TY - JOUR
AU1 - Liu, N.
AU2 - Ding, K.
AU3 - Wang, J. Q.
AU4 - Jia, S. C.
AU5 - Wang, J. P.
AU6 - Xu, T. S.
AB - ABSTRACT Lactic acid bacteria (LAB) and the glutathione (GSH) pathway are protective against aflatoxin, but information on the effect of LAB on aflatoxin metabolism and GSH activity in farm animals is scarce. This study aimed to investigate the effects of LAB and aflatoxin B1 (AFB1) on growth performance, aflatoxin metabolism, and GSH pathway activity using 480 male Arbor Acres broiler chickens from d 1 to 35 of age. Diets were arranged in a 2 × 2 factorial design, including AFB1 at 0 or 40 µg/kg of feed and LAB at 0 or 3 × 1010 cfu/kg of feed, and the LAB was a mixture of equal amounts of Lactobacillus acidophilus, Lactobacillus plantarum, and Enterococcus faecium. The results showed that there were highly significant (P < 0.01) effects of AFB1 toxicity, LAB protection, and their interaction on ADFI, ADG, and G:F of broilers during d 1 to 35. Compared with the AFB1 diet, the LAB diet reduced (P < 0.05) the residues of AFB1 in the liver, kidney, serum, ileal digesta, and excreta on d 14 by 121.5, 80.6, 43.7, 47.0, and 26.5%, respectively, and on d 35 by 40.6, 60.2, 131.7, 37.9, and 32.9%, respectively, whereas the LAB diet increased (P < 0.05) the contents of aflatoxin M1, a metabolite of AFB1, in the liver, kidney, serum, and ileal digesta on d 14 by 98.2, 154.2, 168.6, 19.1, and 34.1%, respectively, and in the kidney and serum on d 35 by 32.6 and 142.2%, respectively. For the activity of the GSH pathway in the liver and duodenal mucosa, there were significant (P ≤ 0.01) effects of LAB and AFB1 on reduced GSH, glutathione S-transferases (GST), and glutathione reductase (GR) on d 14 and 35; compared with the control diet, the LAB diet increased (P < 0.05) GSH, GST, and GR by a range of 11.6 to 86.1%, and compared with the AFB1 diet, the LAB diet increased (P < 0.05) GSH, GST, and GR by a range of 24.1 to 146.9%. In the liver, there were interactions (P < 0.05) on GSH and GST on d 14 and on GSH on d 35; in the mucosa, interactions were significant (P ≤ 0.01) on GSH and GR on d 14 and on GST on d 35. It can be concluded that LAB is effective in the detoxification of AFB1 by modulating toxin metabolism and activating the GSH pathway in animals. INTRODUCTION Aflatoxin contamination of feedstuffs is frequent in some areas with a hot and humid climate and has negative effects on animal production and food security. Aflatoxin B1 (AFB1) is the most toxic form and can be metabolized into less toxic or nontoxic substances, such as aflatoxin M1 (AFM1) and aflatoxin Q1, and a more toxic AFB1 8,9-epoxide (AFBO; Rawal et al., 2010; Dohnal et al., 2014). The AFBO binds to DNA and other critical cellular molecules, causing genotoxicity in the event of inadequate or deficient glutathione (GSH) activity against the toxic group (Gallagher et al., 1996; Guengerich et al., 1996; Klein et al., 2003). Currently, physical adhesion to aflatoxin in feeds is commonly used to decrease aflatoxicity, whereas inducing the antioxidative pathway activity to conjugate more AFBO may be a novel integrated measure for aflatoxin detoxification. Research has demonstrated that lactic acid bacteria (LAB) can confer a certain protection against AFB1 in animals, reflected in improved growth performance and decreased immunotoxicity and oxidative stress (Gratz et al., 2006; Abbès et al., 2016). The GSH pathway can perform vital functions in animal health through reactions with various electrophilic metabolites (Martensson et al., 1990; Wu et al., 2004; Novaes et al., 2013). However, literature is unavailable regarding the effect of LAB on the GSH pathway based on an AFB1–contaminated diet in farm animals. Therefore, hypothetically, the LAB can modulate the metabolism of AFB1 and activate GSH protection against AFB1 damage. The objectives are to investigate the effect of LAB on AFB1 residue and its main metabolite AFM1, the concentration of GSH, and the activities of glutathione S-transferases (GST) and glutathione reductase (GR) in broiler chickens. MATERIALS AND METHODS The Lactic Acid Bacteria Strains, Aflatoxin B1, and Diets The LAB strains in this study are permitted to be used in the feed additive industry in P.R. China (Announcement of Ministry of Agriculture of P.R. China, No.2045-2013) and were obtained from Hongxiang Biological Feed Laboratory at Henan University of Science and Technology (Luoyang, P.R. China). The strains Lactobacillus acidophilus (ACCC11073), Lactobacillus plantarum (CICC21863), and Enterococcus faecium (CICC20430) were combined in equal amounts and added at a rate of 3.0 × 1010 cfu/kg of feed. A total of 1.00 g of the strain powder was diluted with 9 mL of 1% tryptic soy broth medium (TSB; QingDao Hopebio-Technology Co., Ltd, Qingdao, P.R. China) and then homogenized. Viable counts of bacteria were conducted by plating serial 10-fold dilutions onto TSB agar plates after the bacteria were anaerobically cultured at 37°C, pH 6.2, for 24 h. The AFB1 was produced using Aspergillus flavus from the China General Microbiological Culture Collection Center (Beijing, P.R. China). A total of 20.0 kg corn meal (mesh size 2.00 mm) was placed in a 100-L container, with 10.0 L distilled water, and then autoclaved. The medium was inoculated with 500-mL A. flavus and incubated at 28°C for 7 d. The incubated corn meal was autoclaved to kill A. flavus, dried, and ground (mesh size 0.425 mm) for the animal feeding experiments. The AFB1 concentrations in the moldy corn meal were estimated to be 4,587 µg/kg using an AFB1 kit and modulated to 40 µg/kg of feed using noncontaminated corn meal (mesh size 2.00 mm) as a diluent in the expense of corn in the formulation. The nutrition levels of the diet were as recommended by Arbor Acres Broiler Management Handbook (Han, et al., 2006) in P.R. China, and the water contents of all ingredients and diets were controlled at under 12%, and the materials were stored in a cool, dry, dark and well-ventilated place. All diets were fed as mash. No antibiotics were used either in feed or water throughout the experiment. The diets are listed in Table 1. Table 1. Ingredients and nutrient levels of basal diet1 (air-dry basis)   Content, g/kg    Content, g/kg  Ingredient  d 1 to 14  d 15 to 35  Nutrient  d 1 to 14  d 15 to 35  Corn  577.2  582.9  CP  214.4  200.1  Soybean meal  250.0  255.0  ME, kcal/kg  2,983.3  3,119.6  Corn gluten meal  60.0  44.0  Crude fiber  26.7  26.3  Full-fat soybeans  50.0  35.0  Ca  10.2  8.6  Soybean oil  10.0  38.0  Available P  5.0  4.2  Lys  2.5  1.3  Lys  12.3  10.7  Met  1.5  1.0  Met  5.1  4.3  Salt  4.0  4.0  Met + Cys  8.4  7.4  Limestone  11.5  17.0  Thr  7.9  7.5  Dicalcium phosphate  21.5  10.0  Trp  2.4  2.1  Choline chloride  1.8  1.8        Premix2  10.0  10.0          Content, g/kg    Content, g/kg  Ingredient  d 1 to 14  d 15 to 35  Nutrient  d 1 to 14  d 15 to 35  Corn  577.2  582.9  CP  214.4  200.1  Soybean meal  250.0  255.0  ME, kcal/kg  2,983.3  3,119.6  Corn gluten meal  60.0  44.0  Crude fiber  26.7  26.3  Full-fat soybeans  50.0  35.0  Ca  10.2  8.6  Soybean oil  10.0  38.0  Available P  5.0  4.2  Lys  2.5  1.3  Lys  12.3  10.7  Met  1.5  1.0  Met  5.1  4.3  Salt  4.0  4.0  Met + Cys  8.4  7.4  Limestone  11.5  17.0  Thr  7.9  7.5  Dicalcium phosphate  21.5  10.0  Trp  2.4  2.1  Choline chloride  1.8  1.8        Premix2  10.0  10.0        1Calculated values (Xiong et al., 2014); Aflatoxin B1 is not detectable (<2 µg/kg). 2Provided, per kilogram of diet, 9,000 IU vitamin A (retinyl acetate), 4,000 IU cholecalciferol, 50 IU vitamin E (DL-tocopheryl acetate), 2 mg vitamin K, 2 mg thiamin, 5 mg riboflavin, 15 mg d-pantothenic acid, 40 mg niacin, 2 mg pyridoxine, 0.1 mg biotin, 0.55 mg folic acid, 0.01 mg vitamin B12, 120 mg manganese, 1.2 mg iodine, 40 mg iron, 16 mg copper, 100 mg zinc, and 0.3 mg selenium. View Large Table 1. Ingredients and nutrient levels of basal diet1 (air-dry basis)   Content, g/kg    Content, g/kg  Ingredient  d 1 to 14  d 15 to 35  Nutrient  d 1 to 14  d 15 to 35  Corn  577.2  582.9  CP  214.4  200.1  Soybean meal  250.0  255.0  ME, kcal/kg  2,983.3  3,119.6  Corn gluten meal  60.0  44.0  Crude fiber  26.7  26.3  Full-fat soybeans  50.0  35.0  Ca  10.2  8.6  Soybean oil  10.0  38.0  Available P  5.0  4.2  Lys  2.5  1.3  Lys  12.3  10.7  Met  1.5  1.0  Met  5.1  4.3  Salt  4.0  4.0  Met + Cys  8.4  7.4  Limestone  11.5  17.0  Thr  7.9  7.5  Dicalcium phosphate  21.5  10.0  Trp  2.4  2.1  Choline chloride  1.8  1.8        Premix2  10.0  10.0          Content, g/kg    Content, g/kg  Ingredient  d 1 to 14  d 15 to 35  Nutrient  d 1 to 14  d 15 to 35  Corn  577.2  582.9  CP  214.4  200.1  Soybean meal  250.0  255.0  ME, kcal/kg  2,983.3  3,119.6  Corn gluten meal  60.0  44.0  Crude fiber  26.7  26.3  Full-fat soybeans  50.0  35.0  Ca  10.2  8.6  Soybean oil  10.0  38.0  Available P  5.0  4.2  Lys  2.5  1.3  Lys  12.3  10.7  Met  1.5  1.0  Met  5.1  4.3  Salt  4.0  4.0  Met + Cys  8.4  7.4  Limestone  11.5  17.0  Thr  7.9  7.5  Dicalcium phosphate  21.5  10.0  Trp  2.4  2.1  Choline chloride  1.8  1.8        Premix2  10.0  10.0        1Calculated values (Xiong et al., 2014); Aflatoxin B1 is not detectable (<2 µg/kg). 2Provided, per kilogram of diet, 9,000 IU vitamin A (retinyl acetate), 4,000 IU cholecalciferol, 50 IU vitamin E (DL-tocopheryl acetate), 2 mg vitamin K, 2 mg thiamin, 5 mg riboflavin, 15 mg d-pantothenic acid, 40 mg niacin, 2 mg pyridoxine, 0.1 mg biotin, 0.55 mg folic acid, 0.01 mg vitamin B12, 120 mg manganese, 1.2 mg iodine, 40 mg iron, 16 mg copper, 100 mg zinc, and 0.3 mg selenium. View Large Animals and Samples The experimental protocol of this study was approved by the Institutional Committee for Animal Use and Ethics of the Henan University of Science and Technology (Luoyang, P.R. China). A total of 480 1-d-old male Arbor Acres broilers (around 42 g/bird initial BW) were randomly allocated into 4 groups with 6 cages of 20 chicks each. A cage was a replicate. There was no statistical difference for the total BW per replicate between treatments. All replicates of treatments were evenly distributed in 3-layered cages in the chicken house, not considering layer effects. Chicks were given ad libitum access to feed and water throughout the trial. The room temperature was maintained at 34°C for first 5 d and then gradually decreased to 22°C by d 35. The birds received continuous light for the first 24 h followed by 16:8 light:dark for the remainder of the experiment. The birds and feed in each cage were weighed weekly, and the feed efficiency was adjusted for mortality on a cage basis. All the birds were monitored for general health twice a day. On d 14 and 35, the total amount of excreta per replicate was collected and mixed, and 10% was air-dried at 65°C for the determination of AFB1 and AFM1 content. Care was taken during the collection of excreta samples to avoid contamination from feathers and other foreign materials. Then, 5 birds per cage were randomly selected and euthanized by CO2 suffocation and dissected. Blood was immediately drawn from the heart by syringe, and aliquots were placed in centrifuge tubes for serum preparation (Liu et al., 2008). The liver and kidney were removed, and the duodenal mucosa and ileal digesta were collected and pooled per replicate. Partial samples of the liver, kidney, and ileal digesta were air-dried for the measurement of AFB1 and AFM1, and the remaining liver samples and duodenal mucosa were freeze-dried for the detection of GSH and related enzymes. Chemical and Biochemical Analysis The AFB1 contents in the samples were detected by commercial kits (Longke Fangzhou Biotech, Beijing, P.R. China) with a sensitivity of detection of 2 µg/kg, intra-assay CV < 6%, and interassay CV < 11%. Briefly, 0, 2, 5, 10, 20, and 50 µg/L AFB1 standard solutions were used to make the calibration curve, and all of them were included in an ELISA test kit. The AFM1 ELISA kits were purchased from Cusabio Biotech (Wuhan, P.R. China) with a detection range of 0.1 to 8.1 µg/kg, intra-assay CV < 3.1%, and interassay CV < 4.2%. The toxin contents in the feed, liver, kidney, digesta, and excreta were expressed as micrograms per kilogram on an air-dry basis (65°C) and as micrograms per liter in the serum samples. Commercial kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, P.R. China) were used for the detection of reduced GSH (detection range from 0.3 to 147.1 mg/L), GST (detection range from 6.0 to 22.0 units/mL), and GR (detection range from 1.6 to 320 units/L). The units of GSH, GST, and GR were finally calculated and expressed as milligrams per gram, micromoles per minute per gram, and micromoles per minute per gram per gram protein, respectively, in the supernatant of tissue samples to minimize the errors during sample preparation. Three parallel tests with aliquots of the same sample were performed for all samples and all chemical and biochemical analyses. Statistical Analysis Data were analyzed by ANOVA using the GLM procedure of SAS (SAS Inst. Inc., Cary, NC). The model for the factorial design included the LAB, AFB1, and the LAB × AFB1 interaction, and the effects of LAB statistically different from the control or AFB1 treatment were analyzed using the LAB or AFB1 present or not as a class. A cage (replicate) was an experimental unit for the samples of growth performance and excreta, and the mean of 5 birds per cage was the statistical unit for the samples of blood, liver, kidney, digesta, and mucosa. Differences between variables were separated using Duncan's multiple range test. Values in tables are means and pooled SEM. RESULTS Effects of Lactic Acid Bacteria and Aflatoxin B1 on Growth Performance Mortalities of broilers throughout the experiment were <5%, not statistically significant between treatments. During d 1 to 14, the main effects of LAB, AFB1, and their interaction on ADFI, ADG, and G:F were significant (P < 0.05), except for the effects of LAB and the interaction on G:F (Table 2). During d 15 to 35, most effects on the parameters of growth performance were significant (P < 0.01), except for the effect of the interaction on ADFI. During d 1 to 35, all effects on the growth parameters were significant (P < 0.01). Compared with the control diet, the LAB diet had no (P ≥ 0.05) effects on ADG and G:F, except for increased (P = 0.033) ADFI during d 1 to 35. In contrast to the AFB1 diet, the LAB inclusion improved (P < 0.05) ADFI, ADG, and G:F of broilers during each period, except for G:F on d 14, indicating that the main effects of dietary factors on growth performance are mainly attributable to the AFB1 basal diet with the LAB inclusion. Table 2. Effect of dietary factors on the growth performance of broilers   Treatment1    P-value  Item  Control  Control + LAB  AFB1  AFB1 + LAB  SEM  LAB  AFB1  LAB × AFB1  Dietary factors      AFB1, µg/kg  –2  –  40  40              LAB, cfu/kg  0  3 × 1010  0  3 × 1010          d 1 to 14 of age      ADFI, g/bird  36.88  37.15  34.38  36.13b  0.333  0.007  <0.001  0.038      ADG, g/bird  28.68  28.75  25.75  27.56B  0.291  0.004  <0.001  0.007      G:F  0.778  0.774  0.749  0.763  0.008  0.499  0.015  0.260  d 15 to 35 of age      ADFI, g/bird  129.08  129.67  126.96  128.59B  0.249  <0.001  <0.001  0.050      ADG, g/bird  73.69  74.37  69.84  72.60B  0.273  <0.001  <0.001  0.001      G:F  0.571  0.574  0.550  0.565B  0.002  <0.001  <0.001  0.008  d 1 to 35 of age      ADFI, g/bird  92.20  92.66a  89.93  91.61B  0.189  <0.001  <0.001  0.004      ADG, g/bird  55.69  56.12  52.20  54.58B  0.147  <0.001  <0.001  <0.001      G:F  0.604  0.606  0.580  0.596B  0.002  <0.001  <0.001  0.001    Treatment1    P-value  Item  Control  Control + LAB  AFB1  AFB1 + LAB  SEM  LAB  AFB1  LAB × AFB1  Dietary factors      AFB1, µg/kg  –2  –  40  40              LAB, cfu/kg  0  3 × 1010  0  3 × 1010          d 1 to 14 of age      ADFI, g/bird  36.88  37.15  34.38  36.13b  0.333  0.007  <0.001  0.038      ADG, g/bird  28.68  28.75  25.75  27.56B  0.291  0.004  <0.001  0.007      G:F  0.778  0.774  0.749  0.763  0.008  0.499  0.015  0.260  d 15 to 35 of age      ADFI, g/bird  129.08  129.67  126.96  128.59B  0.249  <0.001  <0.001  0.050      ADG, g/bird  73.69  74.37  69.84  72.60B  0.273  <0.001  <0.001  0.001      G:F  0.571  0.574  0.550  0.565B  0.002  <0.001  <0.001  0.008  d 1 to 35 of age      ADFI, g/bird  92.20  92.66a  89.93  91.61B  0.189  <0.001  <0.001  0.004      ADG, g/bird  55.69  56.12  52.20  54.58B  0.147  <0.001  <0.001  <0.001      G:F  0.604  0.606  0.580  0.596B  0.002  <0.001  <0.001  0.001  aStatistically different from the control treatment (P < 0.05). bStatistically different from the AFB1 treatment (P < 0.05). BStatistically different from the AFB1 treatment (P < 0.01). 1LAB = lactic acid bacteria; AFB1 = aflatoxin B1. 2– = not detectable (<2 µg/kg). View Large Table 2. Effect of dietary factors on the growth performance of broilers   Treatment1    P-value  Item  Control  Control + LAB  AFB1  AFB1 + LAB  SEM  LAB  AFB1  LAB × AFB1  Dietary factors      AFB1, µg/kg  –2  –  40  40              LAB, cfu/kg  0  3 × 1010  0  3 × 1010          d 1 to 14 of age      ADFI, g/bird  36.88  37.15  34.38  36.13b  0.333  0.007  <0.001  0.038      ADG, g/bird  28.68  28.75  25.75  27.56B  0.291  0.004  <0.001  0.007      G:F  0.778  0.774  0.749  0.763  0.008  0.499  0.015  0.260  d 15 to 35 of age      ADFI, g/bird  129.08  129.67  126.96  128.59B  0.249  <0.001  <0.001  0.050      ADG, g/bird  73.69  74.37  69.84  72.60B  0.273  <0.001  <0.001  0.001      G:F  0.571  0.574  0.550  0.565B  0.002  <0.001  <0.001  0.008  d 1 to 35 of age      ADFI, g/bird  92.20  92.66a  89.93  91.61B  0.189  <0.001  <0.001  0.004      ADG, g/bird  55.69  56.12  52.20  54.58B  0.147  <0.001  <0.001  <0.001      G:F  0.604  0.606  0.580  0.596B  0.002  <0.001  <0.001  0.001    Treatment1    P-value  Item  Control  Control + LAB  AFB1  AFB1 + LAB  SEM  LAB  AFB1  LAB × AFB1  Dietary factors      AFB1, µg/kg  –2  –  40  40              LAB, cfu/kg  0  3 × 1010  0  3 × 1010          d 1 to 14 of age      ADFI, g/bird  36.88  37.15  34.38  36.13b  0.333  0.007  <0.001  0.038      ADG, g/bird  28.68  28.75  25.75  27.56B  0.291  0.004  <0.001  0.007      G:F  0.778  0.774  0.749  0.763  0.008  0.499  0.015  0.260  d 15 to 35 of age      ADFI, g/bird  129.08  129.67  126.96  128.59B  0.249  <0.001  <0.001  0.050      ADG, g/bird  73.69  74.37  69.84  72.60B  0.273  <0.001  <0.001  0.001      G:F  0.571  0.574  0.550  0.565B  0.002  <0.001  <0.001  0.008  d 1 to 35 of age      ADFI, g/bird  92.20  92.66a  89.93  91.61B  0.189  <0.001  <0.001  0.004      ADG, g/bird  55.69  56.12  52.20  54.58B  0.147  <0.001  <0.001  <0.001      G:F  0.604  0.606  0.580  0.596B  0.002  <0.001  <0.001  0.001  aStatistically different from the control treatment (P < 0.05). bStatistically different from the AFB1 treatment (P < 0.05). BStatistically different from the AFB1 treatment (P < 0.01). 1LAB = lactic acid bacteria; AFB1 = aflatoxin B1. 2– = not detectable (<2 µg/kg). View Large Lactic Acid Bacteria Reduced Aflatoxin B1 and Aflatoxin M1 Residues The LAB diet reduced (P < 0.05) the tissue residues of AFB1 in the liver, kidney, and serum by 54.97, 44.62, and 30.42%, respectively, on d 14 and by 28.89, 37.56, and 56.85%, respectively, on d 35 (Table 3). Additionally, the contents of AFB1 in the ileal digesta and excreta decreased (P < 0.01) by 31.97 and 20.97%, respectively, on d 14 and by 27.46 and 24.76%, respectively, on d 35. These data imply that in the LAB diet, less AFB1 is absorbed into the circulatory system of broilers and more AFB1 is decomposed. Table 3. Effect of dietary factors on the residues of aflatoxins in broilers   AFB1 residue1  AFM12 residue  Item  AFB1  AFB1 + LAB  SEM  AFB1  AFB1 + LAB  SEM  Dietary factors      AFB1, µg/kg  40  40    40  40        LAB, cfu/kg  0  3 × 1010    0  3 × 1010    d 14 of age      Liver, µg/kg  36.61  16.53A  1.247  3.83  7.59A  0.453      Kidney, µg/kg  24.34  13.48A  1.060  5.15  13.09A  0.975      Serum, µg/L  6.64  4.62a  0.490  1.02  2.74A  0.086      Ileal digesta, µg/kg  53.77  36.58A  2.357  10.09  12.02a  0.558      Excreta, µg/kg  40.58  32.07A  1.623  7.42  9.95  0.843  d 35 of age      Liver, µg/kg  52.34  37.22A  1.140  16.33  17.34  1.725      Kidney, µg/kg  41.64  26.00A  1.116  14.13  18.73a  1.323      Serum, µg/L  8.76  3.78A  0.607  1.02  2.47A  0.079      Ileal digesta, µg/kg  77.50  56.22A  4.188  16.21  16.98  1.254      Excreta, µg/kg  67.50  50.79A  2.662  13.58  16.28  0.923    AFB1 residue1  AFM12 residue  Item  AFB1  AFB1 + LAB  SEM  AFB1  AFB1 + LAB  SEM  Dietary factors      AFB1, µg/kg  40  40    40  40        LAB, cfu/kg  0  3 × 1010    0  3 × 1010    d 14 of age      Liver, µg/kg  36.61  16.53A  1.247  3.83  7.59A  0.453      Kidney, µg/kg  24.34  13.48A  1.060  5.15  13.09A  0.975      Serum, µg/L  6.64  4.62a  0.490  1.02  2.74A  0.086      Ileal digesta, µg/kg  53.77  36.58A  2.357  10.09  12.02a  0.558      Excreta, µg/kg  40.58  32.07A  1.623  7.42  9.95  0.843  d 35 of age      Liver, µg/kg  52.34  37.22A  1.140  16.33  17.34  1.725      Kidney, µg/kg  41.64  26.00A  1.116  14.13  18.73a  1.323      Serum, µg/L  8.76  3.78A  0.607  1.02  2.47A  0.079      Ileal digesta, µg/kg  77.50  56.22A  4.188  16.21  16.98  1.254      Excreta, µg/kg  67.50  50.79A  2.662  13.58  16.28  0.923  aStatistically different from the AFB1 treatment (P < 0.05). AStatistically different from the AFB1 treatment (P < 0.01). 1AFB1 = aflatoxin B1; LAB = lactic acid bacteria. 2AFM1 = aflatoxin M1. View Large Table 3. Effect of dietary factors on the residues of aflatoxins in broilers   AFB1 residue1  AFM12 residue  Item  AFB1  AFB1 + LAB  SEM  AFB1  AFB1 + LAB  SEM  Dietary factors      AFB1, µg/kg  40  40    40  40        LAB, cfu/kg  0  3 × 1010    0  3 × 1010    d 14 of age      Liver, µg/kg  36.61  16.53A  1.247  3.83  7.59A  0.453      Kidney, µg/kg  24.34  13.48A  1.060  5.15  13.09A  0.975      Serum, µg/L  6.64  4.62a  0.490  1.02  2.74A  0.086      Ileal digesta, µg/kg  53.77  36.58A  2.357  10.09  12.02a  0.558      Excreta, µg/kg  40.58  32.07A  1.623  7.42  9.95  0.843  d 35 of age      Liver, µg/kg  52.34  37.22A  1.140  16.33  17.34  1.725      Kidney, µg/kg  41.64  26.00A  1.116  14.13  18.73a  1.323      Serum, µg/L  8.76  3.78A  0.607  1.02  2.47A  0.079      Ileal digesta, µg/kg  77.50  56.22A  4.188  16.21  16.98  1.254      Excreta, µg/kg  67.50  50.79A  2.662  13.58  16.28  0.923    AFB1 residue1  AFM12 residue  Item  AFB1  AFB1 + LAB  SEM  AFB1  AFB1 + LAB  SEM  Dietary factors      AFB1, µg/kg  40  40    40  40        LAB, cfu/kg  0  3 × 1010    0  3 × 1010    d 14 of age      Liver, µg/kg  36.61  16.53A  1.247  3.83  7.59A  0.453      Kidney, µg/kg  24.34  13.48A  1.060  5.15  13.09A  0.975      Serum, µg/L  6.64  4.62a  0.490  1.02  2.74A  0.086      Ileal digesta, µg/kg  53.77  36.58A  2.357  10.09  12.02a  0.558      Excreta, µg/kg  40.58  32.07A  1.623  7.42  9.95  0.843  d 35 of age      Liver, µg/kg  52.34  37.22A  1.140  16.33  17.34  1.725      Kidney, µg/kg  41.64  26.00A  1.116  14.13  18.73a  1.323      Serum, µg/L  8.76  3.78A  0.607  1.02  2.47A  0.079      Ileal digesta, µg/kg  77.50  56.22A  4.188  16.21  16.98  1.254      Excreta, µg/kg  67.50  50.79A  2.662  13.58  16.28  0.923  aStatistically different from the AFB1 treatment (P < 0.05). AStatistically different from the AFB1 treatment (P < 0.01). 1AFB1 = aflatoxin B1; LAB = lactic acid bacteria. 2AFM1 = aflatoxin M1. View Large On d 14, compared with the AFB1 diet, the LAB diet increased (P < 0.05) the contents of AFM1 in the liver, kidney, serum, and ileal digesta but did not affect (P ≥ 0.05) the content of AFM1 in the excreta. On d 35, the LAB diet increased (P < 0.05) the contents of AFM1 in the kidney and serum but not (P ≥ 0.05) in other tissues. These data indicate that LAB may increase the transformation of AFB1 into AFM1 in the gastrointestinal lumen and then partially into circulation or that absorbed AFB1 is metabolized to AFM1. Additionally, this transformation is more pronounced for young birds during d 1 to 14 because of the significances of most parameters during that phase. Lactic Acid Bacteria Increased the Activity of the Reduced Glutathione Pathway In the liver, on d 14, the main effects of LAB, AFB1, and their interaction were significant (P < 0.05) on GSH, GST, and GR, except for the effect of the interaction on GR (Table 4); the control diet with LAB inclusion increased (P < 0.05) GSH and GR, whereas the AFB1 diet with LAB inclusion increased (P < 0.05) all 3 parameters. On d 35, the effects of LAB and AFB1 on GSH, GST, and GR were significant (P < 0.01), but the only significant effect of the interaction was on GSH (P < 0.05). The control diet with LAB inclusion increased (P < 0.05) GSH, GST, and GR, and the AFB1 diet with LAB inclusion increased (P < 0.05) GSH and GR. Table 4. Effect of dietary factors on the activity of the glutathione pathway in the liver of broilers   Treatment2    P-value  Item1  Control  Control + LAB  AFB1  AFB1 + LAB  SEM  LAB  AFB1  LAB × AFB1  Dietary factors      AFB1, µg/kg  –3  –  40  40              LAB, cfu/kg  0  3 × 1010  0  3 × 1010          d 14 of age      GSH, mg/g  42.83  47.78a  12.84  31.70B  1.052  <0.001  <0.001  <0.001      GST, units/g  527.60  559.25  173.36  394.28B  24.853  <0.001  <0.001  0.001      GR, units/g  35.99  42.80A  24.30  34.45B  1.268  <0.001  <0.001  0.202  d 35 of age      GSH, mg/g  24.18  32.34A  9.83  22.68B  1.071  <0.001  <0.001  0.041      GST, units/g  248.70  320.32A  174.19  216.72  11.902  <0.001  <0.001  0.236      GR, units/g  24.35  32.94A  18.42  23.48b  1.173  <0.001  <0.001  0.148    Treatment2    P-value  Item1  Control  Control + LAB  AFB1  AFB1 + LAB  SEM  LAB  AFB1  LAB × AFB1  Dietary factors      AFB1, µg/kg  –3  –  40  40              LAB, cfu/kg  0  3 × 1010  0  3 × 1010          d 14 of age      GSH, mg/g  42.83  47.78a  12.84  31.70B  1.052  <0.001  <0.001  <0.001      GST, units/g  527.60  559.25  173.36  394.28B  24.853  <0.001  <0.001  0.001      GR, units/g  35.99  42.80A  24.30  34.45B  1.268  <0.001  <0.001  0.202  d 35 of age      GSH, mg/g  24.18  32.34A  9.83  22.68B  1.071  <0.001  <0.001  0.041      GST, units/g  248.70  320.32A  174.19  216.72  11.902  <0.001  <0.001  0.236      GR, units/g  24.35  32.94A  18.42  23.48b  1.173  <0.001  <0.001  0.148  aStatistically different from the control treatment (P < 0.05). bStatistically different from the AFB1 treatment (P < 0.05). AStatistically different from the control treatment (P < 0.01). BStatistically different from the AFB1 treatment (P < 0.01). 1GSH = glutathione; GST = glutathione S-transferases; GR = glutathione reductase. 2LAB = lactic acid bacteria; AFB1 = aflatoxin B1. 3– = not detectable (<2 µg/kg). View Large Table 4. Effect of dietary factors on the activity of the glutathione pathway in the liver of broilers   Treatment2    P-value  Item1  Control  Control + LAB  AFB1  AFB1 + LAB  SEM  LAB  AFB1  LAB × AFB1  Dietary factors      AFB1, µg/kg  –3  –  40  40              LAB, cfu/kg  0  3 × 1010  0  3 × 1010          d 14 of age      GSH, mg/g  42.83  47.78a  12.84  31.70B  1.052  <0.001  <0.001  <0.001      GST, units/g  527.60  559.25  173.36  394.28B  24.853  <0.001  <0.001  0.001      GR, units/g  35.99  42.80A  24.30  34.45B  1.268  <0.001  <0.001  0.202  d 35 of age      GSH, mg/g  24.18  32.34A  9.83  22.68B  1.071  <0.001  <0.001  0.041      GST, units/g  248.70  320.32A  174.19  216.72  11.902  <0.001  <0.001  0.236      GR, units/g  24.35  32.94A  18.42  23.48b  1.173  <0.001  <0.001  0.148    Treatment2    P-value  Item1  Control  Control + LAB  AFB1  AFB1 + LAB  SEM  LAB  AFB1  LAB × AFB1  Dietary factors      AFB1, µg/kg  –3  –  40  40              LAB, cfu/kg  0  3 × 1010  0  3 × 1010          d 14 of age      GSH, mg/g  42.83  47.78a  12.84  31.70B  1.052  <0.001  <0.001  <0.001      GST, units/g  527.60  559.25  173.36  394.28B  24.853  <0.001  <0.001  0.001      GR, units/g  35.99  42.80A  24.30  34.45B  1.268  <0.001  <0.001  0.202  d 35 of age      GSH, mg/g  24.18  32.34A  9.83  22.68B  1.071  <0.001  <0.001  0.041      GST, units/g  248.70  320.32A  174.19  216.72  11.902  <0.001  <0.001  0.236      GR, units/g  24.35  32.94A  18.42  23.48b  1.173  <0.001  <0.001  0.148  aStatistically different from the control treatment (P < 0.05). bStatistically different from the AFB1 treatment (P < 0.05). AStatistically different from the control treatment (P < 0.01). BStatistically different from the AFB1 treatment (P < 0.01). 1GSH = glutathione; GST = glutathione S-transferases; GR = glutathione reductase. 2LAB = lactic acid bacteria; AFB1 = aflatoxin B1. 3– = not detectable (<2 µg/kg). View Large In the duodenal mucosa, on d 14, the effects of LAB, AFB1, and their interaction on GSH, GST, and GR were significant (P < 0.01), except for the effect of the interaction on GST (Table 5). The control diet with LAB inclusion increased (P < 0.05) GSH and GST, whereas the AFB1 diet with LAB inclusion increased (P < 0.05) GSH, GST, and GR. On d 35, the effects of the dietary factors on GSH and GST were significant (P < 0.01), the effect of the interaction on GST was significant (P < 0.01), and the control diet with LAB inclusion increased (P < 0.05) GST and GR, whereas the addition of LAB to the AFB1 basal diet increased (P < 0.05) GSH and GR. These data imply that exogenous probiotics can induce the detoxification of the GSH pathway in the intestinal lumen of broilers. Table 5. Effect of dietary factors on the activity of the glutathione pathway in the duodenal mucosa of broilers   Treatment2    P-value  Item1  Control  Control + LAB  AFB1  AFB1 + LAB  SEM  LAB  AFB1  LAB × AFB1  Dietary factors      AFB1, µg/kg  –3  –  40  40              LAB, cfu/kg  0  3 × 1010  0  3 × 1010          d 14 of age      GSH, mg/g  30.79  57.31A  22.50  29.20B  2.324  <0.001  <0.001  <0.001      GST, units/g  209.94  259.29a  151.72  191.90b  12.022  0.001  <0.001  0.707      GR, units/g  27.81  29.31  11.59  23.29B  1.013  <0.001  <0.001  <0.001  d 35 of age      GSH, mg/g  31.71  34.45  20.48  25.42B  0.704  <0.001  <0.001  0.134      GST, units/g  335.97  544.41A  179.11  228.10  20.035  <0.001  <0.001  0.001      GR, units/g  27.39  34.76a  16.52  26.01B  1.665  <0.001  <0.001  0.530    Treatment2    P-value  Item1  Control  Control + LAB  AFB1  AFB1 + LAB  SEM  LAB  AFB1  LAB × AFB1  Dietary factors      AFB1, µg/kg  –3  –  40  40              LAB, cfu/kg  0  3 × 1010  0  3 × 1010          d 14 of age      GSH, mg/g  30.79  57.31A  22.50  29.20B  2.324  <0.001  <0.001  <0.001      GST, units/g  209.94  259.29a  151.72  191.90b  12.022  0.001  <0.001  0.707      GR, units/g  27.81  29.31  11.59  23.29B  1.013  <0.001  <0.001  <0.001  d 35 of age      GSH, mg/g  31.71  34.45  20.48  25.42B  0.704  <0.001  <0.001  0.134      GST, units/g  335.97  544.41A  179.11  228.10  20.035  <0.001  <0.001  0.001      GR, units/g  27.39  34.76a  16.52  26.01B  1.665  <0.001  <0.001  0.530  aStatistically different from the control treatment (P < 0.05). bStatistically different from the AFB1 treatment (P < 0.05). AStatistically different from the control treatment (P < 0.01). BStatistically different from the AFB1 treatment (P < 0.01). 1GSH = glutathione; GST = glutathione S-transferases; GR = glutathione reductase. 2LAB = lactic acid bacteria; AFB1 = aflatoxin B1. 3– = not detectable (<2 µg/kg). View Large Table 5. Effect of dietary factors on the activity of the glutathione pathway in the duodenal mucosa of broilers   Treatment2    P-value  Item1  Control  Control + LAB  AFB1  AFB1 + LAB  SEM  LAB  AFB1  LAB × AFB1  Dietary factors      AFB1, µg/kg  –3  –  40  40              LAB, cfu/kg  0  3 × 1010  0  3 × 1010          d 14 of age      GSH, mg/g  30.79  57.31A  22.50  29.20B  2.324  <0.001  <0.001  <0.001      GST, units/g  209.94  259.29a  151.72  191.90b  12.022  0.001  <0.001  0.707      GR, units/g  27.81  29.31  11.59  23.29B  1.013  <0.001  <0.001  <0.001  d 35 of age      GSH, mg/g  31.71  34.45  20.48  25.42B  0.704  <0.001  <0.001  0.134      GST, units/g  335.97  544.41A  179.11  228.10  20.035  <0.001  <0.001  0.001      GR, units/g  27.39  34.76a  16.52  26.01B  1.665  <0.001  <0.001  0.530    Treatment2    P-value  Item1  Control  Control + LAB  AFB1  AFB1 + LAB  SEM  LAB  AFB1  LAB × AFB1  Dietary factors      AFB1, µg/kg  –3  –  40  40              LAB, cfu/kg  0  3 × 1010  0  3 × 1010          d 14 of age      GSH, mg/g  30.79  57.31A  22.50  29.20B  2.324  <0.001  <0.001  <0.001      GST, units/g  209.94  259.29a  151.72  191.90b  12.022  0.001  <0.001  0.707      GR, units/g  27.81  29.31  11.59  23.29B  1.013  <0.001  <0.001  <0.001  d 35 of age      GSH, mg/g  31.71  34.45  20.48  25.42B  0.704  <0.001  <0.001  0.134      GST, units/g  335.97  544.41A  179.11  228.10  20.035  <0.001  <0.001  0.001      GR, units/g  27.39  34.76a  16.52  26.01B  1.665  <0.001  <0.001  0.530  aStatistically different from the control treatment (P < 0.05). bStatistically different from the AFB1 treatment (P < 0.05). AStatistically different from the control treatment (P < 0.01). BStatistically different from the AFB1 treatment (P < 0.01). 1GSH = glutathione; GST = glutathione S-transferases; GR = glutathione reductase. 2LAB = lactic acid bacteria; AFB1 = aflatoxin B1. 3– = not detectable (<2 µg/kg). View Large DISCUSSION Aflatoxin B1 is toxic to farm animals, resulting in millions of dollars in annual losses to producers due to depressed growth rates, increased susceptibility to disease, reduced feed utilization, and other adverse effects. In this study, some similar effects were observed. The AFB1 diet significantly decreased the ADFI, ADG, and G:F of broiler chickens during d 1 to 35 of age. The sensitivity of young animals to AFB1 is associated with their rapid growth, not yet well developed physiological function, inability to defend against toxic xenobiotics, and deficient or inadequate activity of GST. But the dietary AFB1 did not cause different mortalities between treatments in this study, indicating that the toxin level was too low to cause serious death, but the depressed growth performance demonstrated the subclinical symptoms of aflatoxicosis. Recently, a number of in vitro studies have reported that probiotics, including LAB, had antimycotic and antiaflatoxigenic potential (Peltonen et al., 2001; Biernasiak et al., 2006; Roger et al., 2015). Likewise, in vivo experiments demonstrated that LAB was able to detoxify AFB1. Zuo et al. (2013) reported that a combination of Lactobacillus casei with other probiotics in a diet containing AFB1 at 246.8 μg/kg did not improve the growth performance of broilers during d 43 to 73 of age, but a significant effect was found for AFB1 at 446.8 μg/kg, which may be due to higher toxin tolerances for adult broilers. Gratz et al. (2006) showed that the administration of Lactobacillus rhamnosus GG modulated AFB1 uptake and alleviated AFB1–associated growth faltering and liver effects in rats. In this study, the inclusion of LAB showed protective effects on the ADG, ADFI, and G:F based on the AFB1 diet, indicating that the LAB was capable of relieving the subclinical symptoms of aflatoxicosis. The capability was also supported by the interaction of dietary factors on the growth performance, and the LAB effect is dependent on the presence of aflatoxin. Abbès et al. (2016) found that the LAB induced protective effects against AFB1 toxicity or oxidative stress in part through the adhesion of cell wall constituents, namely, physical adhesion, and in part through biodegradation; however, to some extent, the adhesion diminished the bioavailability. Fortunately, it is easy to handle this situation by increasing LAB intake because excessive probiotics have no side effects, but further study about this is needed. For the toxicants in the animal gastrointestinal lumen, some disappear with excreta and others are degraded by exogenous or endogenous microbes or absorbed by the intestinal epithelial cells. The absorbed toxin inevitably results in toxic threats and residues in the tissues of animals. The amount of AFB1 in the tissues or digesta is dependent on the ingested content of toxicants, the physiological status of animals, or the ability of a detoxifier (Bintvihok et al., 2002; Hussain et al., 2010). In this study, compared with the AFB1 diet, the LAB diet reduced AFB1 residues in the liver, kidney, serum, and ileal digesta, indicating that the LAB interfered with the AFB1 absorption. Additionally, the lower residue of AFB1 in the excreta than in the ileal digesta may be due to the degradation of aflatoxin by cecal microbes. The main hydroxylated metabolite of AFB1 is AFM1, whose toxicity is much less than its precursor, and the metabolizing sites involved are the liver, kidney, intestine, and respiratory organs (Fernández et al., 1997; Van Vleet et al., 2002). In this study, compared with the AFB1 diet, LAB diets showed lower levels of AFM1 in the liver and kidney, indicating that less AFB1 was absorbed into the organs. Rather, the higher levels of AFM1 in the digesta showed that more AFB1 was decomposed by the probiotics used in this study. Gratz et al. (2006) reported that a L. rhamnosus strain was able to bind AFB1, to restrict its rapid absorption from the intestine, and to increase fecal AFM1. Similarly, the basolateral AFM1 concentration in Caco-2 cells was increased after incubation with AFB1 in the presence of LAB strains and showed a dose dependence on the probiotics (Gratz et al., 2007). However, in this study, in the AFB1 or LAB diet, there were increased levels of AFB1 and AFM1 in the tissues, digesta, and excreta with age growing of birds. It seems that the detoxifying efficiency of LAB is affected by the physiological age, which needs further study. The mechanism of AFB1 transformation into AFM1 is mainly related to the activation of cytochrome P-450 1A5 in poultry (Gilday et al., 1996; reviewed by Rawal et al., 2010). An in vitro study showed that a L. rhamnosus strain GG reduced AFB1 transport, metabolism, and toxicity in Caco-2 cells via cytochrome P-450 3A4 (Gratz et al., 2007). Furthermore, the LAB could bind and degrade AFM1 in milk and its products. El Khoury et al. (2011) reported that Lactobacillus bulgaricus and Streptococcus thermophilus strains used in the Lebanese dairy industry showed effectiveness in reducing free AFM1 content in liquid culture medium or during yogurt processing. Bovo et al. (2013) found that 7 LAB strains could remove from 41 to 87% of AFM1 in skimmed milk. Therefore, in this study, in the intestinal digesta, the LAB effectively degraded not just AFB1 but AFM1 as well. However, their possible interaction with host enterocytes needs further study. For AFB1 metabolism, another focus is on the highly toxic metabolite AFBO, which reacts with cellular nucleophiles and induces mutations by alkylating DNA (reviewed by Carvajal-Moreno, 2015). The formation of AFBO is related to cytochrome P-450 1A37 and 1A5 in poultry (Gilday et al., 1996; Ourlin et al., 2000). The principal route of AFBO detoxification is through conjugation with endogenous GSH, a reaction catalyzed by GST (Eaton and Bammler, 1999; Kim et al., 2010). Previous studies demonstrated that probiotics could protect DNA against AFBO damage. The concentration of GSH in the mouse spleen was decreased by the oral ingestion of AFB1 and increased by the addition of LAB (Abbès et al., 2016). Fermented milk containing LAB was able to increase GST and GSH in the liver of rats exposed to AFB1 (Kumar et al., 2012). The LAB also protected membranes and DNA from damage in Caco-2 cells (Gratz et al., 2007) and in chickens (Slizewska et al., 2010), although poultry appears to lack functional GST with an affinity toward AFBO (Klein et al., 2000). Indeed, in this study, the GST activity was activated by the LAB in the liver or mucosa on d 14 or 35 and also varied with the age of growing birds in the LAB diet, indicating that the LAB can promote GST production via a host itself or its gut microbes. In the GSH redox cycle, GR catalyzes the reduction of glutathione disulfide to the sulfhydryl form GSH, but it is not as important as the catalytic effect of GST on AFB1 (Tulayakul et al., 2005). However, information is unavailable about the effect of LAB on the GSH pathway in farm animals exposed to AFB1. Interestingly, in this study, in the liver and duodenal mucosa, the LAB showed significantly increased effects on the activity of the GSH pathway. Conversely, the AFB1 decreased the antioxidant activity. Furthermore, on d 14, the LAB effect was more pronounced in the AFB1 basal diet, which resulted in significant interaction effects on these GSH parameters. These data are suggestive of the beneficial modulation of dietary LAB on the GSH pathway when farm animals, especially young ones, are exposed to the toxicity of AFB1. Meanwhile, the role of exogenous GSH from the bacteria in the gut lumen against the toxicity is worthy of further exploration. Conclusions In this study, the toxicity of AFB1 was reflected in decreased growth performance and GSH activity. 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TI - Detoxification, metabolism, and glutathione pathway activity of aflatoxin B1 by dietary lactic acid bacteria in broiler chickens
JF - Journal of Animal Science
DO - 10.2527/jas2017.1644
DA - 2017-10-01
UR - https://www.deepdyve.com/lp/oxford-university-press/detoxification-metabolism-and-glutathione-pathway-activity-of-SqXFvapTsW
SP - 4399
EP - 4406
VL - 95
IS - 10
DP - DeepDyve
ER -