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Suppressive effect of corn bran hemicellulose on liver injury induced by D-galactosamine in rats
Nutrition 21 (2005) 1044 –1051 www.elsevier.com/locate/nut
Basic nutritional investigation
Suppressive effect of corn bran hemicellulose on liver injury induced by D-galactosamine in rats Ayako Daizo, M.S.*, Yukari Egashira, Ph.D., and Hiroo Sanada, Ph.D. Laboratory of Food and Nutrition, Graduate School of Science and Technology, Chiba University, Chiba, Japan Manuscript received August 16, 2004; accepted February 5, 2005.
Abstract
Objective: It is well known that the indigestible oligo- and polysaccharides including dietary fiber are important food components and that they have many physiologic functions. This study examined the effect of water-soluble corn bran hemicellulose (CBH) on the development of D-galactosamine (GalN) hepatitis in rats to obtain some knowledge about new functions of dietary fiber. Methods: Male Wistar rats were fed diets containing 5% CBH for various days (1 to 14 d). On the final day of feeding rats were treated with GalN (400 mg/kg), and their plasma transaminase (aspartate and alanine aminotransferases) activity (6 or 24 h later) and liver glutathione concentration (6 h later) were determined. Results: Ingested CBH suppressed the increase in plasma aspartate and alanine aminotransferase activities 24 h after GalN treatment. Such suppressive effect was observed only 7 d after CBH ingestion and not after 1 or 3 d. In the early phase of the liver injury, at 6 h after GalN treatment, the liver glutathione concentration in the CBH group was significantly higher than that in the control group, and the concentration in the CBH group after GalN injection was almost the same as that in the control group without GalN treatment. Conclusion: Results suggest that dietary CBH suppresses the development of hepatic injury by GalN in rats and that this phenomenon is partly attributable to the increase in hepatic glutathione concentration by CBH. © 2005 Elsevier Inc. All rights reserved.
Keywords:
Corn bran hemicellulose; Liver injury; D-galactosamine; Glutathione
Introduction It is well known that the indigestible oligo- and polysaccharides including dietary fiber are important food components. Because these indigestible saccharides have been reported to affect lipid and carbohydrate metabolism [1] and immunologic function [2], it is considered that they must have an effect on liver function and liver injury. However, research on these indigestible saccharides in relation to liver injury or function has been inadequate. The effects of oligosaccharides and dietary fiber on various protective functions of organisms have recently been reported [3,4], and our previous study showed that intake of oligosaccharides, especially of oligosaccharides containing D-galactose, decreases the increase in plasma transaminase activities in rats
* Corresponding author. Tel.: ⫹81-47-365-1111; fax: 81-47-363-1401. E-mail address: [email protected] 0899-9007/05/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2005.02.009
injected with D-galactosamine (GalN), which causes hepatitis in experimental animals [5]. Moreover, dietary corn bran hemicellulose (CBH) has been shown to prevent the development of orotic acid–induced fatty liver [6]. However, these functions of indigestible dietary polysaccharides have not been investigated in detail. Arabinoxylane hemicellulose is an indigestible polysaccharide that is abundant in cereals commonly consumed by humans, and in the present study we investigated the effect of CBH, mainly consisting of arabinoxylane hemicellulose, on GalN-induced liver injury. The CBH preparation used contained uronic acid (13.5%) and galactose (6.7%) as constituents of dietary fiber (Table 1). Morphologic and pathophysiologic characteristics of GalN-induced liver damage are very similar to those of human viral hepatitis. GalN induces hepatotoxicity by inhibiting RNA and protein synthesis through a decrease in cellular uridine triphosphate concentration, which ultimately leads to liver cell necrosis [7–10]. In contrast, there
A. Daizo et al. / Nutrition 21 (2005) 1044 –1051 Table 1 Composition of the corn bran hemicelluouse preparation Water Protein Lipid Ash Fiber* Other Arabinose Xylulose Galactose Glucose Uronic acid
3.8% 0.6% 0.1% 1.5% 86.0% 8.0% 31.5% 45.7% 6.7% 2.6% 13.5%
* Relative content of constitutional monosaccharide residues.
have been several reports that endotoxin is involved in the induction of this hepatic injury and promotes the secretion of cytokines, such as tumor necrosis factor-␣ and interleukin-6, by Kupffer cells [11,12]. Based on these findings, hepatitis may develop as a result of an overlap between its impairment of hepatocyte metabolism and its immunologic action, and it seems to destroy hepatocytes first by apoptosis and then by necrosis. Because certain probiotic bacterial strains are capable of decreasing bacterial translocation and hepatocellular damage after GalN injection [13], it has been suggested that dietary oligosaccharides have an effect on the microflora and bacterial translocation that results in protection against hepatic injury caused by GalN. Oligosaccharides and dietary fiber may affect immune function, probably through a change in intestinal bacteria or short-chain fatty acids generated by intestinal bacteria, and these dietary substances may affect the development of GalN-induced hepatitis immunologically[14]. Further, it has been reported that thiobarbituric acid reacting substances, which are generated through free radical reactions, increase during the process of necrosis caused by GalN [15], and concentrations of vitamin C and glutathione in the concerned tissues have been shown to decrease under this oxidative stress [16,17]. Bergenin, a constituent of Mallotus japonicus, has also been found to protect rats against GalN-induced hepatitis, and this effect is thought to be attributable in part to maintaining adequate hepatic glutathione levels [18]. The present study assessed the effect of water-soluble CBH on induction of hepatic injury by GalN in relation to the mechanism of its hepatotoxic action. Tissue glutathione levels were measured in addition to plasma transaminase activities as indices of hepatitis to clarify the relation to oxidative stress.
Materials and methods Materials GalN hydrochloride and transaminase C-II test from Wako Pure Chemicals Industries (Osaka, Japan), and so-
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dium pentobarbital (Nembutal) solution from Dinabot (Osaka, Japan) were commercially available. Water-soluble CBH, Cell-ace, was a kind gift from Nihon Shokuhin Kako Co., Ltd. (Tokyo, Japan). Other reagents were purchased from Wako Pure Chemicals Industries. Animals and feeding In our previous preliminary experiments we observed a wide variety of sensitivity to GalN and dietary fiber among rats obtained from different suppliers. Because the clean (pathogen-free) male Wistar rats obtained from Japan SLC (Hamamatsu, Japan) were found to be the most sensitive of the rats examined, 4-wk-old male rats obtained from Japan SLC were used in this study. Rats were kept in an environmentally controlled room at a temperature of 22 ⫾ 1°C on a 12-h light, 12-h dark cycle (light from 7:00 AM to 7:00 PM). Compositions of the mineral and vitamin mixtures used in this study were the same as those of AIN-76 [19]. Three separate experiments were performed. All rats were fed a commercial diet (type CE-2, CLEA, Tokyo, Japan) for 3 d and then a control (Cont) diet (Table 2) for 4 d. Rats were then assigned to one of several groups and fed the experimental diets whose composition is presented in Table 2. All rats were allowed free access to their diets and drinking water except for the period around the time of the GalN injection, as described below. In experiment 1, 15 rats in two groups were fed the Cont diet (n ⫽ 7) or the CBH diet (n ⫽ 8) for 14 d and then injected with GalN (400 mg/kg of body weight) intraperitoneally at 1:00 PM on 14 d. Diets were withheld for 4 h before and after the GalN injection (8 h of deprivation in total). Twenty-four hours after the GalN treatment, blood was drawn from the posterior vena cava into a heparinized syringe under anesthesia with Nembutal (0.13 mL/100 g of body weight), and livers were quickly excised. Blood samples were immediately centrifuged at 3000 rpm for 20 min, and plasma samples were stored at ⫺20° until analyzed. In experiment 2, the effect of feeding duration of the
Table 2 Compositions of the experimental diets (%)
Casein Corn bran hemicellulose Cornstarch* Sucrose DL-methionine Corn oil Vitamin mixture† Mineral mixture† Cellulose Choline bitartrate
Cont
CBH
20 — 40 25 0.3 5 1 3.5 5 0.2
20 5 35 25 0.3 5 1 3.5 5 0.2
CBH, diet containing 5% corn bran hemicellulose; Cont, control diet. * Cornstarch was digestible gelatinized cornstarch. † Composition of the AIN-76 diet (1977).
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CBH diet on the response of rats to the hepatotoxic action of GalN was examined. Because the results of the preliminary experiment suggested that the CBH diet exerted a suppressive effect not only after 14 d of feeding but also after 7 d of feeding, a feeding period of 7 d was used in experiments 2 and 3. Thirty-six rats were assigned to one of three CBH diet groups (18 rats) and three Cont diet groups (18 rats). The CBH diet groups were given the CBH diet for 1, 3, or 7 d after 6, 4, or 0 d, respectively, on the Cont diet (7 d in total). Then GalN was administered to all animals at 1:00 PM on the final day of the CBH diet period to avoid agedependent variations in responses. The corresponding Cont groups were given the Cont diet instead of the CBH diet. All rats were killed 24 h after administration of GalN. GalN injection, blood collection, and dissection were performed in the same manner as in experiment 1. The liver was quickly excised, immediately frozen in liquid nitrogen, and stored at ⫺80°C until analyzed. In experiment 3, the effect of the CBH diet on the concentration of glutathione in rat tissues was assessed 6 h after the GalN injection, in the early phase of liver injury, to elucidate the mechanism of the suppressive action of CBH on GalN-induced hepatitis in relation to oxidative stress. Seventeen rats were fed the Cont diet (n ⫽ 8) or the CBH diet (n ⫽ 9) for 7 d. Fifty percent of the rats in the Cont diet group were then injected with GalN solution or the same volume of saline (Cont/⫺GalN group, n ⫽ 4), and four or five of the rats in the CBH diet group were then injected with GalN solution or the same volume of saline (CBH/ ⫹GalN group, n ⫽ 5), in the same manner as described in experiment 1 at 3:00 PM on the final day of the feeding period. Their diets were removed 4 h before GalN injection, and they were kept fasting thereafter. Six hours after GalN injection, dissection and blood collection were performed as described in experiment 1. The liver and spleen were rapidly excised, immediately frozen in liquid nitrogen, and stored at ⫺80° until analyzed. In all experiments the Cont and CBH diet groups with or without GalN injection are referred to as the Cont/⫹GalN, CBH/⫹GalN, Cont/⫺GalN, and CBH/ ⫺GalN groups, respectively. In experiment 2, the group that was injected with GalN after being fed the CBH diet for 7 d is referred to as the 7-d/CBH/⫹GalN group, and the other groups in experiment 2 are also expressed by the abbreviations in the same manner. The care and treatment of the rats were in accordance with the guidelines prescribed by the Faculty of Horticulture, Chiba University, and National Institutes of Health Guide for the Care and Use of Laboratory Animals [20]. Determination of blood aspartate and alanine aminotransferases Plasma activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in all experimental animals were assayed with commercial assay kits (Transaminase C-II-test, Wako Pure Chemicals Industries).
Table 3 Weight gain, feed efficiency, and plasma AST and ALT activities of animals 24 h after GalN Injection (experiment 1)* Diet Weight gain (g/d) Feed efficiency† (%) Plasma transaminase activity ALT (U/L) AST (U/L)
Cont/ ⫹ GalN (n ⫽ 7) 5.08 ⫾ 0.14 41.68 ⫾ 0.85 484.47 ⫾ 75.28 1354.32 ⫾ 178.88
CBH/ ⫹ GalN (n ⫽ 8) 5.40 ⫾ 0.14 44.15 ⫾ 0.61 202.42 ⫾ 54.45‡ 537.83 ⫾ 157.68‡
ALT, alanine aminotransferase; AST, aspartate aminotransferase; CBH, corn bran hemicellulose diet; Cont, control diet; GalN, D-galactosamine. * All values are means ⫾ standard errors of the mean. † Body weight gain/feed intake. ‡ Significantly different (P ⬍ 0.05) from the corresponding values in the Cont ⫹ GalN group.
Determination of hepatic glycogen Hepatic glycogen was measured in experiment 2 by the anthrone method [21]. Determination of glutathione Concentrations of reduced (GSH) and oxidized (GSSG) forms of glutathione in the liver and spleen were measured in experiment 3. The procedure is based on the initial formation of S-carboxymethyl derivatives of free thiols with iodoacetic acid followed by conversion of free amino groups to 2,4-dinitrophenyl derivatives by reaction with 1-fluoro-2,4-dinitrobenzene. The reaction mixture was chromatographed on a 3-aminopropylsilane– derivatized silica column and eluted with a sodium or ammonium acetate gradient in a water/methanol/acetic acid solvent at pH 4.5 [22]. Statistics All values obtained in experiment 1 were evaluated by Student’s t test. Two-way analysis of variance (ANOVA), and in some instances Student’s t test was used to perform the statistical analyses in experiments 2 and 3. The level of statistical significance was P ⬍ 0.05.
Results Effect of CBH diet fed for14 d after GalN-inducted hepatitis (experiment 1) Body weight gain, feed efficiency, and plasma AST and ALT activities in each group in experiment 1 are presented in Table 3. Body weight gain and feed efficiency values in the Cont and CBH groups were almost the same, respectively. However, plasma AST and ALT activity values in the CBH/⫹GalN group were significantly lower than those
A. Daizo et al. / Nutrition 21 (2005) 1044 –1051 Table 4 Weight gain, feed efficiency, carcass weight, liver weight, and liver ratio of animals 24h after GalN injection (experiment 2)* Cont/⫹GalN Weight gain (g/d) 1d 3d 7d Feed efficiency† (%) 1d 3d 7d Carcass weight (g) 1d 3d 7d Liver weight (g) 1d 3d 7d Liver ratio‡ (%) 1d 3d 7d
CBH/⫹GalN
4.41 ⫾ 0.94 3.66 ⫾ 0.33 4.47 ⫾ 0.12
5.08 ⫾ 0.52 4.38 ⫾ 0.20§ 5.45 ⫾ 0.29§
31.01 ⫾ 6.54 25.34 ⫾ 2.01 31.34 ⫾ 0.65
34.87 ⫾ 2.78 32.71 ⫾ 1.43§ 39.62 ⫾ 1.15§
119.16 ⫾ 3.15 118.00 ⫾ 3.37 116.16 ⫾ 2.88
119.33 ⫾ 3.14 116.50 ⫾ 2.85 117.33 ⫾ 2.47
7.17 ⫾ 0.67 5.88 ⫾ 0.23 5.91 ⫾ 0.21
5.96 ⫾ 0.30 6.32 ⫾ 0.38 7.29 ⫾ 0.69
4.96 ⫾ 0.27 4.98 ⫾ 0.11 5.04 ⫾ 0.17
5.27 ⫾ 0.2 5.43 ⫾ 0.32 6.24 ⫾ 0.49
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7-d/Cont/⫹GalN group, but enzyme activities in 1-d and 3-d/Cont/⫹GalN groups were not significantly different from those in the corresponding CBH/⫹GalN groups. Although enzyme activities were affected by the diets and interaction effects (diets by feeding periods) when analyzed by two-way ANOVA, they were not influenced by feeding period. However, results of one-way ANOVA (with the factor of feeding periods) indicated that plasma AST activity in the CBH groups decreased significantly as the feeding period increased. Moreover, as presented in Table 5, liver glycogen concentration in the 7-d/CBH/⫹GalN group was significantly higher than that in the 7-d/Cont/⫹GalN group. The results of two-way ANOVA also indicated that liver glycogen concentration was significantly affected by the diets but not by the feeding periods. These results indicate that the preventive effect of CBH diet against GalN-induced
CBH, corn bran hemicellulose diet; Cont, control diet; GalN, Dgalactosamine * All values are means ⫾ standard errors of the mean (n ⫽ 6). † Body weight gain/feed intake. ‡ Liver ratio: (Liver weight/Carcass weight) ⫻ 100 § Significantly different (P ⬍ 0.05) from the value in the Cont ⫹ GalN group in the corresponding feeding period.
in the Cont/⫹GalN group, indicating that the CBH diet promoted protective activity against GalN-induced hepatitis. Effect of duration of feeding on the CBH diet (experiment 2) In experiment 2, differences in resistance of rats to GalNinduced hepatitis according to duration of feeding on the CBH diet were examined in relation to hepatic glycogen concentration. Body weight gain, feed efficiency, carcass weight, liver weight, and liver ratio in each group are presented in Table 4. Although mean initial body weights in all groups were almost the same (data not shown), body weight gain and feed efficiency were significantly higher in the 3-d and 7-d/CBH/⫹GalN groups than in the 3-d and 7-d/Cont/ ⫹GalN groups (Student’s t test). No significant differences in carcass weight, liver weight, or liver ratio were observed between the Cont/⫹GalN and CBH/⫹GalN groups irrespective of feeding duration. Analysis by two-way ANOVA (factors of diets and feeding periods) showed that feed efficiency and liver ratio were significantly affected by the diets, and that liver weight was affected by the interaction effect (diets by feeding periods) of the two factors, but none of the parameters listed in Table 4 were significantly altered by feeding period. As shown in Fig. 1, plasma AST and ALT activities were significantly lower in the 7-d/CBH/⫹GalN group than in the
Fig. 1. Effect of duration of CBH diet intake on plasma transaminase activities after GalN treatment (experiment 2). Animals were fed a 5% CBH diet (white bars) or the Cont diet (black bars) for 1, 3, or 7 d before injection of GalN, as described in MATERIALS AND METHODS. Values are expressed as mean ⫾ standard error of the mean, with n ⫽ 5 in the 7-d/Cont/⫺GalN and CBH/⫺GalN groups and n ⫽ 6 in the other groups. * Significantly different (P ⬍ 0.05) from the value in the Cont/⫹GalN groups in the corresponding feeding periods. ALT, alanine aminotransferase; AST, aspartate aminotransferase; CBH, corn bran hemicellulose diet; Cont, control diet; GalN, D-galactosamine.
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Table 5 Glycogen concentration (milligrams) in livers (grams) of animals 24 h after GalN injection (experiment 2)* Diet period
Cont/⫹GalN
CBH/⫹GalN
1d 3d 7d
0.27 ⫾ 0.29 0.60 ⫾ 0.18 2.69 ⫾ 1.37
19.95 ⫾ 8.40 21.32 ⫾ 7.89 35.91 ⫾ 8.59†
CBH, corn bran hemicellulose diet; Cont, control diet; GalN, D-galactosamine * All values are means ⫾ standard errors of the mean (n ⫽ 5 for 7-d CBH ⫹ GalN group; n ⫽ 6 for the other groups). † Significantly different (P ⬍ 0.05) from the value in the Cont ⫹ GalN group in the corresponding feeding period.
hepatitis is observed after successive feeding of the diet for longer than 7 d. Influence of CBH diet on hepatic glutathione concentration 6 h after GalN injection (experiment 3) In experiments 1 and 2 plasma transaminase activities were assayed 24 h after GalN injection. In experiment 3, however, the hepatic glutathione concentration in rats fed the CBH diet was measured 6 h after GalN injection, a relatively early phase of the hepatic injury, to explore the mechanism by which the CBH diet attenuates induction of hepatitis by GalN. Body weight gain, feed efficiency, carcass weight, liver weight, liver ratio, cecum weight, and cecum ratio in each group are presented in Table 6. Body weight gain, feed efficiency, and carcass weight were not significantly different across groups. Liver weight was significantly decreased by the CBH diet but was unaffected by
GalN. In contrast, liver ratio was affected by the CBH diet and GalN and was higher in the Cont/⫹GalN and CBH/ ⫹GalN groups than in the Cont/⫺GalN and CBH/⫺GalN groups. These results obtained 6 h after GalN administration differed from the results obtained at 24 h in experiment 2. Also, the liver ratio in the CBH/⫹GalN and CBH/⫺GalN groups was lower than that in the Cont/⫹GalN and Cont/ ⫺GalN groups. Cecum weight and its ratio to carcass weight were increased by the CBH diet, suggesting fermentation of CBH in the cecum. Plasma AST and ALT activities were affected by GalN treatment but not by diet, and thus the activities in the Cont/⫹GalN and CBH/⫹GalN groups were higher than those in the Cont/⫺GalN and CBH/⫺GalN groups. Table 7 presents hepatic and splenic concentrations of GSH and GSSG of the animals in experiment 3. Concentrations of hepatic GSH and GSSG were decreased by GalN, and the total GSH plus GSSG concentration was increased by the CBH diet. Thus, concentrations of GSH, GSSG, and their sum tended to be lower in the Cont/⫹GalN and CBH/ ⫹GalN groups than in the Cont/⫺GalN and CBH/⫺GalN groups, and concentrations in the CBH/⫺GalN and CBH/ ⫹GalN groups tended to be higher than those in the Cont/ ⫺GalN and Cont/⫹GalN groups. In contrast, splenic concentrations of GSH and GSSG were not significantly affected by GalN or the CBH diet according to the results of ANOVA. In the Cont diet groups, however, concentrations of GSH, GSSG, and GSH plus GSSG (total) were significantly lower in the Cont/⫹GalN group than in the Cont/⫺GalN group according to Student’s t test.
Table 6 Weight gain, feed efficiency, carcass weight, liver weight, liver ratio, cecum weight, cecum ratio, and plasma transaminase activities 6 h after GalN injection (experiment 3)* Cont
Weight gain (g/d) Feed efficiency† (%) Carcass weight (g) Liver weight‡ (g) Ratio (%) Cecum weight§ (g) Ratio (%) ALT (U/L) AST (U/L)
ANOVA#
CBH
⫺GalN
⫹GalN
⫺GalN
⫹GalN
GalN
Diet
GalN ⫻ diet
4.03 ⫾ 0.71 36.93 ⫾ 5.30 121.5 ⫾ 2.25 6.11 ⫾ 0.13 5.03 ⫾ 0.13 1.74 ⫾ 0.08 1.43 ⫾ 0.06 5.75 ⫾ 0.36 30.51 ⫾ 0.86
3.75 ⫾ 0.45 38.29 ⫾ 4.09 120.75 ⫾ 2.28 6.69 ⫾ 0.38 5.54 ⫾ 0.27 2.01 ⫾ 0.14 1.66 ⫾ 0.11 38.51 ⫾ 6.40 94.3 ⫾ 9.32储
4.17 ⫾ 0.15 39.23 ⫾ 1.38 118.75 ⫾ 3.11 5.23 ⫾ 0.19 4.40 ⫾ 0.12 3.58 ⫾ 0.08 3.02 ⫾ 0.13 8.13 ⫾ 1.20 36.43 ⫾ 10.41
4.22 ⫾ 0.34 41.65 ⫾ 1.35 119.00 ⫾ 4.59 5.84 ⫾ 0.43 4.88 ⫾ 0.21 4.74 ⫾ 0.71 4.02 ⫾ 0.60 32.96 ⫾ 12.08 82.30 ⫾ 22.4¶
NS NS NS NS ⬍0.05 NS NS ⬍0.05 ⬍0.05
NS NS NS ⬍0.05 ⬍0.05 ⬍0.05 ⬍0.05 NS NS
NS NS NS NS NS NS NS NS NS
ALT, alanine aminotransferase; ANOVA; analysis of variance; AST, aspartate aminotransferase; CBH, corn bran hemicellulose diet; Cont, control diet; GalN, D-galactosamine; NS, not significant * All values are means ⫾ standard errors of the mean (n ⫽ 5 in the CBH ⫹ GalN group; n ⫽ 4 in the other groups). † Body weight gain/Feed intake ‡ (Liver weight/carcass weight) ⫻ 100. § (Cecum weight/carcass weight) ⫻ 100. 储 Significantly different (P ⬍ 0.05) from the values in the Cont ⫺ GalN group. ¶ Significantly different (P ⬍ 0.05) from the values in the CBH ⫺ GalN group. # Statistical significance was analyzed by two-way ANOVA, using GalN and diet as factors.
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Table 7 Influence of CBH ingestion on glutathione concentration in the liver and spleen 6 h after GalN injection (experiment 3)* Cont
Liver (mol/g) GSH GSSG Total Spleen (mol/g) GSH GSSG Total
ANOVA§
CBH
⫺GalN
⫹GalN
⫺GalN
⫹GalN
GalN
Diet
GalN ⫻ diet
3.26 ⫾ 0.63 0.65 ⫾ 0.16 3.91 ⫾ 0.79
2.11 ⫾ 0.49 0.36 ⫾ 0.09† 2.47 ⫾ 0.58†
4.02 ⫾ 0.36 1.03 ⫾ 0.04 5.50 ⫾ 0.39
2.97 ⫾ 0.15‡ 0.79 ⫾ 0.04‡ 3.76 ⫾ 0.15‡
⬍0.05 ⬍0.05 NS
NS ⬍0.05 ⬍0.05
NS NS NS
1.04 ⫾ 0.05 0.96 ⫾ 0.07 2.00 ⫾ 0.11
0.81 ⫾ 0.13† 0.81 ⫾ 0.09† 1.62 ⫾ 0.21†
1.20 ⫾ 0.13 1.33 ⫾ 0.19 2.52 ⫾ 0.32
1.02 ⫾ 0.12 1.12 ⫾ 0.26 2.14 ⫾ 0.34
NS NS NS
NS NS NS
NS NS NS
ANOVA, analysis of variance; CBH, corn bran hemicellulose diet; Cont, control diet; GalN, D-galactosamine; GSH, reduced glutathione; GSSG, oxidized glutathione; NS, not significant; Total, GSH ⫹ GSSG * All values are means ⫾ standard errors of the mean (n ⫽ 5 in the CBH ⫹ GalN group; n ⫽ 4 in the other groups). † Significantly different (P ⬍ 0.05) from the value in the Cont ⫺ GalN group by Student’s t test. ‡ Significantly different (P ⬍ 0.05) from the value in the CBH ⫺ GalN group by Student’s t test. § Effects of diets and GalN were analyzed for statistical significance by two-way ANOVA.
Discussion Intake of the CBH diet for 14 d was found to prevent GalN-induced hepatic injury, as shown by the significantly lower plasma AST and ALT activities in the CBH/⫹GalN group than in the Cont/⫹GalN group (Table 3). The morphologic features of GalN-induced liver damage have been shown to be very similar to those of human viral hepatitis [23], and the damage generally reflects disturbances of hepatic cell metabolism, which lead to increases in blood levels of AST and ALT, lactate dehydrogenase, and ␥-glutamyl transferase as a result of hepatic cell destruction or changes in membrane permeability. The effect of duration of the CBH diet intake on GalNinduced hepatic injury was examined in experiment 2. Results of Student’s t test showed that a significant suppressive effect of CBH intake on GalN-induced hepatitis was recognized only when CBH diet was given for 7 d, and not for 1 or 3 d. Analysis of the data shown in Fig. 1 by one-way ANOVA showed that only AST activity in the CBH/⫹GalN group was significantly decreased as the feeding period increased and that the other activities were unaffected by the length of the feeding period, probably because of variations in values among rats. Changes in intestinal bacteria microflora associated with CBH intake, which are also generally considered to be same as the effects of many kinds of dietary fiber, are thought to be involved in their action. Because there is a report of variable changes in host-defense function that may be related to the state of intestinal bacteria microflora [24], it is thought that any physiologic alterations associated with the change in intestinal bacteria microflora would probably make rats more resistant to GalN hepatotoxicity and that the change would occur when CBH was ingested for longer than 7 d. Although cecum weight was not measured in experiment 2, it was significantly lower in the CBH/⫺GalN group than in the Cont/⫺GalN group in experiment 3. In addition, Wang et al. [5] reported that
GalN-induced hepatic injury was suppressed when rats had consumed a diet containing raffinose or galactooligosaccharide for 14 d, and that their cecum weight increased in comparison with control rats. Indigestible oligosaccharides in addition to indigestible polysaccharides have been shown to pass through intestine to the cecum and colon, where they ferment to produce short-chain fatty acids, such as lactic, acetic, propionic and butyric acids, and these short-chain fatty acids convert the pH of the cecum and colon to acidic. The suppressive effect of dietary CBH on induction of GalN-induced hepatitis may be attributable in part to the short-chain fatty acids absorbed or to the decrease in enteric gram-negative bacteria, such as Escherichia coli, that possess lipopolysaccharide under acidic conditions. Although translocation of bacteria from the gut into the systemic circulation has been considered to be partly involved in the development of GalN-induced hepatitis [25], the role of short-chain fatty acids in the suppression of hepatitis has never been confirmed. A similar suppressive effect of raffinose and galactooligosaccharide intake has been observed on GalN-induced hepatic injury, but not by fructo-oligosaccharide or gentiooligosaccharide [26], despite the fact that at least the first three of these saccharides have been found to promote proliferation of Bifidobacterium and production of shortchain fatty acids. The glycogen concentration in the liver (Table 5) after GalN treatment seemed to be inversely correlated with plasma AST or ALT activity (Fig. 1), and the concentration was significantly higher in the 7-d/CBH/⫹GalN group than in the 7-d/Cont/⫹GalN group. Deviations in glycogen concentrations in the 1-d and 3-d/CBH/⫹GalN groups were greater than those in the other groups (Table 5), suggesting that the rate of time-dependent reduction in susceptibility to GalN by the CBH diet may vary widely among rats in these periods. The protective effect of dietary CBH against GalNinduced hepatic injury was demonstrated in experiments 1
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and 2, and experiment 3 was performed to clarify the mechanism. In this experiment, we determined the glutathione concentration in the liver because GalN-induced hepatitis is thought to be caused in part by oxidative stress in the liver [16]. We also determined glutathione level in the spleen to explore the relation between immunologic function and protective activity of CBH against GalN-induced hepatic injury because there have been reports suggesting that tumor necrosis factor-␣ [12], endotoxin, and some cytokines [27] are involved in the development of GalN-induced hepatitis. In the present experiment, glutathione levels were measured 6 h after GalN administration, at a relatively early phase of the development of hepatic injury. Manabe et al. [28] estimated that plasma AST and ALT activities begin to increase around 6 h after GalN injection. Thus, the enzyme activities at that time after GalN injection were only slightly higher than those without GalN (Table 6), whereas enzyme activities 24 h after GalN administration in experiments 1 and 2 were significantly increased. The results of ANOVA indicated that plasma AST and ALT activities 6 h after GalN injection was not significantly affected by dietary CBH, in contrast to the results in experiments 1 and 2, in which CBH was shown to decrease the increase in enzyme activity 24 h after GalN injection. Liver weight was decreased by CBH intake but unaffected by GalN injection. Conversely, the liver ratio was increased by GalN injection and decreased by CBH ingestion. Some studies have suggested that liver weight is decreased 24 h after GalN injection as a result of atrophy accompanied by necrosis [26], but the finding that the liver weight had not decreased 6 h after GalN injection in this study (Table 6) demonstrates that atrophy had not yet occurred. Thus, the relation between liver ratios in the Cont/⫹GalN and CBH/⫹GalN groups in experiment 2 appears to be different from their relation in experiment 3, but the reason liver weight was decreased by CBH ingestion is unknown. Although the details of the mechanism of the development of this hepatitis-like injury by GalN have not been elucidated, the mechanism has been reported to involve free radicals or the immune system. Because there are reports suggesting that the intracellular level of glutathione affects immunity [29], free radical production [30], DNA synthesis [31], and cytophylaxis [29], we examined the liver glutathione concentration in experiment 3. Concentrations of GSH or GSSG in the liver in the CBH/⫺GalN and CBH/ ⫹GalN groups were significantly higher than those in the Cont/⫺GalN and Cont/⫹GalN groups (Table 7), indicating that dietary CBH had caused the increase in hepatic glutathione level. Concentration of glutathione in the spleen, however, was not significantly affected by dietary CBH. Although concentrations of GSH and GSSG in the liver appeared to be decreased by GalN, they remained higher in the CBH/⫹GalN group than in the Cont/⫹GalN group. Because oxidative stress induced by GalN in hepatocytes has been reported to cause decreased levels of glutathione in cells [30], the increase in hepatic glutathione level by di-
etary CBH probably attenuates the development of GalNinduced hepatitis by decreasing oxidative stress. However, the fact that glutathione affects lymphocyte function [29] seems to suggest that attenuation of GalNinduced hepatitis is explained by the change in the immune system associated with the alteration in glutathione level. In addition, there are some reports indicating that immune function is altered by oligosaccharide intake [32,33]. Lim et al. [34] reported that acetyl bergenin restored decreased levels of hepatic GSH and decreased glutathione reductase and glutathione S-transferase activities induced by GalN, and that the protective effect of acetyl bergenin on GalN-induced hepatitis may be related to normalization mechanisms by maintaining adequate levels of GSH. The reason dietary CBH increased the hepatic glutathione level is unknown. One plausible mechanism that can be proposed is that the marked increase in proliferation of intestinal bacteria as a result of repeated CBH intake would promote the biological defense system involving the increase in glutathione level. This mechanism should be investigated in the future. In conclusion, the results of this study suggest that dietary CBH suppresses the development of hepatic injury induced by GalN in rats, and that at least part of its effect is attributable to increases in hepatic glutathione level.
References [1] Li J, Kaneko T, Qin LQ, Wang J, Sato A. Long-term effects of high dietary fiber intake on glucose tolerance and lipid metabolism in GK rats: comparison among barley, rice, and cornstarch. Metabolism 2003;52:1206 –10. [2] Yamada K, Tokunaga Y, Ikeda A, Ohkuura K, Kaku-Ohkura S, Mamiya S, et al. Effect of dietary fiber on the lipid metabolism and immune function of aged Sprague-Dawley rats. Biosci Biotechnol Biochem 2003;67:429 –33. [3] BO Lim, Yamada K, Nonaka M, Kuramoto Y, Hung P, M Sugano. Dietary fibers modulate indices of intestinal immune function in rats. J Nutr 1997;127:663–7. [4] Kelly-Quagliana KA, Nelson PD, Buddington, RK. Dietary oligofructose and inulin modulate immune functions in mice. Nutr Res 2003;23:257– 67. [5] Wang B, Egashira Y, Ohota T, Sanada H. Effect of indigestible oligosaccharides on the hepatotoxic action of D-galactosamine in rats. Biosci Biotechnol Biochem 1998;62:1504 –9. [6] Miyasaka H, Sanada H. Alterations of cytoplasmic enzyme activities of liver in rats fed on diets containing orotic acid with dietary fibers. Comp Biochem Physiol 1992;3:555–7. [7] Keppler D, Lesch R, Reutter W, Decker K. Experimental hepatitis induced by D-galactosamine. Exp Mol Pathol 1968;9:279 –90. [8] Decker K, Keppler D, Pausch J. The regulation pyrimidine nucleotide level and its role in experimental hepatitis. Adv Enzyme Regul 1973;11:205–30. [9] Hofmann F, Wilkening J, Nowack J, Decker K. Responses of isolated rat hepatocytes to D-galactosamine and uridine. Hoppe-Seyler Z Physiol Chem 1976;357:427–33. [10] Keppler D, Rudigier J, Bischoff E, Decker K. The trapping of uridine phosphates by D-galactosamine, D-galactosamine and 2-deoxy-Dgalactosamine. A study on the mechanism of galactosamine hepatitis. Eur J Biochem 1970;17:246 –53.
A. Daizo et al. / Nutrition 21 (2005) 1044 –1051 [11] Galun E, Zeira E, Pappo O, Peters M, Rose-Jhon S. Liver regeneration induced by a designer human IL-6/sIL-6R fusion protein reverses severe hepatocellular injury. FASEB J 2000;14:1979 – 87. [12] Jeffrey D, Leonore AH, Kristine V, Carl W. Glutathione levels in antigen-presenting cells modulate Th1 versus Th2 response patterns. Proc Natl Acad Sci USA 1998;6:3071– 6. [13] Adawi D, Ahrne S, Molin G. Effects of different probiotic strains of Lactobacillus and Bifidobacterium on bacterial translocation and liver injury in an acute liver injury model. Int J Food Microbiol 2001;70: 213–20. [14] Francis RJ, Bornet MD, Brouns F. Immune-stimulating and gut health-promoting promoting properties of short-chain fractooligosaccharides. Nutr Rev 2002;60:326 –34. [15] Hu HL, Chen RD. Changes in free radicals, trace elements, and neurophysiological function in rats with liver damage induced by D-galactosamine. Biol Trace Elem Res 1992;34:19 –25. [16] Sun F, Hamagawa E, Tsutui C, Sakaguchi N, Kakuta Y, Tokumaru S, Kojo S. Evaluation of oxidative stress during apoptosis and necrosis caused by D-galactosamine in rat liver. Biochem Pharmacol 2003; 65:101–7. [17] Hu HL, Chen RD, Ma LH. Protective effect of zinc on liver injury induced by D-galactosamine in rat. Biol Trace Elem Res 1992;34: 27–33. [18] Lim HK, Kim HS, Choi HS, Oh S, Jang CG, Choi J, et al. Effects of bergenin, the major constituent of Mallotus japonicus against Dgalactosamine–induced hepatotoxicity in rats. Pharmacology 2001; 63:71–5. [19] American Institute of Nutrition. Report of the American Institute of Nutrition ad hoc committee on standards for nutritional studies. J Nutr 1977;107:1340 – 8. [20] National Research Council. Guide for the care and use of laboratory animals. Publication no. 85-23. Washington, DC: National Institutes of Health; 1985. [21] Stifter S, Dton S, Novic B, Muntiozler E. The estimation of glycogen with the anthrone reagent. Arch Biochem 1950;25:191–7. [22] Reed DJ, Babson JR, Beatty PW, Brodie AE, Ellis WW, Potter DW. High-performance liquid chromatography analysis of nanomole levels of glutathione, glutathione disulfide, and related thiols and disulfides. Anal Biochem 1980;106:55– 62. [23] Decker K, Keppler D. Galactosamine hepatitis: key role of the nucleotide deficiency period in the pathogenesis of cell injury and cell death. Rev Physiol Biochem Pharmacol 1974;71:77–106.
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[24] Ozawa A, Ohnishi N, Ikigai H, Tazume S, Sawamura S, Watanabe K, Izumi S. The role intestinal bacteria in host defense mechanisms against infection. In: Mitsuoka T, editor. Intestinal flora and self-defense. Tokyo: Japan Scientific Societies; 1982, p. 191–211. [25] Grün M, Liehr H, Rasenack U. Significance of endotoxemia in experimental “galactosamine-hepatitis” in the rat. Acta Hepatogastroenterol 1976;23:64 – 81. [26] Wang B, Cheng C, Egashira Y, Ohta T, Sanada H. Effect of oligosaccarides on galactosamine hepatitis in rats. J Jpn Soc Nutr Food Sci 1995;48:181–7. [27] He P, Noda Y, Sugiyama K. Green tea suppresses lipopolysaccharideinduced liver injury in D-galactosamine–sensitized rats. J Nutr 2001; 131:1560 –7. [28] Manabe A, Chen C, Egasura Y, Ohta T, Sanada H. Dietary wheat gluten alleviates the elevation of serum transaminase activities in D-galactosamine–injected rats. J Nutr Sci Vitaminol 1996;42:121– 32. [29] Lawrence BP, Meyer M, Reed, DJ, Kerkvliet NI. Role of glutathione and reactive oxygen intermediates in 2,3,7,8-tetrachlorodibenzo-pdioxin–induced immune suppression in C57Bl/6 mice. Toxicol Sci 1999;52:50 – 60. [30] Park EJ, Zhao YZ, Na M, Bae K, Kim YH, Lee BH, Sohn DH. Protective effects of honokiol and magnolol on tertiary butyl hydroperoxide-or D-galactosamine–induced toxicity in rat primary hepatocytes. Planta Med 2003;69:33–7. [31] Fernandes AP, Holmgren. A. Glutaredoxins: glutathione-dependent redox enzymes with functions far beyond a simple thioredoxin backup system. Antioxid Redox Signal 2004;6:63–74. [32] Francis RJ, Bornet MD, Brouns F. Immune-stimulating and gut health-promoting promoting properties of short-chain fractooligosaccharides. Nutr Rev 2002;60:326 –34. [33] Swanson KS, Grieshop CM, Flickinger EA, Bauer LL, Healy HP, Dawson KA, et al. Supplemental fructooligosaccharides and mannanoligosaccharides influence immune function, ileal and total tract nutrient digestibilities, microbial populations and concentrations of protein catabolites in the large bowel of dogs. J Nutr 2002;132: 980 –9. [34] Lim HK, Kim HS, Choi HS, Oh S, Jang CG, Choi J, et al. Effects of acetylbergenin against D-galactosamine–induced hepatotoxicity in rats. Pharmacol Res 2000;42:471– 4.
Basic nutritional investigation
Suppressive effect of corn bran hemicellulose on liver injury induced by D-galactosamine in rats Ayako Daizo, M.S.*, Yukari Egashira, Ph.D., and Hiroo Sanada, Ph.D. Laboratory of Food and Nutrition, Graduate School of Science and Technology, Chiba University, Chiba, Japan Manuscript received August 16, 2004; accepted February 5, 2005.
Abstract
Objective: It is well known that the indigestible oligo- and polysaccharides including dietary fiber are important food components and that they have many physiologic functions. This study examined the effect of water-soluble corn bran hemicellulose (CBH) on the development of D-galactosamine (GalN) hepatitis in rats to obtain some knowledge about new functions of dietary fiber. Methods: Male Wistar rats were fed diets containing 5% CBH for various days (1 to 14 d). On the final day of feeding rats were treated with GalN (400 mg/kg), and their plasma transaminase (aspartate and alanine aminotransferases) activity (6 or 24 h later) and liver glutathione concentration (6 h later) were determined. Results: Ingested CBH suppressed the increase in plasma aspartate and alanine aminotransferase activities 24 h after GalN treatment. Such suppressive effect was observed only 7 d after CBH ingestion and not after 1 or 3 d. In the early phase of the liver injury, at 6 h after GalN treatment, the liver glutathione concentration in the CBH group was significantly higher than that in the control group, and the concentration in the CBH group after GalN injection was almost the same as that in the control group without GalN treatment. Conclusion: Results suggest that dietary CBH suppresses the development of hepatic injury by GalN in rats and that this phenomenon is partly attributable to the increase in hepatic glutathione concentration by CBH. © 2005 Elsevier Inc. All rights reserved.
Keywords:
Corn bran hemicellulose; Liver injury; D-galactosamine; Glutathione
Introduction It is well known that the indigestible oligo- and polysaccharides including dietary fiber are important food components. Because these indigestible saccharides have been reported to affect lipid and carbohydrate metabolism [1] and immunologic function [2], it is considered that they must have an effect on liver function and liver injury. However, research on these indigestible saccharides in relation to liver injury or function has been inadequate. The effects of oligosaccharides and dietary fiber on various protective functions of organisms have recently been reported [3,4], and our previous study showed that intake of oligosaccharides, especially of oligosaccharides containing D-galactose, decreases the increase in plasma transaminase activities in rats
* Corresponding author. Tel.: ⫹81-47-365-1111; fax: 81-47-363-1401. E-mail address: [email protected] 0899-9007/05/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2005.02.009
injected with D-galactosamine (GalN), which causes hepatitis in experimental animals [5]. Moreover, dietary corn bran hemicellulose (CBH) has been shown to prevent the development of orotic acid–induced fatty liver [6]. However, these functions of indigestible dietary polysaccharides have not been investigated in detail. Arabinoxylane hemicellulose is an indigestible polysaccharide that is abundant in cereals commonly consumed by humans, and in the present study we investigated the effect of CBH, mainly consisting of arabinoxylane hemicellulose, on GalN-induced liver injury. The CBH preparation used contained uronic acid (13.5%) and galactose (6.7%) as constituents of dietary fiber (Table 1). Morphologic and pathophysiologic characteristics of GalN-induced liver damage are very similar to those of human viral hepatitis. GalN induces hepatotoxicity by inhibiting RNA and protein synthesis through a decrease in cellular uridine triphosphate concentration, which ultimately leads to liver cell necrosis [7–10]. In contrast, there
A. Daizo et al. / Nutrition 21 (2005) 1044 –1051 Table 1 Composition of the corn bran hemicelluouse preparation Water Protein Lipid Ash Fiber* Other Arabinose Xylulose Galactose Glucose Uronic acid
3.8% 0.6% 0.1% 1.5% 86.0% 8.0% 31.5% 45.7% 6.7% 2.6% 13.5%
* Relative content of constitutional monosaccharide residues.
have been several reports that endotoxin is involved in the induction of this hepatic injury and promotes the secretion of cytokines, such as tumor necrosis factor-␣ and interleukin-6, by Kupffer cells [11,12]. Based on these findings, hepatitis may develop as a result of an overlap between its impairment of hepatocyte metabolism and its immunologic action, and it seems to destroy hepatocytes first by apoptosis and then by necrosis. Because certain probiotic bacterial strains are capable of decreasing bacterial translocation and hepatocellular damage after GalN injection [13], it has been suggested that dietary oligosaccharides have an effect on the microflora and bacterial translocation that results in protection against hepatic injury caused by GalN. Oligosaccharides and dietary fiber may affect immune function, probably through a change in intestinal bacteria or short-chain fatty acids generated by intestinal bacteria, and these dietary substances may affect the development of GalN-induced hepatitis immunologically[14]. Further, it has been reported that thiobarbituric acid reacting substances, which are generated through free radical reactions, increase during the process of necrosis caused by GalN [15], and concentrations of vitamin C and glutathione in the concerned tissues have been shown to decrease under this oxidative stress [16,17]. Bergenin, a constituent of Mallotus japonicus, has also been found to protect rats against GalN-induced hepatitis, and this effect is thought to be attributable in part to maintaining adequate hepatic glutathione levels [18]. The present study assessed the effect of water-soluble CBH on induction of hepatic injury by GalN in relation to the mechanism of its hepatotoxic action. Tissue glutathione levels were measured in addition to plasma transaminase activities as indices of hepatitis to clarify the relation to oxidative stress.
Materials and methods Materials GalN hydrochloride and transaminase C-II test from Wako Pure Chemicals Industries (Osaka, Japan), and so-
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dium pentobarbital (Nembutal) solution from Dinabot (Osaka, Japan) were commercially available. Water-soluble CBH, Cell-ace, was a kind gift from Nihon Shokuhin Kako Co., Ltd. (Tokyo, Japan). Other reagents were purchased from Wako Pure Chemicals Industries. Animals and feeding In our previous preliminary experiments we observed a wide variety of sensitivity to GalN and dietary fiber among rats obtained from different suppliers. Because the clean (pathogen-free) male Wistar rats obtained from Japan SLC (Hamamatsu, Japan) were found to be the most sensitive of the rats examined, 4-wk-old male rats obtained from Japan SLC were used in this study. Rats were kept in an environmentally controlled room at a temperature of 22 ⫾ 1°C on a 12-h light, 12-h dark cycle (light from 7:00 AM to 7:00 PM). Compositions of the mineral and vitamin mixtures used in this study were the same as those of AIN-76 [19]. Three separate experiments were performed. All rats were fed a commercial diet (type CE-2, CLEA, Tokyo, Japan) for 3 d and then a control (Cont) diet (Table 2) for 4 d. Rats were then assigned to one of several groups and fed the experimental diets whose composition is presented in Table 2. All rats were allowed free access to their diets and drinking water except for the period around the time of the GalN injection, as described below. In experiment 1, 15 rats in two groups were fed the Cont diet (n ⫽ 7) or the CBH diet (n ⫽ 8) for 14 d and then injected with GalN (400 mg/kg of body weight) intraperitoneally at 1:00 PM on 14 d. Diets were withheld for 4 h before and after the GalN injection (8 h of deprivation in total). Twenty-four hours after the GalN treatment, blood was drawn from the posterior vena cava into a heparinized syringe under anesthesia with Nembutal (0.13 mL/100 g of body weight), and livers were quickly excised. Blood samples were immediately centrifuged at 3000 rpm for 20 min, and plasma samples were stored at ⫺20° until analyzed. In experiment 2, the effect of feeding duration of the
Table 2 Compositions of the experimental diets (%)
Casein Corn bran hemicellulose Cornstarch* Sucrose DL-methionine Corn oil Vitamin mixture† Mineral mixture† Cellulose Choline bitartrate
Cont
CBH
20 — 40 25 0.3 5 1 3.5 5 0.2
20 5 35 25 0.3 5 1 3.5 5 0.2
CBH, diet containing 5% corn bran hemicellulose; Cont, control diet. * Cornstarch was digestible gelatinized cornstarch. † Composition of the AIN-76 diet (1977).
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CBH diet on the response of rats to the hepatotoxic action of GalN was examined. Because the results of the preliminary experiment suggested that the CBH diet exerted a suppressive effect not only after 14 d of feeding but also after 7 d of feeding, a feeding period of 7 d was used in experiments 2 and 3. Thirty-six rats were assigned to one of three CBH diet groups (18 rats) and three Cont diet groups (18 rats). The CBH diet groups were given the CBH diet for 1, 3, or 7 d after 6, 4, or 0 d, respectively, on the Cont diet (7 d in total). Then GalN was administered to all animals at 1:00 PM on the final day of the CBH diet period to avoid agedependent variations in responses. The corresponding Cont groups were given the Cont diet instead of the CBH diet. All rats were killed 24 h after administration of GalN. GalN injection, blood collection, and dissection were performed in the same manner as in experiment 1. The liver was quickly excised, immediately frozen in liquid nitrogen, and stored at ⫺80°C until analyzed. In experiment 3, the effect of the CBH diet on the concentration of glutathione in rat tissues was assessed 6 h after the GalN injection, in the early phase of liver injury, to elucidate the mechanism of the suppressive action of CBH on GalN-induced hepatitis in relation to oxidative stress. Seventeen rats were fed the Cont diet (n ⫽ 8) or the CBH diet (n ⫽ 9) for 7 d. Fifty percent of the rats in the Cont diet group were then injected with GalN solution or the same volume of saline (Cont/⫺GalN group, n ⫽ 4), and four or five of the rats in the CBH diet group were then injected with GalN solution or the same volume of saline (CBH/ ⫹GalN group, n ⫽ 5), in the same manner as described in experiment 1 at 3:00 PM on the final day of the feeding period. Their diets were removed 4 h before GalN injection, and they were kept fasting thereafter. Six hours after GalN injection, dissection and blood collection were performed as described in experiment 1. The liver and spleen were rapidly excised, immediately frozen in liquid nitrogen, and stored at ⫺80° until analyzed. In all experiments the Cont and CBH diet groups with or without GalN injection are referred to as the Cont/⫹GalN, CBH/⫹GalN, Cont/⫺GalN, and CBH/ ⫺GalN groups, respectively. In experiment 2, the group that was injected with GalN after being fed the CBH diet for 7 d is referred to as the 7-d/CBH/⫹GalN group, and the other groups in experiment 2 are also expressed by the abbreviations in the same manner. The care and treatment of the rats were in accordance with the guidelines prescribed by the Faculty of Horticulture, Chiba University, and National Institutes of Health Guide for the Care and Use of Laboratory Animals [20]. Determination of blood aspartate and alanine aminotransferases Plasma activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in all experimental animals were assayed with commercial assay kits (Transaminase C-II-test, Wako Pure Chemicals Industries).
Table 3 Weight gain, feed efficiency, and plasma AST and ALT activities of animals 24 h after GalN Injection (experiment 1)* Diet Weight gain (g/d) Feed efficiency† (%) Plasma transaminase activity ALT (U/L) AST (U/L)
Cont/ ⫹ GalN (n ⫽ 7) 5.08 ⫾ 0.14 41.68 ⫾ 0.85 484.47 ⫾ 75.28 1354.32 ⫾ 178.88
CBH/ ⫹ GalN (n ⫽ 8) 5.40 ⫾ 0.14 44.15 ⫾ 0.61 202.42 ⫾ 54.45‡ 537.83 ⫾ 157.68‡
ALT, alanine aminotransferase; AST, aspartate aminotransferase; CBH, corn bran hemicellulose diet; Cont, control diet; GalN, D-galactosamine. * All values are means ⫾ standard errors of the mean. † Body weight gain/feed intake. ‡ Significantly different (P ⬍ 0.05) from the corresponding values in the Cont ⫹ GalN group.
Determination of hepatic glycogen Hepatic glycogen was measured in experiment 2 by the anthrone method [21]. Determination of glutathione Concentrations of reduced (GSH) and oxidized (GSSG) forms of glutathione in the liver and spleen were measured in experiment 3. The procedure is based on the initial formation of S-carboxymethyl derivatives of free thiols with iodoacetic acid followed by conversion of free amino groups to 2,4-dinitrophenyl derivatives by reaction with 1-fluoro-2,4-dinitrobenzene. The reaction mixture was chromatographed on a 3-aminopropylsilane– derivatized silica column and eluted with a sodium or ammonium acetate gradient in a water/methanol/acetic acid solvent at pH 4.5 [22]. Statistics All values obtained in experiment 1 were evaluated by Student’s t test. Two-way analysis of variance (ANOVA), and in some instances Student’s t test was used to perform the statistical analyses in experiments 2 and 3. The level of statistical significance was P ⬍ 0.05.
Results Effect of CBH diet fed for14 d after GalN-inducted hepatitis (experiment 1) Body weight gain, feed efficiency, and plasma AST and ALT activities in each group in experiment 1 are presented in Table 3. Body weight gain and feed efficiency values in the Cont and CBH groups were almost the same, respectively. However, plasma AST and ALT activity values in the CBH/⫹GalN group were significantly lower than those
A. Daizo et al. / Nutrition 21 (2005) 1044 –1051 Table 4 Weight gain, feed efficiency, carcass weight, liver weight, and liver ratio of animals 24h after GalN injection (experiment 2)* Cont/⫹GalN Weight gain (g/d) 1d 3d 7d Feed efficiency† (%) 1d 3d 7d Carcass weight (g) 1d 3d 7d Liver weight (g) 1d 3d 7d Liver ratio‡ (%) 1d 3d 7d
CBH/⫹GalN
4.41 ⫾ 0.94 3.66 ⫾ 0.33 4.47 ⫾ 0.12
5.08 ⫾ 0.52 4.38 ⫾ 0.20§ 5.45 ⫾ 0.29§
31.01 ⫾ 6.54 25.34 ⫾ 2.01 31.34 ⫾ 0.65
34.87 ⫾ 2.78 32.71 ⫾ 1.43§ 39.62 ⫾ 1.15§
119.16 ⫾ 3.15 118.00 ⫾ 3.37 116.16 ⫾ 2.88
119.33 ⫾ 3.14 116.50 ⫾ 2.85 117.33 ⫾ 2.47
7.17 ⫾ 0.67 5.88 ⫾ 0.23 5.91 ⫾ 0.21
5.96 ⫾ 0.30 6.32 ⫾ 0.38 7.29 ⫾ 0.69
4.96 ⫾ 0.27 4.98 ⫾ 0.11 5.04 ⫾ 0.17
5.27 ⫾ 0.2 5.43 ⫾ 0.32 6.24 ⫾ 0.49
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7-d/Cont/⫹GalN group, but enzyme activities in 1-d and 3-d/Cont/⫹GalN groups were not significantly different from those in the corresponding CBH/⫹GalN groups. Although enzyme activities were affected by the diets and interaction effects (diets by feeding periods) when analyzed by two-way ANOVA, they were not influenced by feeding period. However, results of one-way ANOVA (with the factor of feeding periods) indicated that plasma AST activity in the CBH groups decreased significantly as the feeding period increased. Moreover, as presented in Table 5, liver glycogen concentration in the 7-d/CBH/⫹GalN group was significantly higher than that in the 7-d/Cont/⫹GalN group. The results of two-way ANOVA also indicated that liver glycogen concentration was significantly affected by the diets but not by the feeding periods. These results indicate that the preventive effect of CBH diet against GalN-induced
CBH, corn bran hemicellulose diet; Cont, control diet; GalN, Dgalactosamine * All values are means ⫾ standard errors of the mean (n ⫽ 6). † Body weight gain/feed intake. ‡ Liver ratio: (Liver weight/Carcass weight) ⫻ 100 § Significantly different (P ⬍ 0.05) from the value in the Cont ⫹ GalN group in the corresponding feeding period.
in the Cont/⫹GalN group, indicating that the CBH diet promoted protective activity against GalN-induced hepatitis. Effect of duration of feeding on the CBH diet (experiment 2) In experiment 2, differences in resistance of rats to GalNinduced hepatitis according to duration of feeding on the CBH diet were examined in relation to hepatic glycogen concentration. Body weight gain, feed efficiency, carcass weight, liver weight, and liver ratio in each group are presented in Table 4. Although mean initial body weights in all groups were almost the same (data not shown), body weight gain and feed efficiency were significantly higher in the 3-d and 7-d/CBH/⫹GalN groups than in the 3-d and 7-d/Cont/ ⫹GalN groups (Student’s t test). No significant differences in carcass weight, liver weight, or liver ratio were observed between the Cont/⫹GalN and CBH/⫹GalN groups irrespective of feeding duration. Analysis by two-way ANOVA (factors of diets and feeding periods) showed that feed efficiency and liver ratio were significantly affected by the diets, and that liver weight was affected by the interaction effect (diets by feeding periods) of the two factors, but none of the parameters listed in Table 4 were significantly altered by feeding period. As shown in Fig. 1, plasma AST and ALT activities were significantly lower in the 7-d/CBH/⫹GalN group than in the
Fig. 1. Effect of duration of CBH diet intake on plasma transaminase activities after GalN treatment (experiment 2). Animals were fed a 5% CBH diet (white bars) or the Cont diet (black bars) for 1, 3, or 7 d before injection of GalN, as described in MATERIALS AND METHODS. Values are expressed as mean ⫾ standard error of the mean, with n ⫽ 5 in the 7-d/Cont/⫺GalN and CBH/⫺GalN groups and n ⫽ 6 in the other groups. * Significantly different (P ⬍ 0.05) from the value in the Cont/⫹GalN groups in the corresponding feeding periods. ALT, alanine aminotransferase; AST, aspartate aminotransferase; CBH, corn bran hemicellulose diet; Cont, control diet; GalN, D-galactosamine.
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Table 5 Glycogen concentration (milligrams) in livers (grams) of animals 24 h after GalN injection (experiment 2)* Diet period
Cont/⫹GalN
CBH/⫹GalN
1d 3d 7d
0.27 ⫾ 0.29 0.60 ⫾ 0.18 2.69 ⫾ 1.37
19.95 ⫾ 8.40 21.32 ⫾ 7.89 35.91 ⫾ 8.59†
CBH, corn bran hemicellulose diet; Cont, control diet; GalN, D-galactosamine * All values are means ⫾ standard errors of the mean (n ⫽ 5 for 7-d CBH ⫹ GalN group; n ⫽ 6 for the other groups). † Significantly different (P ⬍ 0.05) from the value in the Cont ⫹ GalN group in the corresponding feeding period.
hepatitis is observed after successive feeding of the diet for longer than 7 d. Influence of CBH diet on hepatic glutathione concentration 6 h after GalN injection (experiment 3) In experiments 1 and 2 plasma transaminase activities were assayed 24 h after GalN injection. In experiment 3, however, the hepatic glutathione concentration in rats fed the CBH diet was measured 6 h after GalN injection, a relatively early phase of the hepatic injury, to explore the mechanism by which the CBH diet attenuates induction of hepatitis by GalN. Body weight gain, feed efficiency, carcass weight, liver weight, liver ratio, cecum weight, and cecum ratio in each group are presented in Table 6. Body weight gain, feed efficiency, and carcass weight were not significantly different across groups. Liver weight was significantly decreased by the CBH diet but was unaffected by
GalN. In contrast, liver ratio was affected by the CBH diet and GalN and was higher in the Cont/⫹GalN and CBH/ ⫹GalN groups than in the Cont/⫺GalN and CBH/⫺GalN groups. These results obtained 6 h after GalN administration differed from the results obtained at 24 h in experiment 2. Also, the liver ratio in the CBH/⫹GalN and CBH/⫺GalN groups was lower than that in the Cont/⫹GalN and Cont/ ⫺GalN groups. Cecum weight and its ratio to carcass weight were increased by the CBH diet, suggesting fermentation of CBH in the cecum. Plasma AST and ALT activities were affected by GalN treatment but not by diet, and thus the activities in the Cont/⫹GalN and CBH/⫹GalN groups were higher than those in the Cont/⫺GalN and CBH/⫺GalN groups. Table 7 presents hepatic and splenic concentrations of GSH and GSSG of the animals in experiment 3. Concentrations of hepatic GSH and GSSG were decreased by GalN, and the total GSH plus GSSG concentration was increased by the CBH diet. Thus, concentrations of GSH, GSSG, and their sum tended to be lower in the Cont/⫹GalN and CBH/ ⫹GalN groups than in the Cont/⫺GalN and CBH/⫺GalN groups, and concentrations in the CBH/⫺GalN and CBH/ ⫹GalN groups tended to be higher than those in the Cont/ ⫺GalN and Cont/⫹GalN groups. In contrast, splenic concentrations of GSH and GSSG were not significantly affected by GalN or the CBH diet according to the results of ANOVA. In the Cont diet groups, however, concentrations of GSH, GSSG, and GSH plus GSSG (total) were significantly lower in the Cont/⫹GalN group than in the Cont/⫺GalN group according to Student’s t test.
Table 6 Weight gain, feed efficiency, carcass weight, liver weight, liver ratio, cecum weight, cecum ratio, and plasma transaminase activities 6 h after GalN injection (experiment 3)* Cont
Weight gain (g/d) Feed efficiency† (%) Carcass weight (g) Liver weight‡ (g) Ratio (%) Cecum weight§ (g) Ratio (%) ALT (U/L) AST (U/L)
ANOVA#
CBH
⫺GalN
⫹GalN
⫺GalN
⫹GalN
GalN
Diet
GalN ⫻ diet
4.03 ⫾ 0.71 36.93 ⫾ 5.30 121.5 ⫾ 2.25 6.11 ⫾ 0.13 5.03 ⫾ 0.13 1.74 ⫾ 0.08 1.43 ⫾ 0.06 5.75 ⫾ 0.36 30.51 ⫾ 0.86
3.75 ⫾ 0.45 38.29 ⫾ 4.09 120.75 ⫾ 2.28 6.69 ⫾ 0.38 5.54 ⫾ 0.27 2.01 ⫾ 0.14 1.66 ⫾ 0.11 38.51 ⫾ 6.40 94.3 ⫾ 9.32储
4.17 ⫾ 0.15 39.23 ⫾ 1.38 118.75 ⫾ 3.11 5.23 ⫾ 0.19 4.40 ⫾ 0.12 3.58 ⫾ 0.08 3.02 ⫾ 0.13 8.13 ⫾ 1.20 36.43 ⫾ 10.41
4.22 ⫾ 0.34 41.65 ⫾ 1.35 119.00 ⫾ 4.59 5.84 ⫾ 0.43 4.88 ⫾ 0.21 4.74 ⫾ 0.71 4.02 ⫾ 0.60 32.96 ⫾ 12.08 82.30 ⫾ 22.4¶
NS NS NS NS ⬍0.05 NS NS ⬍0.05 ⬍0.05
NS NS NS ⬍0.05 ⬍0.05 ⬍0.05 ⬍0.05 NS NS
NS NS NS NS NS NS NS NS NS
ALT, alanine aminotransferase; ANOVA; analysis of variance; AST, aspartate aminotransferase; CBH, corn bran hemicellulose diet; Cont, control diet; GalN, D-galactosamine; NS, not significant * All values are means ⫾ standard errors of the mean (n ⫽ 5 in the CBH ⫹ GalN group; n ⫽ 4 in the other groups). † Body weight gain/Feed intake ‡ (Liver weight/carcass weight) ⫻ 100. § (Cecum weight/carcass weight) ⫻ 100. 储 Significantly different (P ⬍ 0.05) from the values in the Cont ⫺ GalN group. ¶ Significantly different (P ⬍ 0.05) from the values in the CBH ⫺ GalN group. # Statistical significance was analyzed by two-way ANOVA, using GalN and diet as factors.
A. Daizo et al. / Nutrition 21 (2005) 1044 –1051
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Table 7 Influence of CBH ingestion on glutathione concentration in the liver and spleen 6 h after GalN injection (experiment 3)* Cont
Liver (mol/g) GSH GSSG Total Spleen (mol/g) GSH GSSG Total
ANOVA§
CBH
⫺GalN
⫹GalN
⫺GalN
⫹GalN
GalN
Diet
GalN ⫻ diet
3.26 ⫾ 0.63 0.65 ⫾ 0.16 3.91 ⫾ 0.79
2.11 ⫾ 0.49 0.36 ⫾ 0.09† 2.47 ⫾ 0.58†
4.02 ⫾ 0.36 1.03 ⫾ 0.04 5.50 ⫾ 0.39
2.97 ⫾ 0.15‡ 0.79 ⫾ 0.04‡ 3.76 ⫾ 0.15‡
⬍0.05 ⬍0.05 NS
NS ⬍0.05 ⬍0.05
NS NS NS
1.04 ⫾ 0.05 0.96 ⫾ 0.07 2.00 ⫾ 0.11
0.81 ⫾ 0.13† 0.81 ⫾ 0.09† 1.62 ⫾ 0.21†
1.20 ⫾ 0.13 1.33 ⫾ 0.19 2.52 ⫾ 0.32
1.02 ⫾ 0.12 1.12 ⫾ 0.26 2.14 ⫾ 0.34
NS NS NS
NS NS NS
NS NS NS
ANOVA, analysis of variance; CBH, corn bran hemicellulose diet; Cont, control diet; GalN, D-galactosamine; GSH, reduced glutathione; GSSG, oxidized glutathione; NS, not significant; Total, GSH ⫹ GSSG * All values are means ⫾ standard errors of the mean (n ⫽ 5 in the CBH ⫹ GalN group; n ⫽ 4 in the other groups). † Significantly different (P ⬍ 0.05) from the value in the Cont ⫺ GalN group by Student’s t test. ‡ Significantly different (P ⬍ 0.05) from the value in the CBH ⫺ GalN group by Student’s t test. § Effects of diets and GalN were analyzed for statistical significance by two-way ANOVA.
Discussion Intake of the CBH diet for 14 d was found to prevent GalN-induced hepatic injury, as shown by the significantly lower plasma AST and ALT activities in the CBH/⫹GalN group than in the Cont/⫹GalN group (Table 3). The morphologic features of GalN-induced liver damage have been shown to be very similar to those of human viral hepatitis [23], and the damage generally reflects disturbances of hepatic cell metabolism, which lead to increases in blood levels of AST and ALT, lactate dehydrogenase, and ␥-glutamyl transferase as a result of hepatic cell destruction or changes in membrane permeability. The effect of duration of the CBH diet intake on GalNinduced hepatic injury was examined in experiment 2. Results of Student’s t test showed that a significant suppressive effect of CBH intake on GalN-induced hepatitis was recognized only when CBH diet was given for 7 d, and not for 1 or 3 d. Analysis of the data shown in Fig. 1 by one-way ANOVA showed that only AST activity in the CBH/⫹GalN group was significantly decreased as the feeding period increased and that the other activities were unaffected by the length of the feeding period, probably because of variations in values among rats. Changes in intestinal bacteria microflora associated with CBH intake, which are also generally considered to be same as the effects of many kinds of dietary fiber, are thought to be involved in their action. Because there is a report of variable changes in host-defense function that may be related to the state of intestinal bacteria microflora [24], it is thought that any physiologic alterations associated with the change in intestinal bacteria microflora would probably make rats more resistant to GalN hepatotoxicity and that the change would occur when CBH was ingested for longer than 7 d. Although cecum weight was not measured in experiment 2, it was significantly lower in the CBH/⫺GalN group than in the Cont/⫺GalN group in experiment 3. In addition, Wang et al. [5] reported that
GalN-induced hepatic injury was suppressed when rats had consumed a diet containing raffinose or galactooligosaccharide for 14 d, and that their cecum weight increased in comparison with control rats. Indigestible oligosaccharides in addition to indigestible polysaccharides have been shown to pass through intestine to the cecum and colon, where they ferment to produce short-chain fatty acids, such as lactic, acetic, propionic and butyric acids, and these short-chain fatty acids convert the pH of the cecum and colon to acidic. The suppressive effect of dietary CBH on induction of GalN-induced hepatitis may be attributable in part to the short-chain fatty acids absorbed or to the decrease in enteric gram-negative bacteria, such as Escherichia coli, that possess lipopolysaccharide under acidic conditions. Although translocation of bacteria from the gut into the systemic circulation has been considered to be partly involved in the development of GalN-induced hepatitis [25], the role of short-chain fatty acids in the suppression of hepatitis has never been confirmed. A similar suppressive effect of raffinose and galactooligosaccharide intake has been observed on GalN-induced hepatic injury, but not by fructo-oligosaccharide or gentiooligosaccharide [26], despite the fact that at least the first three of these saccharides have been found to promote proliferation of Bifidobacterium and production of shortchain fatty acids. The glycogen concentration in the liver (Table 5) after GalN treatment seemed to be inversely correlated with plasma AST or ALT activity (Fig. 1), and the concentration was significantly higher in the 7-d/CBH/⫹GalN group than in the 7-d/Cont/⫹GalN group. Deviations in glycogen concentrations in the 1-d and 3-d/CBH/⫹GalN groups were greater than those in the other groups (Table 5), suggesting that the rate of time-dependent reduction in susceptibility to GalN by the CBH diet may vary widely among rats in these periods. The protective effect of dietary CBH against GalNinduced hepatic injury was demonstrated in experiments 1
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and 2, and experiment 3 was performed to clarify the mechanism. In this experiment, we determined the glutathione concentration in the liver because GalN-induced hepatitis is thought to be caused in part by oxidative stress in the liver [16]. We also determined glutathione level in the spleen to explore the relation between immunologic function and protective activity of CBH against GalN-induced hepatic injury because there have been reports suggesting that tumor necrosis factor-␣ [12], endotoxin, and some cytokines [27] are involved in the development of GalN-induced hepatitis. In the present experiment, glutathione levels were measured 6 h after GalN administration, at a relatively early phase of the development of hepatic injury. Manabe et al. [28] estimated that plasma AST and ALT activities begin to increase around 6 h after GalN injection. Thus, the enzyme activities at that time after GalN injection were only slightly higher than those without GalN (Table 6), whereas enzyme activities 24 h after GalN administration in experiments 1 and 2 were significantly increased. The results of ANOVA indicated that plasma AST and ALT activities 6 h after GalN injection was not significantly affected by dietary CBH, in contrast to the results in experiments 1 and 2, in which CBH was shown to decrease the increase in enzyme activity 24 h after GalN injection. Liver weight was decreased by CBH intake but unaffected by GalN injection. Conversely, the liver ratio was increased by GalN injection and decreased by CBH ingestion. Some studies have suggested that liver weight is decreased 24 h after GalN injection as a result of atrophy accompanied by necrosis [26], but the finding that the liver weight had not decreased 6 h after GalN injection in this study (Table 6) demonstrates that atrophy had not yet occurred. Thus, the relation between liver ratios in the Cont/⫹GalN and CBH/⫹GalN groups in experiment 2 appears to be different from their relation in experiment 3, but the reason liver weight was decreased by CBH ingestion is unknown. Although the details of the mechanism of the development of this hepatitis-like injury by GalN have not been elucidated, the mechanism has been reported to involve free radicals or the immune system. Because there are reports suggesting that the intracellular level of glutathione affects immunity [29], free radical production [30], DNA synthesis [31], and cytophylaxis [29], we examined the liver glutathione concentration in experiment 3. Concentrations of GSH or GSSG in the liver in the CBH/⫺GalN and CBH/ ⫹GalN groups were significantly higher than those in the Cont/⫺GalN and Cont/⫹GalN groups (Table 7), indicating that dietary CBH had caused the increase in hepatic glutathione level. Concentration of glutathione in the spleen, however, was not significantly affected by dietary CBH. Although concentrations of GSH and GSSG in the liver appeared to be decreased by GalN, they remained higher in the CBH/⫹GalN group than in the Cont/⫹GalN group. Because oxidative stress induced by GalN in hepatocytes has been reported to cause decreased levels of glutathione in cells [30], the increase in hepatic glutathione level by di-
etary CBH probably attenuates the development of GalNinduced hepatitis by decreasing oxidative stress. However, the fact that glutathione affects lymphocyte function [29] seems to suggest that attenuation of GalNinduced hepatitis is explained by the change in the immune system associated with the alteration in glutathione level. In addition, there are some reports indicating that immune function is altered by oligosaccharide intake [32,33]. Lim et al. [34] reported that acetyl bergenin restored decreased levels of hepatic GSH and decreased glutathione reductase and glutathione S-transferase activities induced by GalN, and that the protective effect of acetyl bergenin on GalN-induced hepatitis may be related to normalization mechanisms by maintaining adequate levels of GSH. The reason dietary CBH increased the hepatic glutathione level is unknown. One plausible mechanism that can be proposed is that the marked increase in proliferation of intestinal bacteria as a result of repeated CBH intake would promote the biological defense system involving the increase in glutathione level. This mechanism should be investigated in the future. In conclusion, the results of this study suggest that dietary CBH suppresses the development of hepatic injury induced by GalN in rats, and that at least part of its effect is attributable to increases in hepatic glutathione level.
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