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Choi, Kim, Nam, Yun, Jeong, and Lee: Effect of hemin and zinc on experimental colon carcinogenesis in mice

Abstract

Excessive intake of red meat has been associated with colon carcinogenesis. The effect of hemin and zinc on colon carcinogenesis was investigated in male ICR mice. After acclimation for 1 week, five-week-old mice received three s.c. injections (0-2nd weeks of the experiment) of azoxymethane (AOM; 10 mg/kg b.w.) weekly and were treated by 2% dextran sodium sulfate (DSS) in drinking water for the next 1 week to induce aberrant crypt foci (ACF). Mice were fed on AIN-76A purified rodent diet for 6 weeks. There were three experimental groups; control, hemin, and hemin + zinc groups. The daily oral doses of hemin and zinc were 534 mg/kg and 55 mg/kg b.w., respectively. After staining colonic mucosa with 0.2% methylene blue, the ACF, aberrant crypts (AC), and polyps were counted. Lipid peroxidation in liver was evaluated by thiobarbituric acid-reactive substances (TBARS) assay. The numbers of AC, ACF, large ACF (i.e., ≥4 AC/ACF), and polyps in the hemin group were higher than those in the control group. In hemin + zinc group, the numbers of AC, ACF, large ACF were significantly lower compared to the hemin or control groups (p < 0.05), and the number of polyps was also significantly lower compared to that in the hemin group (p < 0.05). The TBARS level in the livers of the hemin + zinc group was significantly lower than that of the hemin group (p < 0.05). These results suggest that hemin enhances colon carcinogenesis and that co-treatment with zinc can protect against the induction of colon carcinogenesis.

INTRODUCTION

Meat is an important food with respect to the nutrients needed for humans. However, excessive red meat intake may be related to colorectal cancer (CRC) [10]. According to the World Health Organization, CRC had the second highest cancer incidence in Korean men and the third highest incidence in Korean women in 2014 [22].
In red meat, iron is combined with protoporphyrin IX which is referred to as heme iron. It has been suggested that hemin [chloroprotoporphyrin IX iron (III)], a ferric form of heme, may be involved in the link between the risk of CRC and red meat intake [19]. Iron can act as a catalyst for free radical formation, therefore excessive intake of red meat can lead to the production of reactive oxygen species (ROS) [8]. ROS can be related to the induction of CRC by attacking the lipid membrane and DNA. In addition, heme iron was reported to induce lipid peroxidation and colonic cytotoxicity [2]. Malondialdehyde (MDA) and 4-hydroxynoneal (4-HNE) are the major aldehyde products of lipid peroxidation [12]. MDA reacts with DNA, and the DNA can then become mutagenic [14]. The 4-HNE product is also mutagenic. Although the mutagenic level of 4-HNE is weaker than the mutagenic level of MDA, 4-HNE is the main toxic product of lipid peroxidation. Moreover, 4-HNE induces apoptosis and necrosis of human colon carcinoma [1]. Heme iron also catalyzes the formation of apparent total nitroso compounds, which can cause DNA damage or mutation.
Azoxymethane (AOM) can initiate CRC by inducing aberrant crypt foci (ACF), and ACF have been defined as precancerous lesions of the colon in experimental models as well as humans [7]. ACF develop into an advanced adenoma with high-grade dysplasia and progress to invasive cancer [21]. As ACF are pre-neoplastic lesions and distinguishable from normal crypts morphologically, ACF can be used as a biomarker of colon carcinogenesis.
While meat can induce colon carcinogenesis, there are some agents that can suppress colon carcinogenesis, including zinc, which binds to tumor suppressor p53 as an important cofactor [4]. In addition, zinc can induce the formation of metallothionein which protects against free radicals [17], and zinc metabolism is closely related to metallothionein. An increase in the amount of available zinc induces the synthesis of thionein through the action of zinc and leads to the formation of metallothionein [11]. Metallothionein is involved in the regulation of the permeability of the inner mitochondrial membrane and affects the regulation of mitochondrial ROS production [20]. Thus, a reduction of metallothionein can result in an increase in ROS and impaired mitochondrial function [9].
There are some studies into the effects of dietary heme in CRC models and the suppressive effect of zinc on colon carcinogenesis. However, the correlation between zinc and hemin in a CRC animal model was not investigated. In this study, we investigated whether hemin enhances the formation of pre-neoplastic lesions in colonic mucosa and zinc decreases hemin-promoted pre-neoplastic lesions induced by AOM/DSS in mice.

MATERIALS AND METHODS

Experimental materials

AOM, hemin, and zinc were obtained from Sigma-Aldrich Inc. (St. Louis, MO, USA). Dextran sodium sulfate (DSS; molecular weight 36,000~50,000) was obtained from MP Biomedical Inc. (USA).

Animals and Experiment diets

Male ICR mice (4 weeks old) were obtained from Orient Bio (Seongnam, Korea) and housed in an isolating polycarbonate cage (5 mice/cage). The temperature and relative humidity of the housing facility were set at 20 ± 2°C and 50 ± 20%, respectively. Light and dark cycles were set at 12 h each, and the intensity of illumination was maintained at 150-300 lux. The AIN-76A purified diet was obtained from Central Laboratory Animal Inc. (Seoul, Korea). During the experimental periods, diets and litter were used after sterilization, and the animal experiment was conducted in compliance with "Guide for Care and Use of Laboratory Animals" of Chungbuk National University and with the approval of IACUC. Body weight (b.w.), food consumption, and water intake were recorded weekly.

Experiment design

After 45 male ICR mice (4 weeks old) were acclimated to their cages for 1 week, the five-week-old mice were divided into three groups: 1) control group, 2) hemin group, and 3) hemin + zinc group. Both AIN-76A purified diet and distilled water were supplied to all mice. Hemin (534 mg/kg b.w.) and zinc (55 mg/kg b.w.) mixed with 1% CMC were orally administrated as appropriate. AOM (10 mg/kg b.w.) was subcutaneously injected to mice three times, at 0, 1st and 2nd weeks of the experimental period, to induce the formation of pre-neoplastic lesions in mouse colon. Distilled water with 2% DSS was provided for 7 days after the 2nd week of the experimental period. The total experimental period was 6 weeks.

Sample collection

After mice were anesthetized and underwent laparotomy, blood was collected from the abdominal aorta by using a syringe. The liver and entire large intestine were harvested and the liver was frozen in liquid nitrogen (-196°C). The large intestine was washed with saline and fixed in 10% neutral buffered formalin.

ACF, AC and polyp count

The formalin-fixed colonic tissues were stained in 0.2% methylene blue solution for a few seconds. Subsequently, the total number of ACF and AC was counted under a microscope (×100), and the number of polyps was counted by an unassisted eye. The total number of ACF, AC, and polyps were recorded as numbers/colon.

Thiobarbituric acid-reactive substances (TBARS) assay

The TBARS assay was conducted for determination of malondialdehyde (MDA) level in liver. After placing a liver sample in a 2 mL tube, 200 μL of PBS was added. Then, the sample was homogenized; the centrifuge process for the homogenized sample was conducted at 4°C and 13,000 rpm for 15 min. After centrifugation, the supernatant liquid of homogenized sample was placed in a conical tube. Then, solutions were used to measure the level of MDA. Sample (100 μL), 100 μL of 8.1% sodium dodecyl sulfate (SDS) solution, and 200 μL of 20% acetic acid solution were mixed together, after which 100 μL of 0.75% 2-thiobarbituric acid (TBA) solution were added, followed by vortexing and spinning down. Incubation at 95°C was then conducted for 30 min. Afterward, the mixed samples were placed on ice for 10 min, followed by centrifugation at 10,000 rpm for 3 min. Finally, the samples were placed in a 96-well plate, and the optical density of the supernatant liquid was measured at 532 nm. Protein quantitative analysis was conducted by using a DC Protein kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA) that was applied by using the Lowry method.

Statistical analysis

Data were expressed as means ± standard deviation (SD). Data were analyzed by applying one-way ANOVA followed by Tukey’s post-test and Kruskal-Wallis test. Significant differences were determined at the p < 0.05 or p < 0.01 level.

RESULTS

Changes in body weight

Mean mouse body weights in the experimental groups increased with time. Although the mouse body weight in hemin + zinc group appeared to be low during the experimental period, there was no significance to the differences in the mean body weights of the experimental groups (Fig. 3).

Total aberrant crypt foci number

Compared to the ACF number in the control group (43.3), the number of ACF in the hemin group (48.4) was slightly higher (Fig. 4). However, the hemin + zinc group had a significantly lower ACF number (25.5) compared to that in the control (p < 0.05) and hemin (p < 0.05) groups (Fig. 4).

Total aberrant crypt number

Compared to the number of AC in the control group (63.1), the hemin group had a higher number (70.9), but the difference was without significance (Fig. 5). By contrast, the number of AC in the hemin + zinc group (39.3) was significantly lower than those in the control and hemin groups (p < 0.05) (Fig. 5).

Total number of large aberrant crypt foci

Compared to the number of large ACF (i.e., ≥4 AC/ACF) in the hemin + zinc group (2.5), the numbers of large ACF in hemin (4.4) and control (3.6) groups were significantly higher (p < 0.05) (Fig. 6).

Polyp numbers

Compared to the number of polyps in the control group (9.1), the number of polyps in the hemin group (10.6) was slightly increased (Fig. 7). However, the number of polyps in the hemin + zinc group (6.8) was significantly lower than that of the hemin group (p < 0.05) (Fig. 7).

TBARS levels in liver

The TBARS level in the livers of the hemin group (15.21 nM/mg protein) was significantly higher than those in the control (12.43 nM/mg protein) and hemin + zinc (12.44 nM/mg protein) groups (p < 0.05) (Fig. 8).

DISCUSSION

This experiment was designed to determine whether hemin enhances colon carcinogenesis and whether zinc reduces colon carcinogenesis which were evaluated by examining colonic ACF induced by AOM+DSS in male ICR mice. The mice were fed on AIN-76A purified diets and distilled water. Hemin (534 mg/kg b.w.) and zinc (55 mg/kg b.w.) mixed with 1% CMC were administrated to each mouse daily for 6 weeks.
Previously, ACF have been defined as putative precancerous lesions of the colon in both experimental models and humans [18]. ACF were identified by microscope after methylene blue staining. They have altered luminal openings and are larger than adjacent normal crypts [3]. In previous study, hemin affected the formation of ACF in rat and a high-hemin diet increased the number of total foci per colon [16]. In the present study, the number of ACF in the hemin group was significantly higher than that in the control group. On the other hand, it has been reported that zinc, by its antioxidant activity, reduces the formation of ACF [5]. In the present study, the number of ACF in hemin + zinc group was significantly less than that in the hemin group.
The ACF were divided into two groups; large ACF (≥4 AC/ACF) and small ACF (≤3 AC/ACF) because large ACF are likely to have a greater tumorigenic potential [13]. Analysis revealed that the number of large ACF in the hemin + zinc group was significantly smaller than that in the hemin group. These results suggest that zinc might contribute to decreasing tumorigenic potential in an AOM/DSS animal model.
Polyps are an abnormal growth of tissue, and a colon polyp is a small clump of cells that forms on the lining of the colon; in addition, they have malignant potential [23]. In this study, the number of polyps in the hemin + zinc group was significantly lower than that in the hemin group. This result suggests that zinc might reduce the malignant potential of heme iron in colon cancer.
Oxidative stress is associated with damages to cellular structures, which can lead to the initiation and promotion of carcinogenesis [6]. Moreover, oxidative stress is one of the factors that can initiate lipid peroxidation and MDA is a product of lipid peroxidation. Zinc is related to preventing the formation of highly reactive oxidants [15]. In this study, the amount of MDA in the hemin + zinc group was significantly smaller than the amount of MDA in the hemin group. These results suggest that zinc might inhibit lipid peroxidation in the body compartments due to its antioxidant activity.
In conclusion, there was a tendency for hemin to induce the formation of ACF and polyps. Hemin also stimulated lipid peroxidation in the liver. However, treatment with zinc decreased the formation of ACF and polyps and inhibited lipid peroxidation. Based on these results, it is suggested that zinc can protect against colon carcinogenesis that has been promoted by hemin.

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Fig. 1.
Experimental design for colon carcinogenesis in mice. AOM; azoxymethane (10 mg/kg b.w. in saline, S.C., weekly 3 times), DSS; dextran sodium sulfate (2% in drinking water for a week), Hemin and zinc mixed with carboxymethyl cellulose were orally treated every day).
jpvm-2019-43-1-6f1.tif
Fig. 2.
Typical aberrant crypt foci stained with methylene blue (x100). A: aberrant crypt focus with 1 crypt, B: aberrant crypt focus with 2 crypts, C: aberrant crypt focus with 3 crypts.
jpvm-2019-43-1-6f2.tif
Fig. 3.
Change in the body weights of mice in experimental groups. Each data point indicates the mean ± SD (n=15).
jpvm-2019-43-1-6f3.tif
Fig. 4.
The number of colonic aberrant crypt foci (ACF) in experimental groups. Each bar means the mean ± SD (n=15). *Significantly different from control group (p<0.05). #Significantly different from hemin group (p<0.05).
jpvm-2019-43-1-6f4.tif
Fig. 5.
The number of colonic aberrant crypt (AC) in experimental groups. Each bar means the mean ± SD (n=15). *Significantly different from control group (p<0.05). #Significantly different from hemin group (p<0.05).
jpvm-2019-43-1-6f5.tif
Fig. 6.
The number of large colonic aberrant crypt foci (ACF) (≥4 AC/ACF) in experimental groups. Each bar means the mean ± SD (n=15). *Significantly different from control group (p<0.05). #Significantly different from hemin group (p<0.05).
jpvm-2019-43-1-6f6.tif
Fig. 7.
The number of polyps in the colonic mucosa in experimental groups. Each bar means the mean ± SD (n=15). #Significantly different from hemin group (p<0.05).
jpvm-2019-43-1-6f7.tif
Fig. 8.
Effect of hemin and hemin + zinc on lipid peroxidation in the liver of mice. *Significantly different from control group (p<0.05). #Significantly different from hemin group (p<0.05).
jpvm-2019-43-1-6f8.tif
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