in predicting response to IFN in chronic hepatitis C patients.

Mitochondrial injury, oxidative stress, and antioxidant gene expression are induced by hepatitis C virus core protein.

BACKGROUND & AIMS: The mechanisms of liver injury in chronic hepatitis C virus (HCV) infection are poorly understood. Indirect evidence suggests that oxidative stress and mitochondrial injury play a role. The aim of this study was to determine if the HCV core protein itself alters mitochondrial function and contributes to oxidative stress. METHODS: HCV core protein was expressed in 3 different cell lines, and reactive oxygen species (ROS) and lipid peroxidation products were measured. RESULTS: Core expression uniformly increased ROS. In 2 inducible expression systems, core protein also increased lipid peroxidation products and induced antioxidant gene expression as well. A mitochondrial electron transport inhibitor prevented the core-induced increase in ROS. A fraction of the expressed core protein localized to the mitochondria and was associated with redistribution of cytochrome c from mitochondrial to cytosolic fractions. Sensitivity to oxidative stress was also seen in HCV transgenic mice in which increased intrahepatic lipid peroxidation products occurred in response to carbon tetrachloride. CONCLUSIONS: Oxidative injury occurs as a direct result of HCV core protein expression both in vitro and in vivo and may involve a direct effect of core protein on mitochondria. These results provide new insight into the pathogenesis of hepatitis C and provide an experimental rationale for investigation of antioxidant therapy.

Steatosis and liver cancer in transgenic mice expressing the structural and nonstructural proteins of hepatitis C virus.

BACKGROUND & AIMS: The aim of this study was to determine whether expression of hepatitis C virus proteins alters hepatic morphology or function in the absence of inflammation. METHODS: Transgenic C57BL/6 mice with liver-specific expression of RNA encoding the complete viral polyprotein (FL-N transgene) or viral structural proteins (S-N transgene) were compared with nontransgenic littermates for altered liver morphology and function. RESULTS: FL-N transcripts were detectable only by reverse-transcription polymerase chain reaction, and S-N transcripts were identified in Northern blots. The abundance of viral proteins was sufficient for detection only in S-N transgenic animals. There was no inflammation in transgenic livers, but mice expressing either transgene developed age-related hepatic steatosis that was more severe in males. Apoptotic or proliferating hepatocytes were not significantly increased. Hepatocellular adenoma or carcinoma developed in older male animals expressing either transgene, but their incidence reached statistical significance only in FL-N animals. Neither was ever observed in age-matched nontransgenic mice. CONCLUSIONS: Constitutive expression of viral proteins leads to common pathologic features of hepatitis C in the absence of specific anti-viral immune responses. Expression of the structural proteins enhances a low background of steatosis in C57BL/6 mice, while additional low level expression of nonstructural proteins increases the risk of cancer.