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Journal o f Food Protection, Vol. 77, No. 8, 2014, Pages 1367-1371 doi: 10.4315/0362-028X.JFP-13-470 C o p yrig h t © , In ternatio nal A sso cia tio n fo r Food Protection
Inhibitory Effects of Deoxynivalenol on Gastric Secretion in Rats YUWEI WANG.'t WENDA W ll,'i XICHUN WANG,2 CHENGHUA HE,1 HUA YUE,3 ZHE REN, 1 a n d HAIBIN ZHANG1* 1College o f Veterinary Medicine, Nanjing Agricultural University, Nanjing, 210095, People’s Republic o f China; 2College o f Animal Science and Technology, Anhui Agricultural University, Hefei, 230036, People’s Republic o f China; and 2Henan Institutes o f Science and Technology, Xinxiang, 453003, People’s Republic o f China MS 13-470: Received 25 October 2013/Accepted 30 March 2014
ABSTRACT Deoxynivalenol (DON) is a common mycotoxin produced by Fusarium sp. in cereals and foods. Ingestion of contaminated foodstuffs can cause digestive disorders in various animals. Many researchers focus on its toxicity and the pathological damage and absorptive function in the intestines. However, the effect of DON on gastric function is still unclear. The objective of the current study was to evaluate the impact of DON on gastric secretion. Rats were gavaged with DON at the dose of 0, 1, 5, and 25 mg/kg of body weight (bw). Gastric fluids were gathered by pylorus ligation 0.5 h after DON exposure. The results indicate that the volume of gastric fluid decreased by 25, 51, and 61% compared with the control, respectively. The pH increased to 3.2, 3.81, and 6.65 in the 1, 5, and 25 mg/kg bw DON group, compared with the control (1.9). To examine the mucosal injuries, the stomach tissues were made into hematoxylin and eosin slides. Histopathology observations suggest that no mucosal lesions were observed until DON exposure at 25 mg/kg bw. Additionally, the gastrin secretion in the fluids and mRNA expression in tissues were determined by the radioimmunoassay and real-time PCR assay, respectively. The results indicated that both significantly decreased in DON-exposed rats compared with the control. Taken together, DON exposure reduced gastric secretion in rats. Low gastrin secretion and mRNA expression play a major role, unless mucosal lesions by high DON exposure are present.
Mycotoxins are common contaminants in foods and pose a great threat to food safety. Deoxynivalenol (DON) is the most common mycotoxin in cereals produced by Fusarium sp. during the growth and storage process (20, 40). It mainly contaminates maize, grain, and other cereals throughout the world (7, 28). DON-positive wheat samples accounted for greater than 60%, with the highest DON contamination at 41,157.13 pg/kg in Jiangsu province of the People’s Republic of China (21). It remains stable during food processing with high temperature, pressure (39), and strong irradiation (31). Thus, it usually enters the food chain and poses a threat to human health. A new survey in the People’s Republic of China indicated that 97.5% of the domestic soy sauces were contaminated by DON (41). DON contamination is also found in other foods, including bread (16). A survey in Croatia indicates an extremely high rate of DON exposure (32-fold higher than the established tolerable daily intake) in pregnant women (13). Therefore, DON poses great threats to food safety and is a cause of great concern for us. Ingestion of DON-contaminated foods can cause many symptoms, including vomiting and gastroenteritis in humans (35) and feed refusal, growth reduction, and even death in animals (2, 32). The digestive tract is a very susceptible target to DON in animals (15). Inhibitory absorption of the nutrients by DON was observed in vitro (27). Additionally, * Author for correspondence: Tel: + 8 6 13905151215; Fax: + 8 6 02584396586; E-mail: [email protected]. t Authors with equal contributions.
the barrier permeability (18) and intestinal morphology changed after DON exposure (4). The stomach is the first digestive organ responsible for preliminary digestion and emptying of foods; its function depends on the gastric secretion and emptying. Delayed gastric emptying by DON has been observed in rodents (14), indicating gastric disorders in rodents. However, the effect of DON on gastric secretion is still unclear. Gastric secretion is reflected through the volume and acidity of gastric fluid. Gastric acid secretion is important for high pepsin activities and the activation of pepsin from pepsinogen (19, 36). Additionally, gastric acid can protect against microorganism invasion by inhibiting overgrowth of bacteria (42). The volume of gastric fluid also can promote the digestion and emptying of foods. Thus, gastric secretion is important for food digestion in the stomach. Gastrin is the main hormone in stimulating gastric secretion, which is released by G cells in the stomach. It regulates gastric acid secretion via the gastrin-enterochromaffin-like (ECL) cell-parietal cell axis (23) or via the cholecystokinin B (CCK-B) receptors in parietal cells directly (22, 38). Gastric fluid is secreted by various glands and cells in the mucosa; thus, the understanding of gastrin secretion and mucosal lesions is essential. The goal of the present study was to evaluate the effects of DON on gastric secretion. Our results indicated that DON inhibited gastric secretion using pylorus-ligated rats. This study can help reveal the mechanism of digestive disorders caused by DON and provide a basis for reducing its toxic effects.
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FIGURE 1. Experimental design fo r collecting gastric fluid. The gastric fluid was collected by pylorus ligation. The letters represent different treatments as follows: (A) fasting and removal o f the bedding, (B) oral gavage with saline and DON, (C) ligation o f the pylorus, and (D) collection o f gastric fluid.
MATERIALS AND METHODS Materials. Pure DON was purchased from Sigma Chemical Co. (St. Louis, MO), and the purity (> 9 8 % ) was verified using thin-layer chromatography. TRI reagents, the PrimeScript RT Reagent Kit with gDNA Eraser, and the SYBR Premix Ex Taq Kit were supplied by Takara Biotechnology Co., Ltd. (Dalian, People’s Republic of China). All primers for PCR were synthesized by Sango Biotech Ltd. (Shanghai, People’s Republic o f China). Folin phenol and other analytical-grade reagents were purchased from Sinopharm Chemical Reagent Co. (Shanghai, People’s Republic of China). Animals. Female Wistar rats (7 weeks old, 200 to 220 g) were obtained from the Comparative Medicine Center of Yangzhou University (Yangzhou, Jiangsu, People’s Republic of China). The environment was maintained at 23 to 27°C and 48 to 54% relative humidity and a 12-h light and dark circle. The rats were housed in polycarbonate cages with stainless steel wire tops. They had free access to a standard laboratory diet and fresh water until treatment. The rats were randomly divided into four groups (six rats per group) and acclimated to the environment for at least 1 week. Food was withdrawn 24 h before toxin administration. Animal welfare was carried out under the Guide for the Care and Use o f Laboratory Animals (Ministry of Science and Technology of the People’s Republic of China, 2006). The experimental procedures were approved by the Animal Ethics Committee of Nanjing Agricultural University. Collection of gastric fluid. The rats were gavaged with DON at 0, 1, 5, and 25 mg/kg o f body weight (bw; diluted with sterilized saline at 200 to 220 ml) and returned to their cages immediately. Pylorus ligation was performed under anesthesia, as previously described (8), 0.5 h later. After pylorus ligation, the rats were returned to cages to regain consciousness. The rats were euthanized under anesthetic, with the stomachs excised, 4 h later. By cutting FIGURE 2. Gastric secretion after DON
exposure in rats. The fluid was gathered by pylorus ligation within 4 h after oral DON exposure. The results are shown as mean ± SEM (n = 6 per group). Parts A and B present, respectively, the volume and pH value o f the gastric fluid. Statistical significance is indicated by different letters (P < 0.05).
along the greater curvature, the gastric contents were collected. Gastric contents were placed into centrifuge tubes and centrifuged at 2,200 x g for 10 min, and the supernatants were separated to assess gastric secretion, pepsin activity, and gastrin level. The whole procedure for collecting the gastric fluid is shown in Figure 1.
Measurement of gastric secretion. The acidity o f the gastric fluid was determined using a Mettler-Toledo FE20K pH meter (Changzhou, People’s Republic o f China). The volume was obtained by calculating the supernatant and the liquid in gastric residue. The volume o f supernatant was determined using a volumetric cylinder. The fluid in the residue was calculated by weight loss after oven drying at 60°C for 3 days. The total volume o f gastric fluid was calculated as the following formula: total volume of gastric fluid (ml) = supernatant volume (ml) + volume of gastric fluid in gastric contents (ml). Determination of peptic activity. The peptic activity in the gastric fluid was determined, as previously described (26). Briefly, 0.1 ml o f gastric juice was added into 0.9 ml o f 1% bovine serum albumin solution (diluted with 0.1 M HC1). The mixture was incubated at 37°C for 20 min and terminated by boiling in water for 5 min. Filtered supernatant (1 ml) was used to determine the concentration of liberated amino acids, as reported. The optical densities at 610 nm were determined against 0.1 M HC1 using a 720 spectrophotometer. Peptic activity (units) was expressed in terms o f micromoles of tyrosine per milliliter o f gastric fluid per minute. The total pepsin activity = peptic activity/ml o f gastric fluid (mU) x volume o f gastric fluid (ml). Histopathology of gastric mucosa. The stomach was washed with fresh water and fixed in 10% formalin overnight after collection o f gastric fluid. They were embedded in paraffin and sectioned into 5-pm slices. The sections were stained with hematoxylin and eosin and examined microscopically. Gastrin in the gastric fluid. Gastrin in gastric fluid was determined using radioimmunoassay. The procedure was per formed according to the instructions of the GAS RIA Kit (HY10167, Beijing, People’s Republic o f China). Gastrin mRNA levels. After 24 h, fasted rats were gavaged with 0 and 5 mg/kg bw DON (diluted with sterilized PBS). The stomachs were excised under anesthesia 2 h later and stored at —80°C until use. The stomach was split into body, pylorus, and fundus parts. Total RNA was isolated from each tissue sample using TRIzol Reagent, according to the manufacturer’s protocol. The cDNA was synthesized using PrimerScript RT reagent kit with gDNA Eraser, following the manufacturer’s instructions. Primers for gastrin mRNA detection were sense, 5'-CCCAAGGTCCGCAACACT-3', and antisense, 5 '-CGCTACGGCGACCAAAGT-3'.
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FIGURE 3. Pepsin activity in the gastric fluid after DON exposure. The results were shown as mean ± SEM (n = 6 per group). Parts A and B present, respectively, the total activity and activity per milliliter o f gastric fluid. Statistical significance is indicated by different letters (P < 0.05).
500-. B
D ose (m g/kg bw)
GAPDH was chosen as the reference gene and amplified using the following primers: sense, 5'-TGGGTGTGAACCACGAGAA3', and antisense, 5 '-GGCATGGACTGTGGTCATGA-3'. Real-time qPCR was performed with the ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA). The protocol was performed according to the instruction of SYBR Premix Ex Taq Kit. The PCR conditions were initial denaturation at 95°C for 30 s followed by 40 circles of 5 s at 95°C and 20 s at 60°C. The data were expressed by the 2~AAO method, as previously described (25).
Statistics. The data were analyzed using SPSS software version 17.0 (SPSS Inc., Chicago, IL) and expressed as the mean + SEM (n = 6). A one-way analysis of variance and a least significant difference test were used to compare the data among groups. Significant differences were established at P < 0.05. RESULTS Gastric secretion determination. The results showed that DON decreased the volume of the gastric fluid. Compared with the control, the total volume decreased by 25, 51, and 61% at 1, 5, and 25 mg/kg bw in the DON group, respectively (Fig. 2A). Also, DON dose dependently decreased the acidity of the gastric fluid. The average pH value increased from 1.90 of the control to 3.2 and 3.81 in the 1 and 5 mg/kg bw DON groups, respectively. After DON exposure at 25 mg/kg bw, the pH value increased to 6.65 (Fig. 2B). Pepsin activity in the gastric fluid. Total pepsin activity was detected using UV detection. Compared with the control, it decreased by 48,71, and 76% after DON treatment at 1, 5, and 25 mg/kg bw, respectively (Fig. 3A). The results indicated that DON dose dependently decreased total pepsin activities in the gastric fluid (P < 0.05). The peptic activity in each milliliter decreased by 8 and 18% in the 5 and 25 mg/kg bw groups, compared with the control group. No significant decrease was observable in the 1 mg/kg bw DON group (P > 0.05; Fig. 3B).
Gastrin secretion. The results showed that DON exposure dose dependently decreased the total gastrin in gastric fluids. Compared with the control, it decreased by 34, 59, and 70% at 1, 5, and 25 mg/kg bw, respectively (Fig. 4A). The gastrin concentration decreased by 13, 16, and 24% at 1,5, and 25 mg/kg bw, respectively (Fig. 4B). Pathological histology of gastric mucosa. In the control group, gastric mucosal stained its integrity with no epithelial exfoliated cells (Fig. 5A). No mucosal lesions in the stomach were observed after DON exposure at 1 and 5 mg/kg bw (Fig. 5B and 5C). However, slight mucosal lesions were observed in the highest DON group, and many deciduous epithelial cells were observed (Fig. 5D). Gastrin mRNA expression. The gastrin mRNA expression in the stomach was determined by the real-time PCR assay. After DON exposure at 5 mg/kg bw, the gastrin mRNA expression decreased significantly in the whole stomach. Compared with the control, it decreased by 88% in the pylorus, 84% in the fundus, and 54% in the body, respectively (Fig. 6). DISCUSSION DON is a common contaminant in cereals and foods and poses great threats to food safety and human health. The digestive tract is susceptible to DON, and in this study, we examined the effect of DON on gastric secretion in rats. To collect gastric fluids, a pylorus ligation assay was used in this experiment. Pylorus ligation is a common assay for gastric fluid collection, as reported previously (8, 23). Rats are unable to vomit, and pylorus ligation does not affect gastric secretion in rats (18); therefore, it is a feasible and accurate method to determine gastric secretion in rats. Briefly, an interval of 0.5 h was set between DON gavage and pylorus ligation. DON is rapidly taken up into plasma and tissues, with a t\/2a (half-life at a phase) of 21.6 min FIGURE 4. The effect o f DON on gastrin level in the gastric fluid. The fluid was gathered within 6 h after oral exposure to 0, 1, 5, and 25 mg/kg bw DON in rats. The data are presented as mean ± SEM (n = 6 per group). Part A presents the effect o f DON on the total gastrin secretion. Part B represents gastrin concentration in the gastric fluid. Statistical significance is indicated by different letters (P < 0.05).
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FIGURE 5. Histopathology o f gastric mucosa after DON expo sure. The lettered parts present different doses o f DON treatment: (A) placebo group with saline, (B) oral gavage at 1 mglkg bw DON, (C) oral gavage at 5 mglkg bw DON, and (D) oral gavage at 25 mglkg bw DON. The arrow indicates exfoliated epithelial cells from gastric mucosa (hematoxylin and eosin stain, bar = 100 pm).
(5 mg/kg bw) and 33.6 min (25 mg/kg bw) (34). Thus, the 0.5-h interval before pylorus ligation can better reflect the gastric secretion in the stomach after DON exposure. Using the pylorus ligation model, we found that both the volume and acidity of gastric fluid decreased in DONexposed rats. Gastric fluid is secreted by various glands and cells in the mucosa. Thus, gastric mucosal lesions can inhibit the secretion of gastric fluid. In this study, no mucosal lesions were observed in the 1 or 5 mg/kg bw DON group. The data suggested that inhibited gastric secretion was not directly induced by mucosal lesions. In contrast, mucosal injury is the main reason for decreased gastric secretion in the 25 mg/kg bw DON group. Gastrin is a major
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hormone in regulating gastric secretion. It stimulates gastric acid secretion via CCK-B receptors in parietal cells (22, 38) or the gastrin-ECL cell-parietal cell axis (24). Both gastrin secretion and mRNA expression were observed in this study. The similar trend with gastric secretion indicated that gastrin played an important role in reducing gastric secretion. DON has a ribotoxic stress response and can inhibit protein synthesis in vivo and in vitro (JO, 12). Hence, the reduced gastrin secretion may be related to inhibited protein synthesis by DON, which needs further research. In this study, reduced pepsin activities by DON were observed. As is reported, pepsin is activated from pepsinogen at a pH < 4 (29) and occupies the highest activity at a pH » 2.0 and is unstable when the pH > 6 (36). Additionally, low acidity also can inhibit the denaturing of proteins (1); thus, reduced gastric acid secretion plays the leading role in decreased pepsin activities. The pathobiology showed no gastric mucosal lesions after DON exposure, except slight damage at 25 mg/kg bw (Fig. 3). This phenomenon is related to the metabolism of DON in animals. First, DON is taken up rapidly with no accumulation found in stomach tissues (33). Additionally, it is absorbed mainly in the jejunum rather than stomach (3). Finally, gastric mucosa can secrete large amounts of mucus, which may play an important role in preventing DON damage here. Reduced gastric secretion here and delayed gastric emptying in rodents (14) both suggest the gastric disorders caused by DON. The results indicated that the digestive function is suppressed by DON in the stomach. Absorptive disorders caused by DON in the intestines have been observed in vitro (6). The disorders are related to changed intestinal structure and reduced transport function of the epithelium (5, 43). Also, the reduced gastric secretion here is another important reason. First, gastric secretion can promote nutrient absorption by stimulating the secretion of small intestine fluid. Additionally, the volume of gastric fluid is also a supporting factor for gastric emptying (30). Gastric secretion also can promote the absorption of metal ions by enhancing the solubility. Fetal skeletal malforma tions (11) and Ca2+ malabsorption (37) by DON may be related to the decreased gastric acidity (9). To summarize, DON exposure inhibited gastric secre tion in rats. It is related to down-regulated gastrin expression at low DON exposure and mucosal lesions at high DON exposure. The detailed regulating mechanism of gastric secretion needs further research. Please note the results apply only to a DON solution; the DON in contaminated foods is not included. ACKNOWLEDGMENTS
Dose (mg/kg bw) FIGURE 6. Gastrin mRNA expression in stomach. The tissues were excised 2 h after exposure to sterilized phosphate-buffered saline and 5 mglkg bw DON. The real-time PCR assay was used to analyze gastrin mRNA expression. The data are presented as mean ± SEM (n = 6 per group). * Represents a significant difference compared with the control group (P < 0.05).
This study was supported by the National Natural Science Foundation of China (A2000748) and Fundamental Research Funds for Central University (KYZ201149). The work was also funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
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Journal o f Food Protection, Vol. 77, No. 8, 2014, Pages 1367-1371 doi: 10.4315/0362-028X.JFP-13-470 C o p yrig h t © , In ternatio nal A sso cia tio n fo r Food Protection
Inhibitory Effects of Deoxynivalenol on Gastric Secretion in Rats YUWEI WANG.'t WENDA W ll,'i XICHUN WANG,2 CHENGHUA HE,1 HUA YUE,3 ZHE REN, 1 a n d HAIBIN ZHANG1* 1College o f Veterinary Medicine, Nanjing Agricultural University, Nanjing, 210095, People’s Republic o f China; 2College o f Animal Science and Technology, Anhui Agricultural University, Hefei, 230036, People’s Republic o f China; and 2Henan Institutes o f Science and Technology, Xinxiang, 453003, People’s Republic o f China MS 13-470: Received 25 October 2013/Accepted 30 March 2014
ABSTRACT Deoxynivalenol (DON) is a common mycotoxin produced by Fusarium sp. in cereals and foods. Ingestion of contaminated foodstuffs can cause digestive disorders in various animals. Many researchers focus on its toxicity and the pathological damage and absorptive function in the intestines. However, the effect of DON on gastric function is still unclear. The objective of the current study was to evaluate the impact of DON on gastric secretion. Rats were gavaged with DON at the dose of 0, 1, 5, and 25 mg/kg of body weight (bw). Gastric fluids were gathered by pylorus ligation 0.5 h after DON exposure. The results indicate that the volume of gastric fluid decreased by 25, 51, and 61% compared with the control, respectively. The pH increased to 3.2, 3.81, and 6.65 in the 1, 5, and 25 mg/kg bw DON group, compared with the control (1.9). To examine the mucosal injuries, the stomach tissues were made into hematoxylin and eosin slides. Histopathology observations suggest that no mucosal lesions were observed until DON exposure at 25 mg/kg bw. Additionally, the gastrin secretion in the fluids and mRNA expression in tissues were determined by the radioimmunoassay and real-time PCR assay, respectively. The results indicated that both significantly decreased in DON-exposed rats compared with the control. Taken together, DON exposure reduced gastric secretion in rats. Low gastrin secretion and mRNA expression play a major role, unless mucosal lesions by high DON exposure are present.
Mycotoxins are common contaminants in foods and pose a great threat to food safety. Deoxynivalenol (DON) is the most common mycotoxin in cereals produced by Fusarium sp. during the growth and storage process (20, 40). It mainly contaminates maize, grain, and other cereals throughout the world (7, 28). DON-positive wheat samples accounted for greater than 60%, with the highest DON contamination at 41,157.13 pg/kg in Jiangsu province of the People’s Republic of China (21). It remains stable during food processing with high temperature, pressure (39), and strong irradiation (31). Thus, it usually enters the food chain and poses a threat to human health. A new survey in the People’s Republic of China indicated that 97.5% of the domestic soy sauces were contaminated by DON (41). DON contamination is also found in other foods, including bread (16). A survey in Croatia indicates an extremely high rate of DON exposure (32-fold higher than the established tolerable daily intake) in pregnant women (13). Therefore, DON poses great threats to food safety and is a cause of great concern for us. Ingestion of DON-contaminated foods can cause many symptoms, including vomiting and gastroenteritis in humans (35) and feed refusal, growth reduction, and even death in animals (2, 32). The digestive tract is a very susceptible target to DON in animals (15). Inhibitory absorption of the nutrients by DON was observed in vitro (27). Additionally, * Author for correspondence: Tel: + 8 6 13905151215; Fax: + 8 6 02584396586; E-mail: [email protected]. t Authors with equal contributions.
the barrier permeability (18) and intestinal morphology changed after DON exposure (4). The stomach is the first digestive organ responsible for preliminary digestion and emptying of foods; its function depends on the gastric secretion and emptying. Delayed gastric emptying by DON has been observed in rodents (14), indicating gastric disorders in rodents. However, the effect of DON on gastric secretion is still unclear. Gastric secretion is reflected through the volume and acidity of gastric fluid. Gastric acid secretion is important for high pepsin activities and the activation of pepsin from pepsinogen (19, 36). Additionally, gastric acid can protect against microorganism invasion by inhibiting overgrowth of bacteria (42). The volume of gastric fluid also can promote the digestion and emptying of foods. Thus, gastric secretion is important for food digestion in the stomach. Gastrin is the main hormone in stimulating gastric secretion, which is released by G cells in the stomach. It regulates gastric acid secretion via the gastrin-enterochromaffin-like (ECL) cell-parietal cell axis (23) or via the cholecystokinin B (CCK-B) receptors in parietal cells directly (22, 38). Gastric fluid is secreted by various glands and cells in the mucosa; thus, the understanding of gastrin secretion and mucosal lesions is essential. The goal of the present study was to evaluate the effects of DON on gastric secretion. Our results indicated that DON inhibited gastric secretion using pylorus-ligated rats. This study can help reveal the mechanism of digestive disorders caused by DON and provide a basis for reducing its toxic effects.
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FIGURE 1. Experimental design fo r collecting gastric fluid. The gastric fluid was collected by pylorus ligation. The letters represent different treatments as follows: (A) fasting and removal o f the bedding, (B) oral gavage with saline and DON, (C) ligation o f the pylorus, and (D) collection o f gastric fluid.
MATERIALS AND METHODS Materials. Pure DON was purchased from Sigma Chemical Co. (St. Louis, MO), and the purity (> 9 8 % ) was verified using thin-layer chromatography. TRI reagents, the PrimeScript RT Reagent Kit with gDNA Eraser, and the SYBR Premix Ex Taq Kit were supplied by Takara Biotechnology Co., Ltd. (Dalian, People’s Republic of China). All primers for PCR were synthesized by Sango Biotech Ltd. (Shanghai, People’s Republic o f China). Folin phenol and other analytical-grade reagents were purchased from Sinopharm Chemical Reagent Co. (Shanghai, People’s Republic of China). Animals. Female Wistar rats (7 weeks old, 200 to 220 g) were obtained from the Comparative Medicine Center of Yangzhou University (Yangzhou, Jiangsu, People’s Republic of China). The environment was maintained at 23 to 27°C and 48 to 54% relative humidity and a 12-h light and dark circle. The rats were housed in polycarbonate cages with stainless steel wire tops. They had free access to a standard laboratory diet and fresh water until treatment. The rats were randomly divided into four groups (six rats per group) and acclimated to the environment for at least 1 week. Food was withdrawn 24 h before toxin administration. Animal welfare was carried out under the Guide for the Care and Use o f Laboratory Animals (Ministry of Science and Technology of the People’s Republic of China, 2006). The experimental procedures were approved by the Animal Ethics Committee of Nanjing Agricultural University. Collection of gastric fluid. The rats were gavaged with DON at 0, 1, 5, and 25 mg/kg o f body weight (bw; diluted with sterilized saline at 200 to 220 ml) and returned to their cages immediately. Pylorus ligation was performed under anesthesia, as previously described (8), 0.5 h later. After pylorus ligation, the rats were returned to cages to regain consciousness. The rats were euthanized under anesthetic, with the stomachs excised, 4 h later. By cutting FIGURE 2. Gastric secretion after DON
exposure in rats. The fluid was gathered by pylorus ligation within 4 h after oral DON exposure. The results are shown as mean ± SEM (n = 6 per group). Parts A and B present, respectively, the volume and pH value o f the gastric fluid. Statistical significance is indicated by different letters (P < 0.05).
along the greater curvature, the gastric contents were collected. Gastric contents were placed into centrifuge tubes and centrifuged at 2,200 x g for 10 min, and the supernatants were separated to assess gastric secretion, pepsin activity, and gastrin level. The whole procedure for collecting the gastric fluid is shown in Figure 1.
Measurement of gastric secretion. The acidity o f the gastric fluid was determined using a Mettler-Toledo FE20K pH meter (Changzhou, People’s Republic o f China). The volume was obtained by calculating the supernatant and the liquid in gastric residue. The volume o f supernatant was determined using a volumetric cylinder. The fluid in the residue was calculated by weight loss after oven drying at 60°C for 3 days. The total volume o f gastric fluid was calculated as the following formula: total volume of gastric fluid (ml) = supernatant volume (ml) + volume of gastric fluid in gastric contents (ml). Determination of peptic activity. The peptic activity in the gastric fluid was determined, as previously described (26). Briefly, 0.1 ml o f gastric juice was added into 0.9 ml o f 1% bovine serum albumin solution (diluted with 0.1 M HC1). The mixture was incubated at 37°C for 20 min and terminated by boiling in water for 5 min. Filtered supernatant (1 ml) was used to determine the concentration of liberated amino acids, as reported. The optical densities at 610 nm were determined against 0.1 M HC1 using a 720 spectrophotometer. Peptic activity (units) was expressed in terms o f micromoles of tyrosine per milliliter o f gastric fluid per minute. The total pepsin activity = peptic activity/ml o f gastric fluid (mU) x volume o f gastric fluid (ml). Histopathology of gastric mucosa. The stomach was washed with fresh water and fixed in 10% formalin overnight after collection o f gastric fluid. They were embedded in paraffin and sectioned into 5-pm slices. The sections were stained with hematoxylin and eosin and examined microscopically. Gastrin in the gastric fluid. Gastrin in gastric fluid was determined using radioimmunoassay. The procedure was per formed according to the instructions of the GAS RIA Kit (HY10167, Beijing, People’s Republic o f China). Gastrin mRNA levels. After 24 h, fasted rats were gavaged with 0 and 5 mg/kg bw DON (diluted with sterilized PBS). The stomachs were excised under anesthesia 2 h later and stored at —80°C until use. The stomach was split into body, pylorus, and fundus parts. Total RNA was isolated from each tissue sample using TRIzol Reagent, according to the manufacturer’s protocol. The cDNA was synthesized using PrimerScript RT reagent kit with gDNA Eraser, following the manufacturer’s instructions. Primers for gastrin mRNA detection were sense, 5'-CCCAAGGTCCGCAACACT-3', and antisense, 5 '-CGCTACGGCGACCAAAGT-3'.
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FIGURE 3. Pepsin activity in the gastric fluid after DON exposure. The results were shown as mean ± SEM (n = 6 per group). Parts A and B present, respectively, the total activity and activity per milliliter o f gastric fluid. Statistical significance is indicated by different letters (P < 0.05).
500-. B
D ose (m g/kg bw)
GAPDH was chosen as the reference gene and amplified using the following primers: sense, 5'-TGGGTGTGAACCACGAGAA3', and antisense, 5 '-GGCATGGACTGTGGTCATGA-3'. Real-time qPCR was performed with the ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA). The protocol was performed according to the instruction of SYBR Premix Ex Taq Kit. The PCR conditions were initial denaturation at 95°C for 30 s followed by 40 circles of 5 s at 95°C and 20 s at 60°C. The data were expressed by the 2~AAO method, as previously described (25).
Statistics. The data were analyzed using SPSS software version 17.0 (SPSS Inc., Chicago, IL) and expressed as the mean + SEM (n = 6). A one-way analysis of variance and a least significant difference test were used to compare the data among groups. Significant differences were established at P < 0.05. RESULTS Gastric secretion determination. The results showed that DON decreased the volume of the gastric fluid. Compared with the control, the total volume decreased by 25, 51, and 61% at 1, 5, and 25 mg/kg bw in the DON group, respectively (Fig. 2A). Also, DON dose dependently decreased the acidity of the gastric fluid. The average pH value increased from 1.90 of the control to 3.2 and 3.81 in the 1 and 5 mg/kg bw DON groups, respectively. After DON exposure at 25 mg/kg bw, the pH value increased to 6.65 (Fig. 2B). Pepsin activity in the gastric fluid. Total pepsin activity was detected using UV detection. Compared with the control, it decreased by 48,71, and 76% after DON treatment at 1, 5, and 25 mg/kg bw, respectively (Fig. 3A). The results indicated that DON dose dependently decreased total pepsin activities in the gastric fluid (P < 0.05). The peptic activity in each milliliter decreased by 8 and 18% in the 5 and 25 mg/kg bw groups, compared with the control group. No significant decrease was observable in the 1 mg/kg bw DON group (P > 0.05; Fig. 3B).
Gastrin secretion. The results showed that DON exposure dose dependently decreased the total gastrin in gastric fluids. Compared with the control, it decreased by 34, 59, and 70% at 1, 5, and 25 mg/kg bw, respectively (Fig. 4A). The gastrin concentration decreased by 13, 16, and 24% at 1,5, and 25 mg/kg bw, respectively (Fig. 4B). Pathological histology of gastric mucosa. In the control group, gastric mucosal stained its integrity with no epithelial exfoliated cells (Fig. 5A). No mucosal lesions in the stomach were observed after DON exposure at 1 and 5 mg/kg bw (Fig. 5B and 5C). However, slight mucosal lesions were observed in the highest DON group, and many deciduous epithelial cells were observed (Fig. 5D). Gastrin mRNA expression. The gastrin mRNA expression in the stomach was determined by the real-time PCR assay. After DON exposure at 5 mg/kg bw, the gastrin mRNA expression decreased significantly in the whole stomach. Compared with the control, it decreased by 88% in the pylorus, 84% in the fundus, and 54% in the body, respectively (Fig. 6). DISCUSSION DON is a common contaminant in cereals and foods and poses great threats to food safety and human health. The digestive tract is susceptible to DON, and in this study, we examined the effect of DON on gastric secretion in rats. To collect gastric fluids, a pylorus ligation assay was used in this experiment. Pylorus ligation is a common assay for gastric fluid collection, as reported previously (8, 23). Rats are unable to vomit, and pylorus ligation does not affect gastric secretion in rats (18); therefore, it is a feasible and accurate method to determine gastric secretion in rats. Briefly, an interval of 0.5 h was set between DON gavage and pylorus ligation. DON is rapidly taken up into plasma and tissues, with a t\/2a (half-life at a phase) of 21.6 min FIGURE 4. The effect o f DON on gastrin level in the gastric fluid. The fluid was gathered within 6 h after oral exposure to 0, 1, 5, and 25 mg/kg bw DON in rats. The data are presented as mean ± SEM (n = 6 per group). Part A presents the effect o f DON on the total gastrin secretion. Part B represents gastrin concentration in the gastric fluid. Statistical significance is indicated by different letters (P < 0.05).
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FIGURE 5. Histopathology o f gastric mucosa after DON expo sure. The lettered parts present different doses o f DON treatment: (A) placebo group with saline, (B) oral gavage at 1 mglkg bw DON, (C) oral gavage at 5 mglkg bw DON, and (D) oral gavage at 25 mglkg bw DON. The arrow indicates exfoliated epithelial cells from gastric mucosa (hematoxylin and eosin stain, bar = 100 pm).
(5 mg/kg bw) and 33.6 min (25 mg/kg bw) (34). Thus, the 0.5-h interval before pylorus ligation can better reflect the gastric secretion in the stomach after DON exposure. Using the pylorus ligation model, we found that both the volume and acidity of gastric fluid decreased in DONexposed rats. Gastric fluid is secreted by various glands and cells in the mucosa. Thus, gastric mucosal lesions can inhibit the secretion of gastric fluid. In this study, no mucosal lesions were observed in the 1 or 5 mg/kg bw DON group. The data suggested that inhibited gastric secretion was not directly induced by mucosal lesions. In contrast, mucosal injury is the main reason for decreased gastric secretion in the 25 mg/kg bw DON group. Gastrin is a major
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hormone in regulating gastric secretion. It stimulates gastric acid secretion via CCK-B receptors in parietal cells (22, 38) or the gastrin-ECL cell-parietal cell axis (24). Both gastrin secretion and mRNA expression were observed in this study. The similar trend with gastric secretion indicated that gastrin played an important role in reducing gastric secretion. DON has a ribotoxic stress response and can inhibit protein synthesis in vivo and in vitro (JO, 12). Hence, the reduced gastrin secretion may be related to inhibited protein synthesis by DON, which needs further research. In this study, reduced pepsin activities by DON were observed. As is reported, pepsin is activated from pepsinogen at a pH < 4 (29) and occupies the highest activity at a pH » 2.0 and is unstable when the pH > 6 (36). Additionally, low acidity also can inhibit the denaturing of proteins (1); thus, reduced gastric acid secretion plays the leading role in decreased pepsin activities. The pathobiology showed no gastric mucosal lesions after DON exposure, except slight damage at 25 mg/kg bw (Fig. 3). This phenomenon is related to the metabolism of DON in animals. First, DON is taken up rapidly with no accumulation found in stomach tissues (33). Additionally, it is absorbed mainly in the jejunum rather than stomach (3). Finally, gastric mucosa can secrete large amounts of mucus, which may play an important role in preventing DON damage here. Reduced gastric secretion here and delayed gastric emptying in rodents (14) both suggest the gastric disorders caused by DON. The results indicated that the digestive function is suppressed by DON in the stomach. Absorptive disorders caused by DON in the intestines have been observed in vitro (6). The disorders are related to changed intestinal structure and reduced transport function of the epithelium (5, 43). Also, the reduced gastric secretion here is another important reason. First, gastric secretion can promote nutrient absorption by stimulating the secretion of small intestine fluid. Additionally, the volume of gastric fluid is also a supporting factor for gastric emptying (30). Gastric secretion also can promote the absorption of metal ions by enhancing the solubility. Fetal skeletal malforma tions (11) and Ca2+ malabsorption (37) by DON may be related to the decreased gastric acidity (9). To summarize, DON exposure inhibited gastric secre tion in rats. It is related to down-regulated gastrin expression at low DON exposure and mucosal lesions at high DON exposure. The detailed regulating mechanism of gastric secretion needs further research. Please note the results apply only to a DON solution; the DON in contaminated foods is not included. ACKNOWLEDGMENTS
Dose (mg/kg bw) FIGURE 6. Gastrin mRNA expression in stomach. The tissues were excised 2 h after exposure to sterilized phosphate-buffered saline and 5 mglkg bw DON. The real-time PCR assay was used to analyze gastrin mRNA expression. The data are presented as mean ± SEM (n = 6 per group). * Represents a significant difference compared with the control group (P < 0.05).
This study was supported by the National Natural Science Foundation of China (A2000748) and Fundamental Research Funds for Central University (KYZ201149). The work was also funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
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