including Kurozu-M were started from a week before the initial administration of DEN (Sigma, St. Louis). Administrations of DEN (150 mg/kg/week) were performed by intraperitoneal injec- tion 3 times during 3 weeks. The concentration of iron in basal CE-2 diet was 32.2 mg/100 g (data from manufacturer) and that of CE-2 diet containing 2% Kurozu-M, used as the experimental diet in this study, was calculated to be 31.7 mg/100 g (based on iron concentration of 4.8 mg/100 g in Kurozu-M). Preliminary examinations showed that liver tumors more than 10 mm in size at the major axis were present in all rats (n = 5) of the control group at 12 weeks after initial administration of DEN. Serum examinations. At 16 weeks after initial administra- tion of DEN, serum levels of albumin, transaminases (AST and ALT), hyaluronic acid, tumor necrosis factor (TNF)-α (ELISA), interleukin (IL)-2 (ELISA) were measured (SRL Inc., Tokyo, Japan) in half of the rats (each group; n = 8) of both groups. Histology and immunostaining. Immediately after collec- tion of serum, the same rats (each group; n = 8) were sacrificed under anesthesia induced with isoflurane (Wako Pure Chemicals, Osaka, Japan) and the livers were resected. The presence of liver tumors was confirmed macroscopically. Moreover, microscopic examination of liver tissues by hematoxylin-eosin (HE) staining was performed as usual. Further, glutathione S-transferase placental form (GST-P) was examined as a marker of HCC and pre- cancerous lesions. Procedures of GST-P staining were as follows. Livers were fixed, embedded in paraffin and sectioned. Sections of 3 μm thickness were treated with rabbit anti-rat GST-P antibody (MBL International Co., Nagoya, Japan) and then with secondary antibody and avidin-biotin complex (Ultratech HRP Kit; Beckman Coulter Inc., Ontario, Canada). The sites of peroxidase binding were visualized with diaminobenzidine. Sections were counter- stained with hematoxylin. Evaluation of GST-P-positive foci was performed based on size (tranverse diameter) and number per slice (transverse axis) of the right lobe of liver in all 16 rats. Semi-quantitative analysis of GST-P-positive foci was done using Adobe Photoshop CS3 Extended. All evaluations were performed by a pathologist who was blinded as to the treatment the animals had received. MMP2 assay. Levels of activity of MMP-2 were assayed with a commercial kit (Amersham Pharmacia Biotech, Buckinghamshire, UK). Tissues of HCCs were homogenized by centrifugation, and the supernatant was used as the sample. Samples were added to wells coated with MMP-2 antibody and incubated. Each sample was activated with 0.5 mM aminophenylmercuric acetate (APMA). The absorbance was read at 405 nm on a microplate reader. The method used was as described in our previous report.(3) MMP9 assay. Standards and samples were run in the same manner as described for MMP-2 on a microplate coated with MMP-9 antibody, and 1 mM APMA was used for activation. The absorbance was read at 405 nm on a microplate reader. The method used was as described in our previous report.(3) Survival rates. The remaining rats (each group; n = 8) in both groups were continued on the same diets and under same conditions. Survival was examined during 48 weeks after the initial administration of DEN. Differences in the survival rates of the non-sacrificed rats (each group; n = 8) among the two groups after initial DEN administration were analyzed by the Kaplan- Meier method. Statistical analysis. Statistical analysis of serum parameters, MMP-2 and MMP-9 levels, and numbers and sizes of GST-P- positive foci in the liver among rats of the two groups (each group; n = 8) was done with the unpaired t test. The criterion of significance was p<0.05. Levels of serum albumin, transaminases, hyaluronic acid, TNF-α, IL-2 and MMP-2 and MMP-9 levels were given as mean and standard deviation. 32 Results Serum examinations. Levels of serum albumin (g/dl) were 4.3 ± 0.2 in the control group and 4.2 ± 0.2 in the Kurozu-M group. Levels of serum AST/ALT (IU/l) were 236 ± 67/206 ± 51 in the control group and 209 ± 64/183 ± 49 in the Kurozu-M group. There was no significant difference in the levels of albumin or AST/ALT between the two groups. Moreover, the levels of serum hyaluronic acid (ng/ml) were 37.8 ± 3.8 in the control group, and 35.1 ± 2.8 in the Kurozu-M group. There was no significant difference in the level of hyaluronic acid between the two groups. As regards the levels of cytokines, the levels of TNF-α (pg/ml) were 3.4 ± 1.7 in the control group and 3.1 ± 1.8 in the Kurozu-M group. Levels of IL-2 (U/ml) were 1.6 ± 0.6 in the control group and 1.4 ± 0.4 in the Kurozu-M group. There were no significant differences in the levels of either of the cytokines between the two groups. Histology and GSTP staining. In macroscopic examination of the resected liver, white tumors with a size of more than 10 mm at the major axis were apparent in all rats in the control group (Fig. 1). Such large tumors were not found in the Kurozu-M group. In microscopic examination of HE-stained liver sections, focal necrosis of liver cells and invasion of inflammatory cells in portal areas (corresponding to mild chronic hepatitis in humans) were found in both groups. However, fibrosis or piecemeal necrosis was not obvious. Moreover, there were no marked background changes of liver tissues in the two groups (Fig. 2 a and b). As for GST-P staining, the numbers of GST-P-positive foci were 12.2 ± 6.8 in the control group, and 10.7 ± 5.3 in the Kurozu-M group. There was no significant difference between the two groups. However, large HCC with a diameter of more than 10 mm was noted in liver in all animals of the control group. On the other hand, in the Kurozu-M group, no lesion of more than 3 mm in diameter was found in any liver. The size (mm) of GST-P-positive foci in the Kurozu-M group (0.89 ± 0.46) was significantly (p<0.001) reduced compared with the control (7.35 ± 5.31) (Fig. 3 and 4). MMP2 and MMP9 levels in tumor tissues. Total MMP-2 amounted to 6.7 ± 2.6 ng/mg protein in the control group and 3.6 ± 1.7 ng/mg protein in Kurozu-M group. The corresponding values for activated MMP-2 were 0.28 ± 0.12 ng/mg protein and 0.12 ± 0.06 ng/mg protein, respectively. Total MMP-9 amounted to 20.6 ± 4.6 ng/mg protein in the control group and 12.0 ± 2.7 ng/ mg protein in Kurozu-M group. The corresponding values for activated MMP-9 were 1.17 ± 0.38 ng/mg protein and 0.51 ± Fig. 1. Photograph of a representative resected liver tumor. Multiple white tumors were apparent in the control group. The diameter of the tumor shown, at the major axis, was 14 mm. doi: 10.3164/jcbn.10112 ©2011 JCBN