The authors have declared that no competing interests exist.
Conceived and designed the experiments: MW XX. Performed the experiments: MX HX JH. Analyzed the data: MW JY JD TL XX. Contributed reagents/materials/analysis tools: MX GL BT KY YY TL. Wrote the paper: MW HX WR AO OA.
Deoxynivalenol (DON) has various toxicological effects in humans and pigs that result from the ingestion of contaminated cereal products. This study was conducted to investigate the protective effects of dietary supplementation with glutamic acid on piglets challenged with DON. A total of 20 piglets weaned at 28 d of age were randomly assigned to receive 1 of 4 treatments (5 piglets/treatment): 1) basal diet, negative control (NC); 2) basal diet +4 mg/kg DON (DON); 3) basal diet +2% (g/g) glutamic acid (GLU); 4) basal diet +4 mg/kg DON +2% glutamic acid (DG). A 7-d adaptation period was followed by 30 days of treatment. A metabolite analysis using nuclear magnetic resonance spectroscopy (1H-NMR)-based metabolomic technology and the determination of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities for plasma, as well as the activity of Caspase-3 and the proliferation of epithelial cells were conducted. The results showed that contents of low-density lipoprotein, alanine, arginine, acetate, glycoprotein, trimethylamine-N-oxide (TMAO), glycine, lactate, and urea, as well as the glutamate/creatinine ratio were higher but high-density lipoprotein, proline, citrate, choline, unsaturated lipids and fumarate were lower in piglets of DON treatment than that of NC treatment (
The trichothecene mycotoxin deoxynivalenol (DON) is often found as a contaminant in agricultural staples, and the toxic effects of DON have been well-characterized in humans as well as pigs which is the most susceptible animal
While many strategies have been developed to reduce the toxic effects of DON, including physical adsorption, chemical decomposition and microbial detoxification
Substantial effort is being directed toward the study of metabolomics, which provides a useful systematic approach to understanding the global metabolic responses of living systems to influence such as disease, nutrition and environment
This study was conducted in accordance with the Chinese Guidelines for Animal Welfare and was approved by the Animal Care and Use Committee of the Chinese Academy of Sciences (Beijing, China)
The mold strain
A total of 20 piglets (Duroc × Landrace × Large Yorkshire) weaned at 28 d of age were randomly assigned to receive 1 of 4 treatments (5 piglets/treatment): 1) basal diet, negative control (NC); 2) basal diet +4 mg/kg DON (DON); 3) basal diet +2% (g/g) glutamic acid (GLU); 4) basal diet +4 mg/kg DON +2% (g/g) glutamic acid (DG). The basal diets were prepared from corn, soybean meal, wheat bran, limestone, CaHPO4, salt, and additive premix to meet or exceed the nutritional requirements for growing pigs as recommended by the NRC (1998) (
Item | NC | DON | GLU | DG |
Ingredients, % | ||||
Corn | 61.25 | 61.25 | 60.03 | 60.03 |
Soybean | 15.79 | 15.79 | 15.47 | 15.47 |
Extruded-soybean | 10.00 | 10.00 | 9.80 | 9.80 |
Fish meal | 5.00 | 5.00 | 4.90 | 4.90 |
Wheat bran | 3.00 | 3.00 | 2.94 | 2.94 |
Soybean oil | 1.74 | 1.74 | 1.71 | 1.71 |
Premix |
1.00 | 1.00 | 0.98 | 0.98 |
Limestone powder | 0.98 | 0.98 | 0.96 | 0.96 |
Calcium hydrogen phosphate | 0.78 | 0.78 | 0.76 | 0.76 |
Salt | 0.37 | 0.37 | 0.36 | 0.36 |
Glutamic acid | 0.00 | 0.00 | 2.00 | 2.00 |
Lys·HCl (98%) | 0.09 | 0.09 | 0.09 | 0.09 |
Analyzed chemical composition | ||||
DM, % | 89.85 | 89.84 | 89.83 | 89.82 |
CP, % | 18.90 | 18.91 | 18.96 | 18.97 |
Crude ash, % | 6.79 | 6.78 | 6.77 | 6.75 |
Calculated DE, kcal/kg | 3400 | 3400 | 3400 | 3400 |
NC = uncontaminated basal diet, DON = basal diet contaminated with deoxynivalenol (4 mg/kg), GLU = uncontaminated basal diet supplemented with 2% glutamic acid; DG = DON diet supplemented with 2% glutamic acid.
Providing the following amount of vitamins and minerals per kilogram on an as-fed basis: Zn (ZnO), 50 mg; Cu (CuSO4), 20 mg; Mn (MnO), 55 mg; Fe (FeSO4), 100 mg; I (KI), 1 mg; Co (CoSO4), 2 mg; Se (Na2SeO3), 0.3 mg; vitamin A, 8,255 IU; vitamin D3, 2,000 IU; vitamin E, 40 IU; vitamin B1, 2 mg; vitamin B2, 4 mg; pantothenic acid, 15 mg; vitamin B6, 10 mg; vitamin B12, 0.05 mg; vitamin PP, 30 mg; folic acid, 2 mg; vitamin K3, 1.5 mg; biotin, 0.2 mg; choline chloride, 800 mg; and vitamin C, 100 mg. The premix did not contain additional copper, zinc, antibiotics, or probiotics.
The experiment was arranged as a randomized design, and pigs were allowed free access to water throughout the experimental period. After an adaptation period of 7 days, piglets were fed their respective diets 3 times per day (at 8:00, 13:00 and 18:00) for a 30-d period. Fifteen and 30 d after the initiation of treatment, 10 mL of blood was collected from a jugular vein into a collection tube with heparin sodium 2 h after feeding, and centrifuged at 1000×g for 10 min at 4°C to obtain plasma samples, which were stored at −80°C for further analysis. On d 30, piglets were anesthetized with sodium pentobarbital and exsanguinated. The small intestine was excised, and rinsed thoroughly with ice-cold physiological saline solution, and the jejunum and ileum were dissected out. Two-centimeter segments of the mid-jejunum and mid-ileum were cut and fixed in 4% formaldehyde for measurements of crypt cell proliferation. In addition, samples of the jejunal and ileal mucosa were immediately snap-frozen in liquid N and stored at −80°C for the determination of Caspase-3.
Glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) activities were measured using spectrophotometric kits in accordance with the manufacturer's instructions (Nanjing Jiancheng Biotechnology Institute, Nanjing, China).
After tisssue samples were subjected to dehydration, embedding, and sectioning, crypt cell proliferation was determined using proliferating cell nuclear antigen (PCNA) as described by Xu et al. (2003).
1H NMR spectroscopic measurement of plasma samples was conducted as described previously
Free induction decays (FID) were multiplied by an exponential window function of 1.0 Hz prior to Fourier transformation and corrected for phase and baseline distortions using TopSpin 2.0 (Bruker). Chemical shifts were referenced to the peak of the anomeric proton of α-glucose at δ 5.23. NMR spectra (δ 0.5–8.5) were binned with regions 0.002 ppm wide and automatically integrated with the AMIX package (v.3.8.3, Bruker Biospin, Germany). The region at δ 4.55–5.13 was removed to avoid the effects of imperfect water suppression. Consequently, the spectra over the ranges δ 0.5–4.55, and δ 5.13–8.5 were selected and reduced to 3663 regions, each of which was 0.002ppm wide. Each integral region was normalized to the sum of all integral regions for each spectrum prior to pattern recognition analysis.
An overview of the data distribution and intersample similarities (e.g., clusterings and outliers) for each serum sample was first investigated by PCA (principal component analysis) using Simca-P 11.0 software (Umetrics, Sweden). NMR spectral data were further analyzed using the OPLS-DA (orthogonal projection to latent structure with discriminant analysis) method with unit variance scaling. Since the OPLS-DA results for the BPP-LED spectra of serum are similar to those for standard 1D spectra, the analysis of BPP-LED spectra will not be discussed in the
All statistical analyses were performed using the SAS software package (Version 9.2; SAS Institute, Cary, NC, USA). Data were subjected to a Proc Mixed analysis of variance-covariance followed by Tukey's multiple comparisons test. Data are expressed as the mean ± standard error of the mean.
Oxidative stress has been shown to be involved in the progression of DON-induced injuries, and investigations have found that dietary supplementation with DON increases the production of reactive oxygen metabolities, such as hydroxyl radical, hydrogen peroxide, and superoxide
A: SOD activity in each group at day 15 and 30. B: GSH-Px activity in each group at day 15 and 30. Dietary treatments were NC, an uncontaminated basal diet, DON, the basal contaminated with 4mg/kg deoxynivalenol, GLU, uncontaminated basal diet with 2% glutamic acid supplementation, and DG, deoxynivalenol-contaminated (4 mg/kg) basal diet with 2% glutamic acid supplementation. Data are presented as means ± SEM, n = 5 for treatments, with a-d used to indicate statistically significant difference (
A decrease in the production of PCNA labeling index has been reported in piglets treated with DON
Item | Diet | SEM | ||||
NC | DON | GLU | DG | |||
Jejunum | 39.83a | 6.15c | 37.83a | 29.48b | 3.48 | |
Ileum | 61.88a | 22.63c | 57.58a | 50.13b | 4.01 |
Values with different letters within the same row are different (
Dietary treatments were NC, an uncontaminated basal diet, DON, the basal contaminated with 4mg/kg deoxynivalenol, GLU, uncontaminated basal diet with 2% glutamic acid supplementation, and DG, deoxynivalenol-contaminated (4 mg/kg) basal diet with 2% glutamic acid supplementation (n = 5).
Proliferating cell nuclear antigen labeling was defined as the ratio of positive cells to total cells in each section.
Item | Diet | SEM | ||||
NC | DON | GLU | DG | |||
Jejunum | 5.74b | 7.60a | 5.53b | 5.50b | 0.24 | |
Ileum | 5.42b | 7.33a | 5.63b | 5.75b | 0.22 |
Values with different letters within the same row are different (
Dietary treatments were NC, an uncontaminated basal diet, DON, the basal contaminated with 4mg/kg deoxynivalenol, GLU, uncontaminated basal diet with 2% glutamic acid supplementation, and DG, deoxynivalenol-contaminated (4 mg/kg) basal diet with 2% glutamic acid supplementation (n = 5).
1H NMR spectra of biological fluids and tissues provide a unique fingerprint of the metabolic state of an organism along with considerable information on the nature of the drug or toxin to which an animal has been exposed
The spectra in the aromatic region were magnified four times (A) (δ 5.7–8.5) or eight times (B) (δ 5.7–8.5) compared to the aliphatic region (δ 0.6–5.4). Keys for metabolites are given in
Visual inspection of the 1H NMR spectra revealed visible differences in plasma metabolites among piglets in the NC, DON, GLU and DG groups. For example, the concentrations of LDL, alanine, arginine, glycoprotein, TMAO, lactate and urea were higher, while those of HDL, proline, citrate, unsaturated lipids and fumarate were lower in the plasma from the DON group compared with that from the NC group. To perform a more detailed analysis of metabolic differences among the piglets in the four groups, multivariate data analyses were performed using PCA and OPLS-DA.
PCA of plasma CMPG and standard 1D spectral data of piglets in the four groups showed clear clustering (data not shown). Further analysis using OPLS-DA indicated that the concentrations of plasma HDL, proline, acetate, citrate, unsaturated lipids, and fumarate were decreased (
Dietary treatments were NC, an uncontaminated basal diet, DON, the basal contaminated with 4 mg/kg deoxynivalenol, GLU, uncontaminated basal diet with 2% glutamic acid supplementation, and DG, deoxynivalenol-contaminated (4 mg/kg) basal diet with 2% glutamic acid supplementation.
Metabolites | Relative integrals (%) |
SEM | |||||
NC | DON | GLU | DG | ||||
HDL(δ0.83) | CPMG | 1.971b | 1.751c | 2.270a | 2.007ab | 0.091 | <0.01 |
Standard 1D | 1.926a | 1.814b | 1.948a | 1.848b | 0.014 | <0.01 | |
LDL (δ0.84) | CPMG | 1.010b | 1.266a | 0.964b | 1.261a | 0.036 | <0.01 |
Standard 1D | 1.001b | 1.062a | 0.992b | 1.051a | 0.008 | <0.01 | |
Alanine (δ1.48) | CPMG | 1.033b | 1.237a | 0.980b | 1.016b | 0.026 | <0.01 |
Standard 1D | 1.013b | 1.040a | 1.003bc | 0.993c | 0.005 | <0.01 | |
Arginine (δ1.63) | CPMG | 0.502b | 0.658a | 0.499b | 0.610ab | 0.022 | <0.01 |
Standard 1D | 1.249b | 1.314a | 1.248b | 1.304a | 0.009 | <0.01 | |
Acetate (δ1.92) | CPMG | 0.172b | 0.218a | 0.194b | 0.173b | 0.005 | <0.01 |
Standard 1D | 0.310b | 0.318a | 0.320ab | 0.318ab | 0.005 | 0.168 | |
Proline(δ2.00) | CPMG | 1.204a | 0.987b | 1.185a | 1.083ab | 0.024 | <0.01 |
Standard 1D | 1.478a | 1.408c | 1.486a | 1.450b | 0.008 | <0.01 | |
Glycoprotein (δ2.05) | CPMG | 2.190b | 2.766a | 2.254b | 2.630a | 0.063 | <0.01 |
Standard 1D | 1.461c | 1.593a | 1.460c | 1.513b | 0.014 | <0.01 | |
Citrate (δ2.52) | CPMG | 0.196a | 0.166b | 0.187a | 0.197a | 0.004 | <0.01 |
Standard 1D | 0.463a | 0.451b | 0.464a | 4.681a | 0.002 | <0.01 | |
TMAO (δ3.26) | CPMG | 0.575b | 0.811a | 0.549b | 0.613b | 0.029 | <0.01 |
Standard 1D | 0.169b | 0.227a | 0.184b | 0.183b | 0.006 | <0.01 | |
Glycine (δ3.56) | CPMG | 1.179b | 1.439a | 1.237b | 1.242b | 0.028 | <0.01 |
Standard 1D | 0.343b | 0.380a | 0.347b | 0.337b | 0.005 | <0.01 | |
Lactate (δ4.11) | CPMG | 2.462bc | 3.817a | 2.316c | 3.154ab | 0.158 | <0.01 |
Standard 1D | 1.289b | 1.549a | 1.234b | 1.260b | 0.034 | <0.01 | |
Unsaturated lipids (δ5.31) | CPMG | 0.706a | 0.576b | 0.712a | 0.678ab | 0.018 | 0.014 |
Standard 1D | 0.757a | 0.640c | 0.781a | 0.679b | 0.014 | <0.01 | |
Urea (δ5.78) | CPMG | 0.178b | 0.271a | 0.178b | 0.244a | 0.010 | <0.01 |
Standard 1D | 0.165c | 0.212a | 0.179b | 0.204a | 0.005 | <0.01 | |
Fumarate (δ6.52) | CPMG | 0.0061a | 0.0033b | 0.0061a | 0.0057a | 0.00003 | <0.01 |
Standard 1D | 0.028a | 0.026b | 0.028a | 0.028a | 0.000 | 0.016 | |
Glutamate/ Creatinine | CPMG | 3.300b | 4.152a | 3.352b | 3.435b | 0.086 | <0.01 |
Standard 1D | 3.265 | 3.253 | 3.252 | 3.248 | 0.023 | 0.995 |
Data are presented as means ± SEM, n = 5 for treatments, with a-c used to indicate statistically significant difference (
Dietary treatments were NC, an uncontaminated basal diet, DON, the basal contaminated with 4 mg/kg deoxynivalenol, GLU, uncontaminated basal diet with 2% glutamic acid supplementation, and DG, deoxynivalenol-contaminated (4 mg/kg) basal diet with 2% glutamic acid supplementation (n = 5).
Among various mycotoxins, including DON, aflatoxin B1, zearalenone, fumonisin, fusariotoxin T2, and ochratoxin A, DON is encountered at the highest concentrations in the cereal foods worldwide
Due to its emetic effects, DON has been associated with human gastroenteritis
To further our understanding of the biological phenomena observed in the four treatment groups, we decided to perform targeted metabolomic analyses on a series of metabolites from the main metabolic pathways. These metabolites are the end-products or intermediates of cellular processes and therefore reflect the global integrated response of an organ or entire biological system to pathophysiologic stimuli
Another intriguing observation from the current study is that the DON group had an elevated concentration of TMAO and a greater glutamate/creatinine ratio, which suggested renal medullary injury and hepatic failure
Taken together, our findings demonstrated that DON induces oxidative stress, causes epithelial cell apoptosis, and induces energy, lipid and amino acid metabolism disorders. Furthermore, dietary supplementation with glutamic acid decreases oxidative stress, promotes intestinal epithelial cell proliferation, and regulates the metabolism disorders induced by DON, indicating that glutamic acid may be a useful nutritional supplement for regulating DON-induced injury.
We offer our profound admiration and respect to the many dedicated and hard-working researchers in this field and in our lab.