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Original article
Association of hepatic oxidative stress and iron dysregulation with HCC development after interferon therapy in chronic hepatitis C
  1. Shintaro Nanba1,
  2. Fusao Ikeda1,
  3. Nobuyuki Baba2,
  4. Koichi Takaguchi2,
  5. Tomonori Senoh2,
  6. Takuya Nagano2,
  7. Hiroyuki Seki1,
  8. Yasuto Takeuchi1,
  9. Yuki Moritou1,
  10. Tetsuya Yasunaka1,
  11. Hideki Ohnishi1,3,
  12. Yasuhiro Miyake1,
  13. Akinobu Takaki1,
  14. Kazuhiro Nouso1,3,
  15. Yoshiaki Iwasaki4,
  16. Kazuhide Yamamoto1
  1. 1Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
  2. 2Department of Internal Medicine, Kagawa Prefectural Central Hospital, Takamatsu, Japan
  3. 3Department of Molecular Hepatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
  4. 4Health Service Center, Okayama University, Okayama, Japan
  1. Correspondence to Dr Fusao Ikeda, Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1, Shikata-cho, Okayama 700-8558, Japan; fikeda{at}md.okayama-u.ac.jp

Abstract

Background Oxidative stress may play pathogenic roles in the mechanisms underlying chronic hepatitis C (CHC). The impact of excessive oxidative stress and iron dysregulation on the development of hepatocellular carcinoma (HCC) after interferon therapy has not been established.

Methods We investigated the impact of oxidative stress and iron deposition on HCC development after therapy with pegylated interferon (PegIFN)+ribavirin in CHC patients. Systemic and intracellular iron homeostasis was evaluated in liver tissues, peripheral blood mononuclear cells and sera.

Results Of 203 patients enrolled, 13 developed HCC during the 5.6-year follow-up. High hepatic 8-hydroxy-2-deoxyguanosine (8-OHdG) levels were significantly associated with HCC development in multivariate analysis (p=0.0012) which was also significantly correlated with severity of hepatic iron deposition before therapy (p<0.0001). Systemic and intracellular iron regulators of hepcidin and F-box and leucine-rich repeat protein 5 (FBXL5) expression levels were significantly suppressed in CHC patients (p=0.0032 and p=0.016, respectively) despite their significantly higher levels of serum iron and ferritin compared with controls. However, intracellular iron regulators of FBXL5 and iron regulatory proteins were regulated in balance with hepatic iron deposition. Significant correlations were observed among IL-6, bone morphogenetic protein 6, hepcidin and ferroportin, as regards systemic iron regulation.

Conclusions Measurement of hepatic oxidative stress before antiviral therapy is useful for the prediction of HCC development after interferon therapy. Low baseline levels of the intracellular iron regulators of FBXL5 in addition to a suppressed hepcidin level might be associated with severe hepatic iron deposition in CHC patients.

Trial registration number UMIN 000001031.

  • MOLECULAR PATHOLOGY
  • IRON METABOLISM
  • VIRUS
  • CYTOPATHOLOGY
  • HEPATITIS

https://doi.org/10.1136/jclinpath-2015-203215

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    Introduction

    Worldwide, hepatitis C virus (HCV) is a major cause of chronic hepatitis and progressive liver fibrosis leading to cirrhosis and hepatocellular carcinoma (HCC).1 The development of direct-acting antiviral agents (DAAs) has led to great improvements in HCV therapy, and more than 90% of patients with chronic hepatitis C (CHC) are expected to achieve a sustained virological response (SVR). However, the development of HCC after antiviral therapy remains a problem for CHC patients, so it would be useful to be able to evaluate the possibility of HCC development after interferon therapy.

    There is increasing evidence that oxidative stress may play a pathogenic role in the mechanisms underlying HCV-associated liver injury. Increased oxidative stress is observed in liver biopsy specimens from CHC patients.2 Studies with mice transgenic for the HCV core and non-structural 3 and 5A genes demonstrated direct induction of oxidative stress by HCV protein in the liver.3–7 Excessive hepatic iron deposition through oxidative stress might cause progressive hepatitis and liver fibrosis.8 ,9 However, the effects of hepatic iron deposition through oxidative stress on HCC development in CHC patients and the underlying mechanisms have not been fully established.

    Systemic iron homeostasis is controlled predominantly through the regulation of iron acquisition, while iron loss occurs only through exfoliation and blood loss.10 Serum iron levels are balanced by duodenal iron absorption and by iron release from the macrophages and the liver.11 These systemic iron fluxes are precisely regulated by hepcidin, a small peptide hormone released by the liver. Serum iron sensitises bone morphogenetic protein (BMP), and the basal hepcidin level is regulated by signalling through the BMP pathway. Inflammation also induces hepcidin transcription through the interleukin 6 (IL-6) pathway. Hepcidin inactivates ferroportin, the major transmembrane transporter transferring iron out of cells, to reduce systemic iron. The serum hepcidin level is reported to be significantly lower in CHC patients than in healthy controls, and this low level is thought to cause excessive iron accumulation.12

    The main regulators of intracellular iron homeostasis in the liver and macrophages are F-box and leucine-rich repeat protein 5 (FBXL5) and its downstream iron-regulatory protein (IRP).11 ,13 FBXL5 is a ubiquitin. It recognises both IRP1 and IRP2 and promotes their degradation in the proteasome in iron-replete cells, whereas in iron-deficient cells, IRP1 regulates several proteins at the post-transcriptional level for intracellular iron storage. Iron-dependent degradation of IRP1 and IRP2, mediated by FBXL5, is also involved in the maintenance of appropriate intracellular iron concentrations. Excessive iron accumulation is recognised as hepatic iron deposition, and results in intracellular free iron and reactive oxygen species. Serum ferritin, the major iron storage protein, may correlate with intracellular free iron.

    The importance of excessive hepatic oxidative stress and iron dysregulation for the prediction of HCC development after antiviral therapy in CHC patients is not well understood at present. In terms of iron dysregulation, the associations between iron regulators other than hepcidin and excessive iron accumulation and the mechanisms underlying the systemic and intracellular dysregulation of iron homeostasis in CHC patients are not known. Therefore the present study investigated the impact of hepatic oxidative stress and iron metabolism on the development of HCC after therapy with pegylated interferon (PegIFN)+ribavirin. We also comprehensively evaluated changes in systemic and intracellular iron regulators in the serum, peripheral blood mononuclear cells (PBMCs) and livers of CHC patients to clarify the mechanism of dysregulated iron metabolism in chronic HCV infection.

    Methods and materials

    Patients

    A total of 203 Japanese patients with CHC were enrolled in the study. All patients received 24–48 weeks of antiviral therapy with standard doses of PegIFN α-2a (180 μg/week) or 2b (1.5 μg/body weight/week) with ribavirin (600–1000 mg/day) at Okayama University Hospital or Kagawa Prefectural Central Hospital from January 2005 to November 2012. Therapy outcomes were evaluated according to the practice guidelines of the Japan Society of Hepatology.14 Patients with hepatitis B virus co-infection, HIV co-infection or autoimmune liver disease were excluded from the study, as were patients who developed HCC before antiviral therapy or within 1 year after antiviral therapy. A daily alcohol intake of <20 g, 20–80 g or >80 g was defined as no, moderate or heavy alcohol consumption, respectively. Of the 203 CHC patients, 60 whose liver tissues and PBMCs were available were evaluated for systemic and intracellular iron homeostasis, and the results were compared with those of 20 healthy control volunteers enrolled by the Department of Gastroenterology and Hepatology of Okayama University. Subjects with no relevant medical history, no alcohol abuse and serum ferritin levels within the normal range were included. Gene expression was evaluated only in sera and PBMCs. The study was performed in accordance with the Helsinki Declaration, and the protocols were approved by the ethics committees of the participating institutes. This study was registered in the University Hospital Medical Information Network Clinical Trials Registry (UMIN 000001031). All patients provided informed consent before enrolment in the study.

    HCC diagnosis and follow-up

    In accordance with the clinical practice manual of the Japan Society of Hepatology,14 HCC was diagnosed using ultrasonography, CT, MRI, hepatic angiography and/or tumour biopsy, in combination with the detection of serum levels of α fetoprotein and des-gamma-carboxy prothrombin. The follow-up period was defined as the time between the cessation of interferon therapy and HCC diagnosis or the latest confirmation of survival.

    Histological evaluation

    Liver histology was evaluated for all patients before therapy. Liver fibrosis stage and hepatitis activity grade were assigned according to the criteria of Desmet et al.15 Iron deposition in liver tissue was assessed by Perl's Prussian blue staining. The severity of iron deposition was assessed using criteria published by Rowe et al.16

    Immunohistochemistry of hepatic 8-hydroxy-2-deoxyguanosine

    8-Hydroxy-2-deoxyguanosine (8-OHdG) induces a point mutation in daughter DNA strands, and its measurement is considered a useful marker of oxidative stress.17 We performed semiquantitative immunohistochemical analysis of hepatic 8-OHdG for all patients before therapy. This analysis was performed using an avidin–biotin–peroxidase complex technique after microwave antigen retrieval, as described.18 In brief, 4-µm-thick sections were successively treated with blocking solution, 1 μg/mL anti-8-OHdG monoclonal antibody (Japan Institute for the Control of Aging, Fukuroi, Japan) or normal mouse immunoglobulin G (Dako, Glostrup, Denmark), biotinylated secondary antibody and a peroxidase–avidin complex (Envision Plus kit; Dako, Japan). 8-OHdG immunoreactivity was mainly observed in the nuclei of hepatocytes. Immunohistological data were quantified using criteria described in previous reports.19 ,20 Stained cells in three predefined areas around the portal vein were counted at high-powered magnification with Adobe Photoshop CS6. The percentage occupied by positive cells relative to the total area was calculated in five randomly selected visual fields; the values are presented here as averages±SD.

    Genotyping of a single nucleotide polymorphism

    Genomic DNA was extracted from whole-blood samples by means of the QIAamp DNA Mini Kit according to the manufacturer's protocol (Qiagen, Tokyo). The single nucleotide polymorphism (SNP) rs8099917 IL28B was genotyped using TaqMan predesigned SNP genotyping assays, as recommended by the manufacturer (Applied Biosystems, Tokyo). Two point mutations of C282Y and H63D in the HFE gene of the haemochromatosis protein, a key limiting factor in duodenal iron absorption, were tested with restriction fragment length polymorphism methods using primers and restriction enzymes as described.21 The SNP genotypes of all samples were obtained using these methods.

    Quantification of factors associated with iron metabolism

    Iron metabolism in liver tissue and PBMCs was evaluated for 60 patients. Expression levels of the iron regulators of hepcidin, ferroportin, IRP1, IRP2 and FBXL5 were quantified with real-time PCR; total RNA was extracted from liver tissue and PBMC samples with the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. Real-time PCR was performed on the Roche LightCycler 480 System (Roche Diagnostics, Mannheim, Germany) using SYBR Green I Master (Roche). The PCR conditions consisted of an initial reverse transcription step at 50°C for 10 min, followed by an initial denaturation step at 95°C for 5 min, and 40 amplification cycles at 95°C for 15 s, 57°C for 30 s and 72°C for 30 s, and a final extension step at 72°C for 1 min. The primers used in the assays were as described.12 ,22 ,23 The expression level of β-actin was used for internal control for each sample. Serum hepcidin and its regulators of BMP-6 and IL-6 were examined with ELISAs according to the manufacturer's instructions: hepcidin with the EIA Kit for hepcidin (Peninsula Laboratories, San Carlos, California, USA), BMP-6 with the ELISA Kit for BMP-6 (USCN Life Science, Wuhan, China) and IL-6 with the Quantikine HS Human IL-6 Immunoassay (R&D Systems, Minneapolis, Minnesota, USA).

    Statistical analysis

    Data are expressed as averages±SD. We used stepwise logistic regression to identify the factors associated with SVR-= to interferon therapy. We also used stepwise logistic regression to identify factors associated with the intensity of 8-OHdG immunochemical staining. The factors associated with HCC development after therapy were analysed in proportional hazards models. Iron regulators in liver tissue and PBMC samples were compared using the Wilcoxon rank sum test. Correlations between factors related to iron metabolism in CHC patients were evaluated with Pearson’s correlation coefficient. A value of p<0.05 was considered significant. The statistical analyses were performed with JMP V.11 software (SAS Institute, Cary, North Carolina, USA).

    Results

    Characteristics of the patients enrolled in the study

    The characteristics of the enrolled patients are shown in table 1. There was no significant difference between the blood tests of patients with HCV genotypes 1 and 2. Liver histology revealed similar distributions. There were no significant differences in serum ferritin, hepatic iron deposition or hepatic 8-OHdG between HCV genotypes 1 and 2 (p=0.11, p=0.37 and p=0.41, respectively). Interferon therapy resulted in SVR in 105 patients (52%): 68 patients with HCV genotype 1 (47%) and 37 patients with HCV genotype 2 (64%). During follow-up after interferon therapy (median period of 5.6 years), HCC was observed in 13 patients: in none within 1 year of follow-up, in 5 patients within 3 years of follow-up, in 6 within 3–5 years of follow-up, and in the remaining patient after approximately 6 years of follow-up. All patients maintained good liver function without cirrhosis during the follow-up period.

    View this table:
    Table 1

    Characteristics of the 203 patients with chronic hepatitis C

    Associations of hepatic 8-OHdG and iron deposition with the outcome of PegIFN+ribavirin therapy

    Stepwise logistic regression analysis was performed to clarify the associations of hepatic 8-OHdG and iron deposition with outcome following PegIFN+ribavirin therapy (table 2).

    View this table:
    Table 2

    Factors associated with interferon therapy outcomes for CHC patients

    Significant factors associated with SVR to therapy were shown by multivariate analysis to be the IL28B SNP TT genotype, low α fetoprotein levels and early liver fibrosis stages for patients with HCV genotype 1 (p=0.0010, p=0.022 and p=0.010, respectively), and low titre of HCV RNA and IL28B SNP TT genotype for patients with HCV genotype 2 (p=0.021 and p=0.0065, respectively). Hepatic 8-OHdG and hepatic iron deposition showed no significant associations with SVR to therapy for patients with HCV genotype 1 or 2.

    Association of 8-OHdG and iron deposition with HCC development after PegIFN+ribavirin therapy

    We compared the patient characteristics related to HCC development after PegIFN+ribavirin therapy using proportional hazard models (table 3). Age (p=0.036), male gender (p=0.011), alcohol consumption (p=0.025), SVR (p=0.0027), low platelet count (p<0.0001), high α fetoprotein level (p=0.0054), advanced liver fibrosis stage (p<0.0001), excessive iron deposition (p=0.016) and hepatic 8-OHdG level (p<0.0001) demonstrated significant associations with HCC development in the univariate analysis. In the multivariate analysis using the same significant factors, alcohol consumption (p=0.048), low platelet count (p=0.0008), high α fetoprotein level (p=0.045), advanced liver fibrosis stage (p=0.0022) and hepatic 8-OHdG level (p=0.0012) were significantly associated with HCC development.

    View this table:
    Table 3

    Proportional hazard models for the development of hepatocellular carcinoma after interferon therapy in CHC patients

    When 8-OHdG was excluded as a factor in multivariate analysis, SVR was shown to be a significant predictor of HCC development with a risk ratio of 0.0044–0.60 (p=0.0098), consistent with previous reports. Receiver operating characteristic curves were constructed to determine the best cut-off values for 8-OHdG as a factor in HCC development after PegIFN+ribavirin therapy (figure 1).

    Figure 1
    Figure 1

    Receiver operating characteristic curve to determine the best cut-off values for 8-hydroxy-2-deoxyguanosine (8-OHdG). Levels of 0.42% were the best cut-off values with an area under the curve (AUC) of 0.86, with sensitivity of 0.93 and specificity of 0.71.

    CHC patient characteristics associated with hepatic 8-OHdG

    We conducted a stepwise logistic regression analysis to clarify the factors associated with high levels of hepatic 8-OHdG in CHC patients. As shown in table 4, we used the average level of hepatic 8-OHdG in CHC patients as the cut-off value. Male gender (p=0.0073), high levels of haemoglobin (p=0.017), ferritin (p<0.0001) and severe hepatic iron deposition (p<0.0001) were significantly associated with high levels of 8-OHdG in the univariate analysis. Severe hepatic iron deposition was selected as the most significant factor associated with high levels of 8-OHdG in the multivariate analysis (p<0.0001). When the analysis was limited to markers that could be examined in the blood, serum ferritin level was significantly associated with hepatic 8-OHdG levels for CHC patients (p<0.0001) in a stepwise logistic regression analysis. Serum ferritin levels also reflected the degree of hepatic iron deposition (p<0.0001).

    View this table:
    Table 4

    Stepwise logistic regression analysis of factors associated with hepatic 8-OHdG in CHC patients

    Comparison of systemic and intracellular iron regulator levels in CHC patients and healthy controls

    As shown in figure 2, we compared the levels of iron regulators between CHC patients and healthy controls. Serum iron levels were significantly higher in CHC patients compared to controls (p=0.041; Wilcoxon rank sum test). The levels of BMP-6, which should be sensitised by serum iron, were also significantly higher in CHC patients (p<0.0001). A significant difference was also observed in serum IL-6 levels (p=0.0001). Although these up-regulations in CHC patients should increase hepcidin secretion, hepcidin expression levels in PBMCs and hepcidin serum levels were both significantly lower in CHC patients than in controls (p=0.0032 and p=0.0023, respectively). Regarding the expression levels of intracellular iron regulators in PBMCs, serum ferritin, which should reflect intracellular iron deposition, showed significantly higher levels in CHC patients compared to controls (p=0.036). The PBMCs of CHC patients were suspected to be iron-replete, but the FBXL5 levels were significantly suppressed in CHC patients compared to controls (p=0.016). IRP1 and IRP2 levels showed no significant difference between CHC patients and healthy controls (p=0.18 and p=0.67, respectively).

    Figure 2
    Figure 2

    Comparison of the levels of iron regulators in chronic hepatitis C (CHC) patients and healthy controls. CHC patients’ and healthy controls’ serum levels of iron (A), ferritin (B), IL-6 (C), BMP-6 (D) and hepcidin (E) and their intracellular expression levels (in PBMCs) of hepcidin (F), FBXL5 (G), IRP1 (H) and IRP2 (I). Expression levels are expressed as the ratio to the β-actin mRNA value as the internal control for each sample. Iron regulators in liver tissue and PBMC samples were compared with the Wilcoxon rank sum test. The median value of each category is shown under each graph. BMP-6, bone morphogenetic protein 6; CHC, chronic hepatitis C; HC, healthy control; IRP1, iron-regulatory protein 1; IRP2, iron-regulatory protein 2; PBMC, peripheral blood mononuclear cell.

    Changes in the levels of iron regulators associated with severity of hepatic iron deposition in CHC patients

    We evaluated the correlations of systemic iron regulators with serum iron and ferritin in CHC patients (figure 3). We found that serum levels of iron and ferritin corresponded significantly with hepcidin serum levels (p=0.0024 and p=0.0023, respectively; Spearman rank-order correlation), which also corresponded to hepcidin expression level in the liver (p=0.0082). Hepcidin expression level in the liver was associated with serum levels of IL-6 but not with BMP-6 (p=0.0015 and p=0.067, respectively). Hepcidin expression level in the liver was inversely associated with the expression level of ferroportin, the intracellular iron exporter (p=0.045). Serum levels of iron and ferritin corresponded to BMP-6 serum levels (p=0.0044 and p=0.0037, respectively). Regarding the expression levels of intracellular iron regulators in the liver, the degree of hepatic iron deposition was significantly associated with FBXL5 level (p=0.0013, figure 4). The degree of hepatic iron deposition was also significantly associated with serum ferritin level (p<0.0001). FBXL5 level was correlated significantly with IRP1 and IRP2 levels (p=0.029 and p=0.0013, respectively). Comparisons between the patient groups with HCV genotypes 1 and 2 showed that the intracellular iron regulators of FBXL5, IRP1 and IRP2 did not differ with hepatic expression level. Hepatic expression levels of hepcidin and ferroportin as well as serum IL-6 and BMP-6 showed no significant differences as regards systemic iron regulation.

    Figure 3
    Figure 3

    Correlation of systemic iron regulator levels in chronic hepatitis C (CHC) patients. The correlations of iron and ferritin to serum BMP-6 (A and B) and hepcidin (C and D) are shown. The associations of serum levels of hepcidin (E), IL-6 (F), BMP-6 (G), and the expression levels of hepatic ferroportin (H) and FBXL5 (I) were also compared to the expression levels of hepcidin in the liver of CHC patients. Expression levels are expressed as the ratio to the β-actin mRNA value as the internal control for each sample. BMP-6, bone morphogenetic protein 6.

    Figure 4
    Figure 4

    Correlation of intracellular iron regulator levels in CHC patients. The associations of the degree of hepatic iron deposition with intracellular expression levels of IRP1 (A), IRP2 (B) and FBXL5 (C) in the liver and serum ferritin levels (D) in CHC patients are shown. The correlations of FBXL5 expression levels in the liver were also evaluated for IRP1 (E) and IRP2 (F) expression levels. Expression levels are expressed as the ratio to the β-actin mRNA value as the internal control for each sample. IRP1, iron-regulatory protein 1; IRP2, iron-regulatory protein 2.

    Point mutation H63D in the HFE gene did not affect hepatic iron deposition in Japanese CHC patients

    All 60 CHC patients were tested for the point mutations C282Y and H63D in the HFE gene, a key limiting factors in duodenal iron absorption. Only two patients had H63D mutations, and their iron deposition in the liver was grades 0 and 1, suggesting this mutation has a minor effect on excessive iron deposition in Japanese CHC patients. None of CHC patients had the C282Y mutation.

    Discussion

    The present study comprehensively investigated the impact of hepatic oxidative stress and iron metabolism on the development of HCC after antiviral PegIFN+ribavirin therapy in CHC patients, and is the first to demonstrate that an increased hepatic 8-OHdG level before antiviral therapy is a significant factor predicting HCC development after antiviral therapy. 8-OHdG has been considered a useful marker of oxidative stress, and has been implicated in a number of pathologies.24–27 In the natural history of CHC, an increased 8-OHdG level in the liver is closely related to excessive hepatic iron deposition.19 Hepatic iron deposition is also associated with the severity of hepatitis and liver fibrosis.28 ,29 HCC patients have significantly higher oxidative stress than cirrhotic patients.30 Consistent with these reports, the results in the present study showed that increased hepatic 8-OHdG levels accompanying hepatic iron deposition before antiviral therapy might cause HCC development after antiviral therapy. In addition, the significant correlations among hepatic 8-OHdG, hepatic iron deposition and serum ferritin indicated that oxidative stress conditions in the liver can be estimated by measuring the serum ferritin level. Further analysis is needed to evaluate the differences in hepatic 8-OHdG and hepatic iron deposition at the time of HCC development in patients with and without SVR.

    Previous reports on therapy with IFN-α or IFN+ribavirin in patients with the HCV genotype 1b showed that non-SVR patients had significantly higher hepatic iron deposition than those with SVR.24 ,31 ,32 The impact of iron deposition on PegIFN+ribavirin therapy remained unclear. The present study demonstrated that hepatic 8-OHdG and hepatic iron deposition showed no significant associations with SVR to therapy for patients with HCV genotype 1 or 2. Similarly, Kohjima et al33 reported that there were no differences in hepatic iron deposition between patients with and without SVR to PegIFN+ribavirin, and that SVR patients showed significantly lower transcription and protein expression of hepcidin and ferroportin than non-SVR patients. Improvement in the therapeutic efficacy of antiviral therapy might lessen the effects of various patient conditions associated with therapeutic outcome.

    The systemic iron store and inflammation activate hepcidin transcription through BMP and IL-6 pathways.34–36 In the present study, CHC patients had significantly higher serum levels of IL-6 and BMP-6 compared to healthy controls. Although increased hepcidin would be expected due to high levels of these proteins, the expression levels of hepcidin in CHC patients’ PBMCs and their serum hepcidin levels were paradoxically suppressed. Significantly lower expression levels of FBXL5, the main regulator of intracellular iron metabolism, were also observed in CHC patients. Poor liver function can be ruled out as the cause of low levels of iron regulators, because all the CHC patients in the present study had good liver function without liver cirrhosis. There are several reports showing low hepcidin levels in CHC patients,10 ,12 but the present study is the first to indicate that insufficient baseline levels of the intracellular iron regulator of FBXL5 in addition to suppressed hepcidin level might be associated with severe hepatic iron deposition in CHC patients.

    The present study also evaluated the levels of iron regulators in CHC patients with different iron deposition levels. In the IL-6 and BMP pathways in systemic iron homeostasis, we observed significant correlations among alanine aminotransferase (ALT), IL-6 and hepcidin, and among serum iron, BMP-6 and hepcidin. Hepcidin expression levels in the liver were also correlated with expression levels of ferroportin, the iron export transporter. Similarly in intracellular iron homeostasis, the associations between FBXL5 and IRP1 and those between FBXL5 and IRP2 were significant. All of these mechanisms might work well in CHC patients but were suspected to be insufficient for balancing iron concentrations.

    Measuring gene expression may be a less sensitive method of assessing active IRPs due to the mechanisms of post-translational control of IRPs. Fillebeen et al studied intracellular iron metabolism with the HCV replication model using an electrophoretic mobility shift assay for the iron responsive element-binding activities of IRPs in addition to measuring IRP gene expression. They showed up-regulation of IRP2 activity in iron-deficient Huh7.5.1 cells infected with JFH-1,37 which is consistent with the results in the present study that high IRP expression levels were observed in CHC patients with no or very little iron deposition.

    Further analysis is needed to evaluate the direct effects of HCV infection on systemic and intracellular iron homeostasis, and to identify the suppressed mechanisms that affect imbalanced iron homeostasis in HCV replication models.

    In conclusion, oxidative stress in the liver might not affect the outcomes of PegIFN+ribavirin therapy in CHC patients, but is useful for predicting later HCC development. Insufficient baseline levels of iron regulators in both systemic and intracellular iron homeostasis might be associated with severe hepatic iron deposition and oxidative stress in CHC patients, although systemic and intracellular iron regulators adequately balance iron concentrations by recognising serum iron and intracellular iron deposition.

    Take home messages

    • Oxidative stress in the liver may be useful for predicting HCC development after PegIFN+ribavirin therapy in chronic hepatitis C (CHC) patients.

    • Insufficient baseline levels of iron regulators in both systemic and intracellular iron homeostasis may cause severe hepatic iron deposition and oxidative stress in CHC patients.

    • Systemic and intracellular iron regulators balance iron concentrations by recognising serum iron and intracellular iron deposition.

    Acknowledgments

    This work was supported in part by the Research Program of Intractable Disease sponsored by the Ministry of Health, Labour, and Welfare of Japan, and by a Grant-in-Aid (22590733) for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to YI). We thank Chizuru Mori for technical assistance and Toshie Ishi for valuable help with data collection.

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    Supplementary materials

    • Abstract in Japanese

      This web only file has been produced by the BMJ Publishing Group from an electronic file supplied by the author(s) and has not been edited for content.

    Footnotes

    • Handling editor Runjan Chetty

    • Competing interests None declared.

    • Patient consent Obtained.

    • Ethics approval Okayama University approved this study.

    • Provenance and peer review Not commissioned; externally peer reviewed.