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    Effect of Dietary Cholic Acid on Apoptosis and Proliferation of Large Intestinal Epithelial Cells in Irradiation-Exposed Rats

    2010-09-06 03:19:48XUHongISHIZUKASatoshiSONGHuanlu
    食品科學(xué) 2010年19期
    關(guān)鍵詞:膽酸盲腸大腸

    XU Hong,ISHIZUKA Satoshi,SONG Huan-lu

    (1. Beijing Key Laboratory of Food Flavor Chemistry, School of Chemical and Environmental Engineering, Beijing Technology and Business University, Beijing 100048, China;2. Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan)

    Effect of Dietary Cholic Acid on Apoptosis and Proliferation of Large Intestinal Epithelial Cells in Irradiation-Exposed Rats

    XU Hong1,ISHIZUKA Satoshi2,SONG Huan-lu1

    (1. Beijing Key Laboratory of Food Flavor Chemistry, School of Chemical and Environmental Engineering, Beijing Technology and Business University, Beijing 100048, China;2. Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan)

    The effect of dietary cholic acid (CA) on the acute response of the rat large intestinal epithelial cells following a singledose irradiation was investigated. Rats were exposed to whole-body gamma-irradiation after being fed a control or 0.2% CA diet for 10 days. The rats were then sacrificed at 1, 3, 6, 12 h and 24 h post-irradiation. Segments of cecum and distal colon were collected for histochemical analysis. Apoptosis in the epithelial cells of cecum and distal colon was stimulated and peaked at 3 h postirradiation in both dietary groups, however, in CA group, apoptosis was markedly inhibited at 6 h post-irradiation in the distal colon. In epithelial cells of rats fed the control diet, the number of bromodeoxyuridine (BrdU)-incorporated cells began to decrease at 1 h post-irradiation in both distal colon and cecum. Interestingly, the proliferation of the cells was transiently stimulated by CA intake before decreasing. It should be noted that the effect of dietary CA on proliferation and apoptosis in the colonic epithelial cells was more significant than that in the cecal epithelial cells. In conclusion, these results demonstrate that dietary CA alters the regeneration regularity of colonic cells and serves as an inhibitor of apoptosis in the intestinal epithelial cells following DNA damage induced by gamma-irradiation.

    cholic acid (CA);gamma-irradiation;intestinal epithelial cells;proliferation;apoptosis

    Dietary factors are implicated in the etiology of human cancer. In epidemiological observations, high fat diet is positively related to the occurrence of colon cancer[1]. An important way by which fat might exert its effect is the stimulation of bile acid secretion. Bile acids are metabolites of cholesterol metabolism, and function as gut epithelium trophic factors and detergents for the absorption of cholesterol and fatsoluble vitamins. There are different forms of bile acids. Theprimary bile acids, cholic (CA) and chenodeoxycholic (CDCA) acids, are derived via two different metabolic pathways from cholesterol in the liver and transported via bile to the intestine; secondary bile acids such as deoxycholic (DCA) and lithocholic (LCA) acids are formed from CA and CDCA, respectively. The effect of bile acids was thought to influence colon carcinogenesis including mediating loss of colonic surface epithelium[2], DNA damage[3], inducing cell proliferation[4], increasing ornithine decarboxylase activity[5], suppressing the expression of HLA genes[6-8], activating protein kinase C[9], increasing cell membrane permeability[10], regulating gene transcription[11]and inhibiting DNA polymeraseβ[12]. However, studies on CA alone are limited compared to other bile acids such as DCA and LCA.

    Ionizing irradiation is known to induce tumors in the colon[13-15]and to increase the risk of colorectal cancer[16]. Ishizuka et al.[17]reported that irradiation of gamma-ray (60Co) induced aberrant rat colon crypt foci, a biomarker of colorectal cancer. From histochemical point of view, intestinal epithelium is a highly hierarchical organ in which stem cell positions are well defined in terms of the spatial arrangement within the crypt[18]. A few stem cells locate at the base of crypt in large intestine, which are very sensitive even to low dose ionizing irradiation. Irradiation doses as low as 0.01-0.05 Gy can induce apoptosis in the stem cell position. The extreme sensitivity possibly helps eliminate stem cells which undergo significant genome damage, which might Otherwise increase the risk of neoplasia.

    The objective of this study was to investigate the effect of dietary CA on acute response of rat large intestinal epithelial cells to DNA damage, induced by a single dose of gamma-ray at 4 Gy as an ionizing irradiation source and using the histochemical analysis as the main experimental method.

    1 Materials and Methods

    1.1 Animals, reagents and instruments

    This study complied with the Animal Experimental Guides according to the Committee of Experimental Animal Care of Hokkaido University. 3-week-old male WKAH/ HKmSlc (Japan SLC, Inc., Hamamatsu, Shizuoka, Japan) were housed individually in stainless steel wire-bottom cages in an air-conditioned room kept at approximately 23 ℃ and 12 h cycle of light (08:00-20:00): dark (20:00-08:00). After acclimation period of 6 d, the rats were divided into two dietary groups that contained 30 rats per group, and provided ad libitum access to either the control or the CA diet and drinking water for 10 d. The ingredients of the experimental diets were shown in Table 1. Body weight and food intake were measured daily during the whole experimental period.

    BrdU, fluorodeoxyurindine (Sigma Chemical, Steinheim, Germany); OCT compound (Sakura Finetecnical, Tokyo) ; anti-BrdU monoclonal antibody (clone OS94.6, Calbiochem, Cambridge, MA) ; pepsin (Wako Pure Chemical Industries, LTD., Osaka, Japan); biotinylated rabbit anti-mouse IgG+A+M (H+L; Zymed Laboratories, San Francisco, CA); peroxidaseconjugated streptavidin (Cosmo Bio, Tokyo, Japan).

    Cobalt-60 irradiator (Cobalt-60 Teletherapy Apparatus RCR-120-C3, Toshiba Co., Japan).

    Table1 Ingredients of the experimental diets g/kg diet

    1.2 Gamma- irradiation

    At the end of test diet, 25 unanaesthetized rats from each dietary group (50 rats total) were exposed to a whole- body irradiation of 4 Gy (dose rate: 0.39 Gy/min) using cobalt-60 irradiator, in Central Institute of Isotope Science, Hokkaido University. The exposure was performed between 09:00-10:00 .

    1.3 Histochemical analysis of acute response after exposure to gamma-irradiation

    For histochemical studies, ten groups of irradiated rats were sacrificed at 1, 3, 6, 12 h, or 24 h post-irradiation (n=5/ dietary group in each time point), meanwhile two groups (n=5/ dietary group) of non-irradiated rats were sacrificed as the treatment controls, which were described as 0 h post-irradiation in the results. One hour prior to sacrifice (just after the exposure in case of 1 h post-irradiation), each rat was injected with a bromodeoxyuridine (BrdU, 15 mg/kg body weight) solution containing 15 mg BrdU and 1.5 mg fluorodeoxyurindine per 1 mL saline. Following sacrifice under anesthesia with sodium pentobarbital, segments of the cecum and distal colon were flushed with saline, embeddedin OCT compound, rapidly frozen in liquid nitrogen, and stored at -80℃. Frozen sections from these samples were prepared and stained with anti-BrdU monoclonal antibody, and fixed in 10% formalin in phosphate buffered saline. The samples were then soaked in 3% hydrogen peroxide in methanol to block endogenous peroxidase activity, treated with 0.4 mg/mL pepsin (0.1 mol/L HCl), and then with 10% normal rabbit serum to reduce nonspecific binding. After incubation with the primary antibody as mentioned above, samples were incubated with biotinylated rabbit anti-mouse Ig(G+A+M). Samples were then incubated with peroxidaseconjugated streptavidin, and 3,3'-diaminobenzidine tetrahydrochloride was used as the chromogen. After BrdU staining, these sections were counterstained with hematoxylin. For detecting apoptosis and mitotic cells, hematoxylin-eosin (HE) staining was performed. Apoptosis was observed on the evidence of morphological characteristics, such as cell shrinkage, chromatin condensation, and nuclear fragmentation[20]. Mitotic cells were identified by means of chromatin condensation in the absence of cytoplasmic and nuclear shrinkage. In many mitotic cells, discrete chromosomal structure can be observed, in addition, mitotic cells appear horizontally displaced from the other epithelial-lining cells, toward the lumen of the intestine. Their cellular morphologies were shown in Fig.1. Finally, numbers of BrdU-incorporated cells, apoptotic cells, mitotic cells in the epithelial layer were scored in every cell position from the bottom to top along longitudinal half crypt axis of the cecum and distal colon, according to the method of Ijiri and Potten[21]. Fifty half-crypts were scored in each individual rat. The index was the percentage calculated by the number of apoptotic epithelium and BrdU-incorporated cells against the total number of epithelial cells in the same crypt, which were expressed as Apop-index and BrdU-index, respectively.

    Fig.1 Images of BrdU staining and HE staining.

    1.4 Statistical analyses

    All statistics were analyzed using JMP software (SAS Institute, Cary, NC). Data are shown as mean±SD. Statistical differences among groups sacrificed at different time points (0, 1, 3, 6, 12, 24h after irradiation) were performed using Tukey-Kramer, s test, and those between the control and CA dietary groups were performed using Student ,s-t test. Differences were considered significant when P<0.05.

    2 Results and Analysis

    2.1 Initial body weight, body weight gain, and food intake

    Table2 Initial body weight, body weight gain, and food intake (n=30)

    Table 2 shows that dietary CA significantly suppressed the food intake and body weight gain of rats, compared to the control diet.

    2.2 Regulation of the number of apoptotic cells

    Fig.2 Changes of the number of apoptotic cells in the distal colon (A) and the cecum (B) after exposure to a single dose of gamma-ray (4 Gy).

    Changes in the number of apoptotic cells after gammairradiation are shown in Fig.2. The highest number of apoptotic cells was observed at 3 h post-irradiation in both dietary groups. But at 6 h post-irradiation, dietary CA significantly attenuated apoptosis in the distal colon. In contrast, the changes of apoptosis in the cecal epithelial cells of rats fed the CA diet were similar to those rats fed the control diet.

    2.3 Regulation of the number of mitotic cells

    Fig.3 Regulation of the number of mitotic cells in the distal colon (A) and the cecum (B) after exposure to a single dose of gamma-ray (4 Gy).

    No significant change in the number of mitosis was observed in the distal colon after exposure to a single dose of gamma-ray at 4 Gy (Fig. 3). In the cecum, irradiation decreased the number of mitotic cells in the rats fed the CA diet until 6 h post-irradiation before steadily recover. However, dietary CA did not have significant effect on mitosis on both sites of large intestine, compared to the control diet.

    2.4 Regulation of the number of BrdU-incorporated cells

    Fig.4 Regulation of the number of BrdU-incorporated epithelial cells in the distal colon (A) and the cecum (B) after exposure to a single dose of gamma-ray (4 Gy).

    In both distal colon and cecum, the number of BrdU-incorporated cells in rats fed the CA diet transiently increased until 1 h post-irradiation (Fig. 4), and then decreased rapidly afterwards. Moreover, the CA intake strongly stimulated cell proliferation at 1, 3, 6 h and 12 h post-irradiation in the distal colon and at 3 h post-irradiation in the cecum, compared to the control diet.

    2.5 Regulation of the total epithelial cell number

    Fig. 5 Regulation of the total number of epithelial cells inthe distal colon (A) and the cecum (B) after exposure to a single dose of gammaray (4 Gy).

    The intake of CA diet increased the total number of epithelial cells, especially in the distal colon (Fig. 5). In thedistal colon, no significant changes were observed in the control groups throughout the course of experiment, while the total epithelial cells in the CA groups transiently increased at 1, 3 h and 6 h post-irradiation. In the cecum, the number of total epithelial cells in rats fed the control diet at 24 h post-irradiation was decreased, and at 1, 3 h and 12 h postirradiation, the number in rats fed the CA diet was increased.

    2.6 Apoptotic cells and BrdU-incorporated cells against the total number of epithelial cells

    Apop-index in the distal colon reached a high level at 3 h and 6 h after irradiation in both dietary groups, and the CA diet had an inhibitory effect on it at both of time points. But no dietary effects on the apoptosis of epithelial cells were observed in the cecum (Table 3). BrdU-index in the distal colon was significantly higher at 1, 3, 6 h and 12 h postirradiation in CA groups than in control groups, while in the cecum, the CA diet only increased at 3 h after irradiation (Table 2).

    Table3 Apoptotic cells and BrdU-incorporated cells against the total number of epithelial cells in the distal colon and the cecum

    3 Discussion

    Each crypt is an active unit of epithelial cells that proliferate and senesce in a certain sequence that maintains its architecture and highly ordered functions. The findings in the present study provide more insight into how dietary CA affects apoptosis and cell cycle in the damaged intestinal epithelium.

    The process of apoptosis is believed to represent programmed or genetically determined self-deletion or suicide involved in individual cells. Potten[22]reported that following exposure, the appearance of new apoptotic cells was extremely rapid in the small intestine, and increase of apoptosis per crypt began to be observed about 1.5 h after irradiation and reaches peak levels between 3 h and 6 h after irradiation. Similarly, as shown in Fig. 2 and Table 3, apoptosis in the cecum and distal colon was also stimulated and the highest number of apoptotic cells was observed at 3 h postirradiation in both dietary groups. Apoptosis has been considered as an important protective mechanism in the stem cell population for effectively recognizing and eliminating the damage[23]. However, dietary CA suppressed apoptosis marginally at 3 h and inhibited it markedly at 6 h postirradiation in the distal colon, compared to the control groups (Fig. 2). Therefore, this process did not seem to operate completely or was defective in the colon of rats fed the CA diet, thus cells with DNA damage had the potential to persist either carrying low levels of undetectable damage, damage that had been repaired, or damage that had been misrepaired. As a consequence, the cells in the colon of rats fed the CA diet might have a greater risk for perpetual genetic errors that might ultimately lead to cancers.

    Many independent studies have indicated that after irradiation, the cell cycle time of the majority of the cells in the crypt is reduced by approximately 20%[24-26]. The rapid changes in various proliferation indices can only occur in cells that are not killed via apoptosis or cells that are not completely reproductively sterilized[23]. Histochemical study showed (Fig. 4) that at 1 h post-irradiation the number of BrdU-incorporated cells in rats fed the control diet began to decrease and was lower than that in non-irradiated rats in both the distal colon and cecum due to the damage induced by gamma-rays. In contrast, the CA diet significantly stimulated proliferation after irradiation (Table 3, Fig. 4). At 1 h post-irradiation, proliferation in the CA group transiently increased in both sites. It should be noted that in the distal colon, the numbers of BrdU-incorporated cells in the CA groups were significantly higher than those in the control groups until 24 h post-irradiation. With this trend, we also found that the total numbers of epithelial cells in both sites of the CA groups were higher than the control groups after gamma-irradiation (Fig. 5). Based on the above findings of Potten[23], these interesting phenomena might all be due to the inhibition effect of dietary CA on apoptosis of damaged cells after irradiation. However, the cell mitosis after irradiation didnot seem to have direct relationship to the changes of apoptosis and proliferation of epithelial cells (Fig. 3).

    Another interesting finding in this study was that the effect of dietary CA on proliferation and apoptosis in the colonic epithelial cells was more prominent than that in the cecal epithelial cells (Fig. 2 and Fig. 4, Table 3). One of the speculations might be the difference in the microbiota between the cecum and colon. In the colon, secondary bile acid DCA is formed from conjugated forms of CA through deconjugation and 7 α-dehydroxylation by the anaerobic bacterial flora[27-28]. Weidema et al.[29]reported that the enhancing effect of CA on tumor formation might due to the formation of DCA. The secondary bile acids, DCA and LCA, were considered as the most important bile acids in the etiology of colon cancer in humans[30].

    In summary, data in the present study suggest that the dietary CA increases the proliferation rate of colonic cells and transiently serves as an inhibitor of apoptosis in rat intestinal epithelia following a single irradiation of 4 Gy, which may lead to the continuous proliferation of the risky cells and eventually result in the increase of colon cancer occurrence.

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    [29]WEIDEMA W F, DESCHENER E E, COHEN B I, et al. Acute effects of dietary cholic acid and methylazoxymethanol acetate on colon epithelial cell proliferation; metabolism of bile salts and neutral sterols in conventional and germfree SD rats[J]. J Natl Cancer Inst, 1985, 74: 665-670.

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    膳食膽酸對輻射后的大鼠大腸上皮細(xì)胞凋亡與增殖的影響

    徐 虹1,石塚敏2,宋煥祿1
    (1.北京工商大學(xué)化學(xué)與環(huán)境工程學(xué)院,食品風(fēng)味化學(xué)北京市重點(diǎn)實(shí)驗(yàn)室,北京 100048;2.日本北海道大學(xué)大學(xué)院農(nóng)學(xué)研究科,札幌市 060-8589)

    研究膳食膽酸對大腸上皮細(xì)胞在單劑量輻射之后急性反應(yīng)的影響。各組實(shí)驗(yàn)大鼠在喂食空白膳食或者0.2%膽酸膳食10d之后接受全身γ射線照射,然后分別在輻射后1、3、6、12、24h被處死。盲腸和遠(yuǎn)端結(jié)腸樣品被采集進(jìn)行組織化學(xué)研究。結(jié)果顯示:空白和膽酸膳食組的大鼠,其盲腸和遠(yuǎn)端結(jié)腸上皮細(xì)胞的凋亡均在輻射后被激發(fā),并在輻射后3h后達(dá)到最高峰。然而,膳食膽酸在輻射6h后開始顯著抑制遠(yuǎn)端結(jié)腸上皮細(xì)胞的凋亡。比較空白膳食各組, 在大鼠接受輻射1h后盲腸和遠(yuǎn)端結(jié)腸兩個部位的BrdU標(biāo)記的細(xì)胞數(shù)量都開始減少,而膽酸的攝入?yún)s使上皮細(xì)胞的增殖瞬時增加。比較兩個實(shí)驗(yàn)部位,膳食膽酸的攝入對遠(yuǎn)端結(jié)腸部位上皮細(xì)胞增殖和凋亡的影響比對盲腸部位上皮細(xì)胞的影響更大。綜上所述,膳食膽酸的攝入改變了結(jié)腸細(xì)胞更新規(guī)律,并且對輻射后DNA受損傷的大腸上皮細(xì)胞的凋亡有抑制作用。

    膽酸;射線;腸道上皮細(xì)胞;增殖;凋亡

    TS201.4

    A

    1002-6630(2010)19-0375-06

    2010-04-19

    徐虹(1977—),女,講師,博士,研究方向?yàn)槭称窢I養(yǎng)與安全。E-mail:xuhong@th.btbu.edu.cn

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