Volume 130, Issue 7, Pages 2087-2098 (June 2006) Hepatic Iron Overload Induces Hepatocellular Carcinoma in Transgenic Mice Expressing the Hepatitis C Virus Polyprotein  Takakazu Furutani, Keisuke Hino, Michiari Okuda, Toshikazu Gondo, Sohji Nishina, Akira Kitase, Masaaki Korenaga, Shu–Yuan Xiao, Steven A. Weinman, Stanley M. Lemon, Isao Sakaida, Kiwamu Okita  Gastroenterology  Volume 130, Issue 7, Pages 2087-2098 (June 2006) DOI: 10.1053/j.gastro.2006.02.060 Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 1 Hepatocellular iron accumulation in iron-overloaded FL-N/35 transgenic and nontransgenic mice. (A) Serial changes of hepatic iron concentrations in nontransgenic and FL-N/35 transgenic mice. Hepatic iron concentrations were measured by atomic absorption spectrometry in 10 mice in each group at 6 and 12 months, except for 3 mice at the initiation of feeding. TgM-Fe: FL-N/35 transgenic mice fed the excess-iron diet, non–TgM-Fe: nontransgenic mice fed the excess-iron diet, TgM-C: FL-N/35 transgenic mice fed the control diet, non–TgM-C: nontransgenic mice fed the control diet. a: P < .05 versus c and f, b: P < .05 versus d, c: P < .05 versus d, e: P < .05 versus h, f: P < .05 versus g, and g: P < .05 versus h. Hepatic iron deposits in nontransgenic mice fed the control diet at 12 months (B) (Perls’ Prussian blue, original magnification × 100). Hepatic iron deposits found in FL-N/35 transgenic mice fed excess iron at 12 months (C) (Perls’ Prussian blue, original magnification × 100). Cytoplasmic Fe2+ (D) and Fe3+ (E) deposits observed in hepatocytes in FL-N/35 transgenic mice at 12 months after initiation of iron loading (Iron [II]/Iron [III] Detection kit, original magnification × 400). Gastroenterology 2006 130, 2087-2098DOI: (10.1053/j.gastro.2006.02.060) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 2 Hepatic steatosis and triglyceride content in FL-N/35 transgenic and nontransgenic mice. Hepatic steatosis in mice in each group at 6 months (A) and 12 months (B) after the initiation of feeding (H&E, original magnification × 100). TgM-Fe, non–TgM-Fe, TgM-C, and non–TgM-C; see legend for Figure 1. (C) In addition to macrovesicular steatosis, centrilobular microvesicular steatosis (arrow) was evident in FL-N/35 transgenic mouse at 6 months after initiation of iron loading (H&E, original magnification × 400). ⁎Hepatic central vein. (D) Serial change in hepatic triglyceride content in mice in each group. The results are shown as box plot profiles. The bottom and top edges of the boxes are the 25th and 75th percentiles, respectively. Median values are shown by the line within the box. The number in parentheses represents the number of animals examined in each group: a: P < .01 versus non–TgM-Fe, TgM-C, and non–TgM-C; b: P < .01 versus TgM-C and non–TgM-C; and P < .05 versus non–TgM-Fe. (E) Serum ALT levels in mice in each group at 6 and 12 months after initiation of feeding. The results are shown as box plot profiles. The number in parentheses represents the number of animals examined in each group: a: P < .01 versus non–TgM-Fe, TgM-C, and non–TgM-C; b: P < .05 versus non–TgM-C; and c: P < .05 versus TgM-C. Gastroenterology 2006 130, 2087-2098DOI: (10.1053/j.gastro.2006.02.060) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 3 Electron microscopy of the livers of FL-N/35 transgenic and nontransgenic mice. Electron microscopy of the liver of a nontransgenic mouse fed the control diet for 6 months (A) and FL-N/35 transgenic mouse fed excess iron for 6 months (B–D). The nontransgenic mouse shows almost the same-sized mitochondria without significant ultrastructural alterations (A) (×10,000). The transgenic mouse shows irregularly sized and swollen (arrow) mitochondria (B) (×10,000), disorganized cristae (C) (×20,000), and bulging within the intermembrane spaces (D) (×20,000). Gastroenterology 2006 130, 2087-2098DOI: (10.1053/j.gastro.2006.02.060) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 4 In vivo formation of [14C]CO2 from [U-14] palmitic acid in FL-N/35 transgenic and nontransgenic mice. A tracer dose of [U-14] palmitic acid (150 μCi/kg; 0.16 μmol/kg) was administered by gastric intubation in 0.2 mL of corn oil, preceded by fasting for 42 hours. The animal was then placed for 6 hours in a small plastic cage swept by an air flow of 50 mL/min. The outflow was bubbled into 30 mL of monoethanolamine. After 6 hours, 1 mL was removed and counted for [14C] CO2 activity. This experiment was done for 5 mice in each group at 6 months after the initiation of feeding. TgM-Fe, non–TgM-Fe, TgM-C, and non–TgM-C; see legend for Figure 1. *In vivo formation of [14C]CO2 from [U-14] palmitic acid was significantly decreased in FL-N/35 transgenic mice fed the excess-iron diet compared with mice in the 3 other groups (P < .01). Gastroenterology 2006 130, 2087-2098DOI: (10.1053/j.gastro.2006.02.060) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 5 Immunohistochemical detection of hepatic lipid peroxidation products. Immunohistochemical detection of HHE (A) and HNE (C) protein adducts in the livers of mice in each group at 12 months after the initiation of feeding (original magnification × 100). Quantitative analysis of digitized images of HHE- (B) or HNE-stained area (D) in the livers of mice in each group. For each liver, the mean percent area stained positively for HHE- or HNE-protein adducts was calculated for 5 randomly selected views. The numbers in parentheses represent the number of animals examined in each group. a: FL-N/35 transgenic mice fed excess iron showed greater lipid peroxidation products in the liver than mice in the 3 other groups (P < .05). TgM-Fe, non–TgM-Fe, TgM-C, and non–TgM-C; see legend for Figure 1. Gastroenterology 2006 130, 2087-2098DOI: (10.1053/j.gastro.2006.02.060) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 6 Hepatic 8-OHdG levels in FL-N/35 transgenic and nontransgenic mice. Hepatic 8-OHdG levels were quantified with an enzyme-linked immunosorbent assay. The results are shown as box plot profiles. The bottom and top edges of the boxes are the 25th and 75th percentiles, respectively. Median values are shown by the line within the box. TgM-Fe, non–TgM-Fe, TgM-C, and non–TgM-C; see legend for Figure 1. The numbers in parentheses represent the number of animals examined in each group: a: P < .05 versus non–TgM-Fe at 12 months, P < .01 versus TgM-C and non–TgM-C at 12 months, P < .01 versus TgM-Fe at 0 and 6 months; b: P < .01 versus non–TgM-Fe at 0 and 6 months; c: P < .05 versus TgM-C at 0 and 6 months; and d: P < .05 versus non–TgM-C at 0 and 6 months. Gastroenterology 2006 130, 2087-2098DOI: (10.1053/j.gastro.2006.02.060) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 7 Labeling indices of hepatocytic nuclei positively stained for PCNA in FL-N/35 transgenic and nontransgenic mice. (A) Immunohistochemical detection of hepatocytic nuclei positively stained for PCNA in mice at 12 months after initiation of feeding. (B) For the quantitative evaluation of PCNA-positive nuclei, the labeling index for PCNA-positive nuclei was determined by a random evaluation of 1000 cells selected from several areas in hepatic lobules and expressed as a percentage. TgM-Fe, non–TgM-Fe, TgM-C, and non–TgM-C; see legend for Figure 1. The numbers in parentheses represent the number of animals examined in each group: a: P < .01 versus TgM-C and non–TgM-C and b: P < .01 versus TgM-C and non–TgM-C. Gastroenterology 2006 130, 2087-2098DOI: (10.1053/j.gastro.2006.02.060) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 8 Histology of the hepatocellular carcinoma in the iron overloaded FL-N/35 transgenic mouse. (A–C) HCC in a 14-month-old (iron overloading for 12 months) male from the FL-N/35 lineage. The edge of the tumor is clearly discriminated from the surrounding tissue with macro- and microvesicular steatosis (original magnification × 100, A: H&E and B: reticulin stain). Tumor shows increased cell density, increased ratio of nucleus to cytoplasm, loss of reticulin pattern, and markedly thickened trabeculae (original magnification × 400, C: reticulin stain). Gastroenterology 2006 130, 2087-2098DOI: (10.1053/j.gastro.2006.02.060) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions