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ISSN : 2287-7991(Print)
ISSN : 2287-8009(Online)
Journal of the Preventive Veterinary Medicine Vol.36 No.1 pp.36-46
DOI :

Effect of dietary zinc on experimental induction of colonic preneoplastic lesions in mice fed low iron diet

Beom Jun Lee1,†, Bong Su Kang1, Hyunji Park1, Ja Seon Yoon1, Dang-Young Kim1, Jae-Hwang Jeong2, Eun-young Kim3, Sang Yoon Nam1, Young Won Yun1, Jong-Soo Kim1,‡
1College of Veterinary Medicine and Research Institute of Veterinary Medicine, Chungbuk National University
2Department of Biotechnology and Biomedicine, Chungbuk Province College, 3Department of Food Service & Culinary Management, The Graduate School of Kyonggi University
Received 21 Fabruary, 2012, Accepted 9 March, 2012

Abstract

Both iron-deficient and zinc-sufficient diets have been known to be associated with a decreased risk of colon cancer. We investigated that effect of dietary zinc on the formation of colonic aberrant crypt foci (ACF) induced by azoxymethane (AOM) followed by dextran sodium sulfate in iron-deficient mice. Five-week old ICR mice were acclimated for 1 week and fed on iron-deficient diet (4.50 ppm iron) with three different zinc levels (0.01, 0.1, and 1.0 ppm) for 12 weeks. The total number of aberrant crypt (AC) and ACF was measured in the colonic mucosa after methylene blue staining. The total ACF numbers of low Zn (LZn), medium Zn (MZn) and high Zn (HZn) diet groups were 10.00 ± 2.67, 8.78 ± 3.12, and 7.96 ± 2.44, respectively and there were no significant differences among the groups. However, the total AC numbers of HZn (27.07 ± 3.88) and MZn (26.39 ± 5.59) diet groups were significantly low compared to LZn (22.57 ± 5.09) diet group (p<0.01). Cytosolic SOD activity was the highest in LZn diet group. But thiobarbituric acid-reactive substances level in liver was also the highest in LZn diet group compared to other groups. There is no difference in cell proliferation in mucous membrane among the groups, while apoptotic positive cells were increased in the HZn diet group. The high zinc diet exhibited decreased β-catenin-stained areas on the mucous membrane of colon compared to the LZn or MZn diet group. These findings indicate that dietary zinc might exert a modulating effect on development of ACF/AC in the mice with iron-deficient status.

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Introduction

 Colorectal cancer (CRC) is one of the leading causes of cancer death in western countries and in the developed countries [11, 29]. In Korea, the incidence and mortality of CRC have gradually increased in the last decade, becoming the fourth leading cause of cancer deaths [13].

 Several chemical-induced experimental models of colon cancer have been used for evaluation of chemopreventive agents or mechanism studies. The most commonly used experimental colon carcinogens are 1,2-dimethylhydrazine (DMH) and its metabolite azoxymethane (AOM). The DMH or AOM-induced colon cancer shares many similarities to human sporadic colon cancer, including the response to some promotional and preventive agents [4]. The ultimate carcinogenic metabolite of DMH causes methylation of the DNA bases of various organs, including epithelial cells of colonic crypts [19]. Thus, this results in a loss of colonic cells by apoptosis, an increase in proliferation and mutation of colonic epithelial cells [3]. Dextran sodium sulfate (DSS) induces colonic inflammation in rodents and promotes colon carcinogenesis, thereby being used for experimental animal CRC model [21]. AOM treatment during or after DSS administration did not generate a significant number of colonic neoplasm in mice, while DSS administration after AOM treatment could induce a number of colonic neoplasm [29].

 By reason of relatively lengthy developmental process of tumors, preneoplastic lesions can be used as biomarkers for assessing the risk of developing colon cancer or for identifying modulators of colon carcinogenesis in short-term studies [2]. Aberrant crypt foci (ACF) were first reported by Bird in 1987 on a method for directly visualizing altered crypts induced by carcinogens in the intact colon. ACF are the first lesion in the development of colon cancer that can be identified microscopically on methylene blue-stained colon mucosa. ACF are characterized by enlarged and elevated dark crypts and increases pericryptal space [2].

 β-catenin, acting as a structural protein and transcriptional activator-mediating Wnt signaling, is mostly localized at membranes of cell-to-cell border [22]. β-catenin mutation was frequently observed in AOM-induced colon cancer in rodents [9, 10, 28]. When β-catenin mutation occurs, the protein is localized to the cytoplasm or nucleus [12]. Crypts with increased β-catenin expression have been suggested as a more relevant biomarker of colon cancer than ACF [18, 32].

 Iron and zinc are micronutrients that were associated with many diseases including cancers. Many studies reported that iron may induce mutagenic effects mediated through free-radical generation or tumor promotion through nutritional mechanisms [8, 26]. As a transition metal, iron is capable of generating free radicals participating in lipid peroxidation reactions. Such reactions are thought to lead to DNA damage and, in some cases, neoplasia [31].

 Zinc is vital for the function of all living systems. It is an essential trace element important for the stabilization and function of numerous metalloenzymes, especially copperzinc superoxide dismutases (CuZnSOD) [25]. It is demonstrated that zinc plays an important role in the metabolism and interaction of malignant cells [7, 23]. Zinc has a stabilizing effect on membranes possibly by displacing bound transition metal ions and thereby preventing peroxidation of membrane lipids [1]. Moreover, it was founded that zinc had a protective effect against colonic preneoplastic development induced by DMH in rats [5]. However, there is doubt that dietary zinc intake in combination with irondeficient status may have the protective effect against colon carcinogenesis.

 The aim of this study was to identify how dietary zinc had an effect on colonic preneoplastic lesion induced by AOM and DSS in ICR mice fed iron-deficient diet.

Materials and Methods

1. Materials

 AOM was purchased from the Sigma Chemical Company (St Louis MO, USA) and DSS (molecular weight 36,000~ 50,000) was manufactured by MP biomedicals (Solon, OH, USA).

2. Animals

 Male ICR mice (5 weeks old) were obtained from SLC Inc. (Shizuoka, Japan), housed in polycarbonate cages (5 mice/cage). The temperature and relative humidity were maintained at 20 ± 2 ℃ and 50 ± 20%, respectively. Light and dark cycle was at 12 h : 12 h. Mice were allowed access to AIN-93G purified rodent diet (Dyets, Inc., Bethlehem, USA) and water ad libitum. The animal experiment was conducted in accordance with "Guide for care and use of laboratory animals" of Chungbuk National University. After one week of acclimatization, the animals were then taken off chow feed, and fed iron-deficient diet with three levels of zinc. During the experimental period, weekly body weights and feed consumptions were recorded.

3. Experimental designs

 There were four experimental groups including control group and three AOM/DSS treatment groups. Twenty mice were assigned to each treatment group, while ten mice to control group. Mice were fed a low Fe diet (LFe, 4.5 ppm Fe) with three different Zn concentrations including 0.01 ppm (LZn), 0.1 ppm (MZn) and 1.0 ppm (HZn) (Fig. 1). The mice in the control were fed a LFe+MZn diet. The mice in three AOM/DSS treatment groups were fed LFe+LZn, LFe+MZn or LFe+HZn diet.

4. Experimental diets and carcinogen treatment

 The AIN-93G purified rodent diet contained 20% casein, 0.3% L-cystine, 39.7% cornstarch, 13.2% dyetrose, 7% soybean oil, 0.0014% t-butylhydroquinone, 5% cellulose, 3.7% mineral mix, 0.9% ferric citrate, 1% vitamin mix, 0.25% choline bitartrate (Table 1). LZn, MZn and HZn diets contained 0.02%, 0.2% and 2% zinc carbonate, respectively. These diets were fed to animals for 12 weeks.

Fig. 1. Experimental design for colon carcinogenesis in mice fed a low Fe diet. AOM : Azoxymethane (10㎎/㎏ body weight in saline, I.P., weekly 3 times), DSS : Dextran sodium sulfate (2% drinking water for a week). LFe : 4.5 ppm Fe, LZn : 0.01 ppm Zn, MZn : 0.1 ppm Zn and HZn : 1.0 ppm Zn.

 Mice were treated intraperitoneally with AOM (10㎎/㎏ body weight) in saline weekly for the first 3 weeks of experimental period. Additionally, mice received 2% DSS in the drinking water for 7 days after AOM treatment. The mice in the control group were received an injection of saline instead of AOM (Fig. 1).

5. Sample collection

 At 12 weeks, all mice were sacrificed. Before sacrifice, final body weights were measured. After laparotomy, blood was collected by a syringe from the abdominal aorta and immediately transferred into tubes containing K3-EDTA and serum separator tubes (Vacutainer, Becton Drive Franklin Lakes, NJ, USA). The liver, spleen, kidneys, lung, stomach, small intestine and entire large intestine were harvested. One fifth of liver were washed with saline, blotted dry, weighted and then frozen in liquid nitrogen. A half of the large intestine from cecum to anus was longitudinally opened, flushed with saline, and fixed in 10% neutral buffered formalin. The other half was washed with saline, blotted dry and then frozen in liquid nitrogen.

6. Blood analysis

 Blood samples in EDTA tubes were used for analysis of complete blood cell count with Abbott CellDyn-3500 (Abbott Laboratories, Chicago, IL, USA).

7. Histopathological examination

 The tissues fixed in 10% neutral buffered formalin were paraffin-embedded, cut to multiple 4-㎛ sections, and stained with hematoxylin and eosin for histopathological examination under a light microscope (Olympus, Tokyo, Japan).

8. AC and ACF counts

 The colon was removed and washed thoroughly with 0.85% NaCl solution and fixed with 10% neutral phosphate buffered formalin. The colon was then stained with 0.5% methylene blue solution for 30 sec in order to identify AC and ACF. The total numbers of ACF and AC in each focus were counted under a microscope (40~100×). ACF were identified with the following morphological characteristics: 1) the enlarged and elevated crypts than normal mucosa and 2) increased pericryptal space and irregular lumens [15].

Table 1. Composition of the diets with low Fe groups

9. SOD activity assay

 Superoxide dismutase (SOD) activity was measured using a commercial assay kit (Cayman, MI, USA) according to manufacturer’s instructions. This kit utilizes a tetrazolium salt for the detection of superoxide radicals generated by xanthine oxidase and hypoxanthine. One unit of SOD was defined as the amount of enzyme needed to produce 50% dismutation of superoxide radicals. To separate CuZnSOD, liver homogenate was centrifuged at 1,500 × g for 10 min and the supernatant was centrifuged at 10,000 × g for 15 min at 4℃. The resulting 10,000 × g supernatant, containing cytosolic SOD (CuZnSOD), was collected and analyzed for SOD activity using the SOD activity assay kit [14]. The data were represented as SOD unit/㎎ protein.

10. Determination of lipid peroxidation in liver

 The amounts of malondialdehyde (MDA) contained in the tissue homogenate were measured using a thiobarbituric acid reactive substance (TBARS) assay kit (Cayman, MI, USA) according to manufacturer’s instructions. In brief, MDA-TBA adduct was formed by the reaction of MDA and TBA under high temperature (90~100℃) and acidic conditions. The MDA-TBA adduct appeared as pink chromogen and was measured colorimetrically at 532 nm. The data were represented as MDA ㎛/㎎ protein.

11. Immunohistochemistry of PCNA and β-catenin

 Immunohistochemistry for proliferating cell nuclear antigen (PCNA) and β-catenin was performed on 4-㎛ formalin-fixed, paraffin-embedded distal colon sections. The sections were subjected to deparaffinization and hydration prior to quenching of endogenous peroxidase activity (3% H2 O2  in methanol for 15 min). The sections were incubated for 60 min with the primary anti-proliferating cell nuclear antigen mouse monoclonal antibody (diluted 1:200, Santa Cruz, CA, USA) and anti-β-catenin rabbit polyclonal antibody (diluted 1:200, Santa Cruz, CA, USA). Slides were processed with the ABC reagent from Vectastain Elite kit (Vector Laboratories, Burlingame, CA, USA) and developed with the 3,3’-diaminobenzidine, tetrahydrochloride (DAB) chromogen. The sections were counter- stained with mayer hematoxylin. Twenty fields, randomly selected from each slide, were analyzed at 100 × magnification. The numbers of nuclei with positive reactivity for PCNA immunohistochemistry were counted in a total of 3 × 100 cells in 3 different areas of the colonic cancer and expressed as a percentage (mean ± S.D.) [30]. The expression of β-catenin was graded by intensity of nuclear staining accompanied by loss of membranous staining as negative (localization limited to membrane), focal positive (positive cells clustered in focal area) and diffuse positive (positive cells distributed diffusely), described by Kobayashi et al. [12]. Cytoplasmic immunoreactivity was not considered in the present study because the expression was variable and not clearly related to the shift from membranous to nuclear staining.

12. TUNEL assay

 Levels of apoptosis in distal colon tissue were determined using the TdT-mediated dUTP nick-end labeling (TUNEL) method. The 4-㎛ formalin-fixed, paraffin-embedded tissue sections from the distal colon were processed according to manufacturer's instructions for the ApopTag peroxidase in situ apoptosis detection kit (TUNEL; Vector Laboratories, Burlingame, CA, USA). The numbers of nuclei with positive reactivity for TUNEL assay were counted in a total of 3 × 100 cells in 3 different areas of the colonic mucosa and expressed as a percentage (mean ± S.D.) [30].

13. Statistical analysis

 Data were expressed as means ± standard deviation (S.D.). The data were analyzed with SAS version 9.1.2 software (SAS Institute, Cary, NC, USA). Data except ACF, PCNA and TUNEL assay were analysed by one-way analysis of variance and a significant difference among treatment groups were evaluated by Tukey-Kramer test. Data of ACF, PCNA and TUNEL assay were analyzed by Dunn’s multiple comparison test after Kruskal-Wallis’ nonparametric ANOVA. For all comparisons, p values < 0.05 and 0.01 were considered statistically significant.

Results

1. Final body weights of mice and blood analysis

 All AOM/DSS-treated groups showed a significantly lower body weight compared with control group at 12 weeks (Table 2) (p<0.01). However, there were no significant differences among AOM/DSS-treated groups at 12 weeks (Table 2).

 AOM/DSS treatments lowered red blood cell count (RBC), hemoglobin (Hb) and hematocrit (HCT) values significantly or slightly compared the control (Table 2). HZN group showed significantly high levels in white blood cell (WBC) RBC, HB, HCT, mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) compared LZn group (Table 2).

2. Total AC and ACF counts

 As shown in Fig. 2, the total AC number of HZn group (22.57 ± 5.09) was significantly low compared with MZn (26.39 ± 5.59) or LZn (27.07 ± 3.88) group (p<0.05). There were no significant differences in the total ACF numbers of LZn (10.00 ± 2.67), MZn (8.78 ± 3.12) and HZn (7.96 ± 2.44) group (Fig. 2).

3. Counts of ACF with various numbers of AC

 MZn (6.11 ± 3.39) and HZn groups (6.43 ± 2.61) showed a decrease in the number of small ACF (≤3 AC/ ACF) compared with LZn (7.27 ± 2.99) group but there were no significant differences among them (Fig. 3).

 The number of large ACF (≥4 AC/ACF), which is suggested to possess a greater tumorigenic potential [16], was significantly low in HZn group (1.52 ± 0.59 ACF/colon)compared with LZn group (2.73 ± 0.88 ACF/colon) or MZn group (2.67 ± 0.91 ACF/colon) (p<0.01) (Fig. 3).

Table 2. Final body weights and differential blood cell counts in ICR mice fed the low Fe diet

4. SOD activity in the liver

 SOD activities in the liver were 10.38 ± 1.89 U/㎎ pro-tein for the control group, 16.59 ± 2.27 U/㎎ protein for the LZn group, 11.70 ± 2.44 U/㎎protein for the MZn group, and 10.79 ± 1.91 U/㎎ protein for the HZn group, respectively (Fig. 4). LZn group showed a significantly increased activity of SOD compared with the other groups (p<0.05).

Fig 2. Effect of zinc on colonic aberrant crypt and aberrant crypt foci formation in mice fed the low Fe diet. AC: aberrant crypt, ACF: aberrant crypt foci, AOM: azoxymethane,DSS: dextran sodium sulfate, LFe: low Fe diet (4.5 ppm), LZn: low Zn diet (0.01 ppm), MZn: medium Zn diet (0.1 ppm), HZn: high Zn diet (1.0 ppm). Each bar represents mean ± S.D. a,b Means with different letter were significantly different (p<0.05).

Fig 3. Effect of zinc on colonic aberrant crypt formation in mice fed the low Fe diet. AC: aberrant crypt, ACF: aberrant crypt foci, AOM: azoxymethane, DSS: dextran sodium sulfate, LFe: low Fe diet (4.5 ppm), LZn: low Zn diet (0.01 ppm), MZn: medium Zn diet (0.1 ppm), HZn: high Zn diet (1.0 ppm). Each bar represents mean ± S.D. x,y Means with different letter were significantly different (p<0.01).

Fig 4. Effect of zinc on superoxide dismutase activity in the liver of mice fed the low Fe diet. AOM: azoxymethane, DSS: dextran sodium sulfate, LFe: low Fe diet (4.5 ppm), LZn: low Zn diet (0.01 ppm), MZn: medium Zn diet (0.1 ppm), HZn: high Zn diet (1.0 ppm). Each bar represents mean ± S.D. a,b Means with different letter were significantly different (p<0.05).

5. Lipid peroxidation levels in liver

 TBARS levels in the liver were 0.88 ± 0.12 ㎛/㎎ protein for the control group, 1.98 ± 0.44 ㎛/㎎ protein for the LZn group, 1.07 ± 0.31 ㎛/㎎ protein for the MZn group, 1.21 ± 0.33 ㎛/㎎ protein for the HZn group, respectively (Fig. 5). The LZn group showed a significantly increased level of TBARS compared with the other groups (p<0.05).

6. Histopathology

 Tissue section of control group displayed normal colonic architecture with no sign of apparent abnormality. However, there was thickening of epithelium and mixed conformation of dysplasia and hyperplasia in AOM/DSS-treated groups (Fig. 6).

7. Changes in proliferation and apoptosis

 The PCNA and TUNEL stains were carried to observe changes in proliferation and apoptosis (Fig. 7 and 8). As shown in Table 3 and Fig. 7, the levels of PCNA in LZn and MZn groups were significantly up-regulated compared with the control group (p<0.05), but HZn group had no significant increase in the PCNA level compared with the control group. As shown in Table 3 and Fig. 8, the mice that were fed HZn diet exhibited a significantly higher count of brown-color apoptotic bodies compared with the control, LZn, or MZn group (p<0.05).

Fig 5. Effect of zinc on lipid peroxidation in the liver of mice fed the low Fe diet. AOM: azoxymethane, DSS: dextran sodium sulfate, LFe: low Fe diet (4.5 ppm), LZn: low Zn diet (0.01 ppm), MZn: medium Zn diet (0.1 ppm), HZn: high Zn diet (1.0 ppm). Each bar represents the mean ± S.D. a,b Means with different letter were significantly different (p<0.05).

Fig. 6. Histopathology of colonic epithelium in mice fed the low Fe diet by H&E staining. (A) Control group, (B) AOM+DSS+LZn group, (C) AOM+DSS+MZn group, (D) AOM+DSS+HZn group. AOM/DSS treatment induced hyperplasia of epithelium and mucous membrane.

Table 3. Effect of Zn on cell proliferative and apoptotic indices of mice fed the low Fe diet

Fig. 7. Immunohistochemistry of PCNA in the colon of mice fed the low Fe diet. (A) Control group, (B) AOM+DSS+LZn group, (C) AOM+DSS+MZn group, (D) AOM+DSS+HZn group.

Fig. 8. TUNEL assay for apoptotic nuclei in distal colon sections of mice fed the low Fe diet. (A) Control group, (B) AOM+DSS+LZn group, (C) AOM+DSS+MZn group, (D) AOM+DSS+HZn group.

8. Immunohistochemistry of β-catenin

 A variation of the nuclear staining was detected in the epithelial cells of colonic mucosa (Fig. 9). The colonic mucosa of control group did not showed the nuclear localization of β-catenin (Fig. 9A). The strong cytoplasmic and nuclear β-catenin immunoreactivity was observed on the epithelial cells of LZn and MZn group (Fig. 9B & 9C). On the other hand, the nuclear β-catenin expression in HZn group was weaker than LZn and MZn groups (Fig. 9D).

Fig. 9. Immunohistochemistry of β-catenin on distal colon sections of mice fed the low Fe diet. (A) Control group, (B) AOM+DSS+LZn group, (C) AOM+DSS+MZn group, (D) AOM+DSS+HZn group.

Discussion

 Colon is the most common site for malignancies of gastrointestinal tract and colorectal cancer is the second leading cause of cancer death in male of developed countries [11]. Moreover, colon cancer does not appear to be a result of aging, but is intrinsically associated with dietary pattern. Red and processed meat consumption promotes the risk of colorectal cancer, and this promotion is found to correlate closely with dietary Fe [6]. Numerous interests have been raised recently about food components, with the aim of corroborating its preventive or carcinogenic effects. The current study elucidated how low iron and various zinc levels in diet influence on AOM/DSS-induced colonic preneoplastic lesions in the mouse model of colon carcinogenesis.

 In the present study, the low Fe diet was formulated as the concentration of 4.5 ppm Fe that was only about 10% of that medium Fe diet (45 ppm Fe). Fe analysis in liver confirmed that low dietary Fe was supplied uniformly regardless of AOM/DSS treatment or Zn concentration. Commonly, it is known that Fe deficient status induce microcytic hypochromic anemia. Fe deficiency with carcinogen treatment is associated enhancement of lipid peroxidation like Fe overloaded status [20].

 ACF are putative preneoplastic lesions that have been detected in human colon cancer and experimental animals treated with chemical carcinogen. ACF can be identified microscopically on the surface of the whole mount colon mucosa after methylene blue staining. They are distinguished from normal crypts by their larger size, darker staining and increased pericryptal space [2]. In previous study, Dani et al. [5] reported that zinc inhibited the formation of ACF suggesting the potential of zinc in suppressing the progression of preneoplasia to malignant neoplasia and the suppressing effect of zinc could be explained by its putative antioxidant activity. In present study, mice without AOM/DSS treatment showed no evidence of ACF formation in the colon. The AC numbers of HZn groups was founded to be significantly low compared with LZn groups. In addition, the number of large ACF (≥4 AC/ACF), which is suggested to possess a greater tumorogenic potential [16], was significantly decreased in HZn groups compared with LZn groups. These results suggest that dietary supplement of zinc induces an inhibition of preneoplastic lesion formation.

 Colon carcinogenesis is a pathological consequence of persistent oxidative stress, leading to DNA damage and multiple genetic changes that may be caused by overproduction of reactive oxygen species in cancer-related genes [3]. In this study SOD was measured to identify antioxidant status in chemically induced colon carcinogenesis. CuZnSOD has been shown to have a protective effect against lipid peroxidation [27]. The activity of SOD in LZn group was higher than MZn or HZn group. However, LZn group showed the highest level of TBARS compared with MZn or HZn group. These findings may indicate that LZn diet could affect SOD activity/㎎ protein but the LZn diet could not the entire levels of SOD in the liver or body, thereby the LZn group showed a high TBARS level in the liver compared the other group. These results implied that high dietary zinc is an effective means of reducing lipid peroxidation and has a possible protective action on colon carcinogenesis.

 PCNA, a marker of cell proliferation, is a non histone nuclear protein and associated with DNA synthesis phase of the cell cycle [24]. In present study, proliferative index of control group was significantly decreased compared with AOM/DSS-treated groups. But HZn group showed no significant increase compared with the control group. From these results, HZn diet might ameliorate the AOM/DSSpromoted proliferation of colonic epithelial cells.

 TUNEL assay was performed in order to confirm the association between Zn and apoptosis of cells. The epithelium of the mice fed HZn diet represented a markedly higher count of apoptotic bodies compared control and other AOM/DSS-treated groups. These results indicate that high dietary zinc might induce upregulation of programmed cell death.

 β-catenin is a important factor in Wnt signal transduction and thereby regulating transcription of genes related to proliferation and development of cells [17, 18]. In colon carcinogenesis, β-catenin accumulates in nucleus and/or cytosol. Takahashi et al. [28] reported that alteration of β- catenin may play an important role in causing early dysplastic change. In colonic epithelial cells of control group, β-catenin is mostly localized at membranes of cell-to-cell border like normal epithelium of colonic mucosa. LZn and MZn groups showed an increase of nuclear β-catenin expression compared with HZn groups. Especially in LZn group, strong β-catenin expression was seen in the nucleus and cytoplasm. These results indicated that dietary supplement of zinc repressed β-catenin mutation.

 In conclusion, these findings indicate that dietary Zn might exert a protective effect on colon carcinogenesis in ICR mice fed iron deficient diet, resulting from inhibition of AC formation, lipid peroxidation, β-catenin mutation, and proliferation in colonic mucosa.

Acknowledgements

 This work was supported by National Research Foundation Grant funded by the Ministry of Education, Science and Technology (NRF-2008-313-E00628).

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