Systemic Metabolic Responses of Broiler Chickens ... - ACS Publications

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Systemic Metabolic Responses of Broiler Chickens and Piglets to Acute T‑2 Toxin Intravenous Exposure Qianfen Wan,† Qinghua He,‡ Xianbai Deng,§ Fuhua Hao,† Huiru Tang,∥ and Yulan Wang*,†,⊥ †

Chinese Academy of Sciences (CAS) Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Centre for Magnetic Resonance, Wuhan Institute of Physics and Mathematics, University of Chinese Academy of Sciences, Wuhan, Hubei 430071, People’s Republic of China ‡ Department of Food Science and Engineering, College of Chemistry and Environmental Engineering, Shenzhen University, Shenzhen, Guangdong 518060, People’s Republic of China § College of Veterinary Medicine, South China Agricultural University, Guangzhou, Guangdong 510642, People’s Republic of China ∥ State Key Laboratory of Genetic Engineering, Collaborative Innovation Center for Genetics and Development, Metabolomics and Systems Biology Laboratory, School of Life Sciences, Fudan University, Shanghai 200433, People’s Republic of China ⊥ Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou, Zhejiang 310058, People’s Republic of China S Supporting Information *

ABSTRACT: The aim of this study is to thoroughly investigate the toxicity mechanism of mycotoxin T-2 toxin and to further understand the endogenous metabolic alterations induced by T-2 toxin. To achieve this, a nuclear magnetic resonance (NMR)based metabonomics approach was used to analyze the metabolic alterations induced by a single intravenous injection of T-2 toxin (0.5 mg/kg of body weight) in piglets and broiler chickens. A range of metabolites in the plasma, liver, kidney, and spleen of broiler chickens and plasma of piglets was changed following T-2 toxin injection. For example, a rapid increase of amino acids together with a significant reduction of glucose and lipid occurred in the plasma of broiler chickens and piglets following T-2 toxin treatment. A significant accumulation of amino acids and modulated nucleotides were detected in the liver, kidney, and spleen of T-2 toxin-treated broiler chickens. These data indicated that T-2 toxin caused endogenous metabolic changes in multiple organs and perturbed various metabolic pathways, including energy, amino acid, and nucleotide metabolism, as well as oxidative stress. We also observed elevated levels of tryptophan in the T-2 toxin-treated broiler chickens, which may explain the reported neurotoxic effects of T-2 toxin. These findings provide important information on the toxicity of T-2 toxin and demonstrate the power of the NMR-based metabonomics approach in exploring the toxicity mechanism of xenobiotics. KEYWORDS: T-2 toxin, metabonomics, broiler chicken, piglet

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INTRODUCTION T-2 toxin, first isolated from Fusarium tricinctum in 1968,1 is a member of type A subgroup of trichothecenes and present in a wide range of cereal crops and animal feedstuffs. As a result of its high toxicity, T-2 toxin directly or indirectly poses a health threat to humans and animals when T-2 toxin-contaminated cereal crops or animal feed are consumed. For example, T-2 toxin is the main cause of alimentary toxic aleukia (ATA) in humans.2 The clinical symptoms of T-2 intoxication are anorexia, loss of weight, weakness, vomiting, bloody feces, and ataxia.3 There are many studies reporting multiple toxic effects of T-2 toxin, namely, hematotoxicity, immunotoxicity, and genotoxicity.4 Previous work demonstrated that T-2 toxin is capable of causing severe damage through several toxicity mechanisms to tissues where cells are actively and rapidly dividing, such as spleen, thymus, and lymph nodes.5 The main known toxicity mechanism of T-2 toxin is its inhibitory effect on protein synthesis because of its high affinity for the 60S ribosomal subunit and resulting inhibition of peptidyl transferase activity.6−8 Additionally, some research reported that T-2 toxin can affect the biosynthesis of DNA and RNA.9,10 © 2015 American Chemical Society

Moreover, researchers observed an increase of malondialdehyde and a significant reduction of glutathione (GSH) in the liver of broiler chicks after 7 days of exposure to T-2 toxin, implying that oxidative stress and lipid peroxidation are associated with T-2 exposure.11 The use of electron paramagnetic resonance (EPR) spin-trapping technique successfully confirmed the generation of the hydroxyl radicals in T-2 toxin-exposed yeast.12 The observed increase levels of glutathione disulfide (GSSG) in rat thymus revealed by our previous work13 further demonstrate oxidative stress induced by T-2 toxin treatment. The oxidative stress induced by T-2 toxin exposure results in DNA damage-associated apoptosis, which has been reported in lymphoid organs,14 granulose cells,15 and human cervical cancer cells.16 Furthermore, T-2 toxin has an effect on mitochondrial function through inhibiting the activity of succinate dehydrogenase.17 Previous work also demonstrates great species discrepancy among animals exposed to T-2 toxin.18 Livestock Received: Revised: Accepted: Published: 714

October 21, 2015 December 28, 2015 December 30, 2015 December 30, 2015 DOI: 10.1021/acs.jafc.5b05076 J. Agric. Food Chem. 2016, 64, 714−723

Article

Journal of Agricultural and Food Chemistry

°C for 10 min (1610g), the plasma were isolated and then kept at −80 °C for future NMR detection. All the animal experiments performed in this study were approved by the Institutional Animal Care Use Committee of South China Agricultural University. Sample Preparation and 1H NMR Spectroscopy. Plasma samples for NMR spectroscopic analysis were prepared by mixing 200 μL of plasma with 400 μL of phosphate buffer saline solution (45 mM, K2HPO4/NaH2PO4 ≈ 4:1) containing 50% D2O and 0.9% NaCl. Deuterated water acts as a lock field solvent. After mixing and centrifugation, 550 μL of plasma samples were transferred to a 5 mm NMR tube for further NMR detection. About 50 mg of tissue samples (liver, kidney, and spleen) were separately weighted and homogenized with 600 μL of cold methanol/ water (2:1) using a TissueLyser (Qiagen Tissuelyser II, Hilden, Germany) at 20 Hz for 90 s. After sonication in an ice bath 5 times (60 s sonication and 60 s break), the homogenates were then centrifuged at 4 °C and 14489g for 10 min. The supernatants were collected, whereas the pellets were further extracted twice using the same procedure. The combined three extracts were lyophilized after removal of methanol in a rotary evaporator. The obtained tissue extracts were resuspended in 600 μL of phosphate buffer (0.15 M, K2HPO4/ NaH2PO4 ≈ 4:1, pD of 7.4, 0.001% TSP, 0.01% NaN3, and 50% D2O). After mixing and centrifugation at 4 °C and 16099g for 10 min, 550 μL of supernatant was transferred to a 5 mm NMR tube for future NMR analysis. All 1H NMR spectra of plasma and tissue extracts (liver, kidney, and spleen) were acquired at 298 K on a Bruker Avance III 600 MHz NMR spectrometer equipped with an inverse detection cryoprobe (Bruker Biospin, Rheinstetten, Germany). For tissue extracts, a onedimensional first increment of nuclear Overhauser effect spectrometry (NOESY) pulse sequence [RD−G1−90°−t1−90°−tm−G2−90°−acq, where RD = 2 s and tm = 100 ms] with water presaturation was used to acquire the NMR spectra. For spectra of plasma samples, a watersuppressed Carr−Purcell−Meiboom−Gill (CPMG) pulse sequence [RD−90°−(τ−180°−τ)n−acq, where τ = 350 μs and n = 100] was employed. The CPMG pulse sequence can retain NMR signals from small metabolites and attenuate signals from macromolecules with a shorter spin−spin relaxation time. For all of the spectra of tissue extracts and plasma samples, a total of 64 scans were recorded into a spectrum with 32 000 data points and a width of 20 ppm. An exponential weighting function with a line broadening of 1 Hz was applied to all of the spectra before Fourier transformation. For the purpose of metabolite identification, a series of two-dimensional (2D) NMR spectra were acquired as described previously,31 namely, 1H Jresolved spectroscopy (JRES), 1H−1H correlation spectroscopy (COSY), 1H−1H total correlation spectroscopy (TOCSY), 1H−13C heteronuclear single-quantum correlation (HSQC), and 1H−13C heteronuclear multiple-bond correlation (HMBC). NMR Spectrum Processing and Multivariate Data Analysis. After phase and baseline distortions being manually corrected, the spectra of tissue extracts and plasma samples were referenced to TSPd4 (δ 0.000) and the anomeric proton of α-glucose (δ 5.233), respectively. The δ 0.5−8.5 region for plasma, δ 0.5−9.0 region for spleen extracts, and δ 0.5−9.5 region for liver and kidney extracts were reduced into regions of 0.002 ppm wide and integrated using the Amix software (version 2.1, Bruker Biospin, Rheinstetten, Germany). To eliminate interference from imperfect water suppression, the regions between δ 4.40 and 5.20 for plasma, between δ 4.53 and 5.50 for spleen extracts, and between δ 4.53 and 5.20 for liver and kidney extracts were discarded. Additionally, the regions (δ 1.16−1.18 and 3.62−3.68 ppm) containing the ethanol signals were discarded from plasma spectra. The remaining regions were separately normalized to the total sum of intensity for the plasma spectra and to the weight of wet tissues for tissue extract spectra. The data were imported into SIMCA-P software (version 11.0, Umetrics, Umea, Sweden) for subsequent multivariate data analysis. Principal component analysis (PCA) of mean-centered NMR spectroscopic data was performed to provide a graphical overview of data distribution to explore toxicity trends and identify outliers. The

and poultry are major animal protein sources for human; hence, the investigation of the effects of T-2 toxin on those animals has important implications for human health. Metabonomics is a powerful approach to delineate the global metabolic profiling of living organisms and to capture the subtle metabolic alterations associated with physiological and pathological stimulations.19,20 Nuclear magnetic resonance (NMR)-based metabonomics, with its simplicity of sample preparation and robust reproducibility,21,22 has been successfully applied in the area of toxicology. For instance, NMR-based metabonomics was applied to investigate the endogenous metabolic alterations induced by bromobenzene, and a novel molecule, 5-oxoproline, was discovered in the intact liver, plasma, and urine.23 Moreover, the metabonomics investigation of hydrazine toxicity revealed 2-aminoadipate as a metabolite most predominantly changed in the urine and plasma, explaining the neurotoxicity of hydrazine.24 Additionally, NMR-based metabonomics has also been applied to the toxicology investigation of aflatoxin B1,25 cyadox,26 mequindox,27 CCl4,28 perfluorododecanoic acid,29 and deoxynivalenol.30 In this study, we used NMR-based metabonomics to investigate the global metabolic alterations caused by T-2 toxin in the plasma of broiler chickens and piglets and in multiple organs of broiler chickens. The aims of this investigation were to elucidate the endogenous metabolic changes induced by T-2 toxin exposure, explore the underlying mechanisms of T-2 toxin toxicity, and consequently provide important information for food safety assessment.

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MATERIALS AND METHODS

Animal Model and Sample Collection. A total of 22 female broiler chickens, aged 4 weeks, were bought from a commercial chicken-breeding farm in Guangzhou and maintained in an environment-controlled laboratory of the South China Agricultural University. All animals had free access to water and feed. After 2 weeks of acclimation, 22 broiler chickens were randomly divided into two groups, namely, the control group (n = 10) and the treatment group (n = 12). The treatment group broiler chickens received a single intravenous administration (through the right side wing vein of the broiler chickens) of T-2 toxin (Fermentek, Ltd., Jerusalem, Israel) dissolved in 50% ethanol saline solution at a dose of 0.5 mg/kg of body weight, whereas control group broiler chickens received an intravenous injection of ethanol saline solution only. The blood samples of T-2 toxin-treated broiler chickens (1 mL per animal per time point) were collected via puncture of the left side wing vein into heparinized Eppendorf tubes at 0.5, 1, 2, 3, and 4 h postdose. Blood samples for the control group were collected at 4 h postethanol saline injection. After centrifugation of blood samples at 4 °C for 10 min (1610g), the plasma samples were isolated and kept at −80 °C until subsequent NMR analysis. Every three broiler chickens in the treatment group were separately sacrificed after 1, 2, 3, and 4 h T-2 toxin injection, whereas control broiler chickens were sacrificed at 4 h post-dose. Liver, kidney, and spleen were excised, snap-frozen in liquid nitrogen, and stored at −80 °C until subsequent NMR analysis. Because the number of tissue samples collected at a single time point was not enough for multivariate data analysis, we combined the tissue samples collected at all of the time points as a treated group (n = 12). A total of 7 piglets (25 days old) were used for the experiment at a local commercial swine-breeding farm. After a short time of acclimation, the piglets received a single intravenous administration (through the ear vein) of T-2 toxin at a dose of 0.5 mg/kg of body weight. T-2 toxin dissolved in 50% ethanol saline solution. Blood samples (1 mL per animal per time point) were collected from each piglet by puncture of the jugular vein at 0.5, 1, 1.5, 2, 4, 6, 12, 24, 36, 48, 60, and 72 h post-dose. After centrifugation of blood samples at 4 715

DOI: 10.1021/acs.jafc.5b05076 J. Agric. Food Chem. 2016, 64, 714−723

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Figure 1. Typical 600 MHz 1H NMR spectra of plasma (P1a and P2a), liver (L1 and L2), kidney (K1 and K2), and spleen (S1 and S2) extracts from the control broiler chickens and the T-2 toxin-treated broiler chickens and 1H NMR spectra of plasma from the T-2 toxin-treated piglets at 0.5 h (P1b) and 4 h (P2b) post-dose. The dotted regions were relatively expanded 16, 3, or 4 times, as indicated in panels. Key: 1, isoleucine; 2, leucine; 3, valine; 4, 3-hydroxybutyrate; 5, lactate; 6, threonine; 7, alanine; 8, lysine; 9, arginine; 10, glutamate; 11, glutamine; 12, proline; 13, glutathione (oxidized); 14, succinate; 15, malate; 16, aspartate; 17, asparagine; 18, trimethylamine (TMA); 19, creatine; 20, choline; 21, hypotaurine; 22, phosphocholine (PC); 23, glycerophosphocholine (GPC); 24, phosphorylethanolamine (PE); 25, taurine; 26, β-glucose; 27, α-glucose; 28, betaine; 29, myo-inositol; 30, glycine; 31, uridine; 32, uridine monophosphate (UMP); 33, uridine diphosphate (UDP); 34, inosine; 35, uracil; 36, fumarate; 37, tyrosine; 38, phenylalanine; 39, xanthine; 40, hypoxanthine; 41, formate; 42, adenosine monophosphate (AMP); 43, inosine monophosphate (IMP); 44, nicotinamide; 45, nicotinamide adenine dinucleotide phosphate (NADP); 46, nicotinamide adenine dinucleotide (NAD); 47, methionine; 48, dimethylglycine; 49, tryptophan; 50, citrate; 51, pyruvate; 52, histidine; 53, lipid; 54, β-alanine; 55, N-acetyl-glycoproteins; and 56, urea. PCA trajectory plot was generated from averaging PCA scores at each time point, which represents the shifting of metabolic space following toxic exposure over time. To identify the metabolic differentiation between the control group and the treatment group, projection to latent structure discriminant analysis (PLS-DA) and orthogonal projection to latent structure discriminant analysis (OPLS-DA) were performed using unit variance-scaled NMR data. A 7-fold crossvalidation and a permutation test were applied to assess the validity of constructed PLS-DA models. OPLS-DA models were further validated

using the cross validation−analysis of variance (CV−ANOVA) test based on discrimination significance at the levels of p < 0.05. To visualize significant metabolites contributing to the discrimination, loadings plots were generated using the back-transformed loadings from OPLS-DA incorporated with color-coded absolute coefficient values (|r|) of the corresponding loadings in MATLAB (version 7.1, Mathworks, Inc., Natick, MA). In the coefficient loading plots, metabolites with red color signify more significant contributions to class separation than those with blue color. According to the sample 716


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Figure 2. PCA trajectory plots obtained from the plasma spectra of (A) broiler chickens and (B) piglets, following T-2 toxin treatment.

Figure 3. OPLS-DA scores (left) and coefficient plots (right) obtained from comparison of 1H NMR spectra of plasma between controls and T-2 toxin-treated broiler chickens at 1 h post-dose and comparison of 1H NMR spectra of piglet plasma between plasma spectra obtained at 0.5 h postdose and that of 4 h post-dose. number in each group, the cutoff value for the coefficients was chosen on the basis of discrimination significance (p < 0.05). To further quantitate dynamic changes of metabolites over T-2 toxin exposure time, the relative concentrations of important metabolites for each time point were calculated using the formula (CT − CC)/CC, where CT represents the averaged metabolite concentrations in the T-2 toxin treatment group and CC refers to that in the control group.

and myo-inositol), organic acid, choline metabolites (choline, phosphocholine, and glycerophosphocholine), purines, and pyrimidines. Multivariate Data Analysis of NMR Data. The PCA trajectory of plasma profiles (Figure 2) showed that the profiles of T-2 toxin-treated broiler chickens speedily deviated from the controls and the recovery of metabolic profiles was not observed at 4 h post-dose (Figure 2A), whereas the profiles of piglets appeared to have recovered at 3 days post-exposure (Figure 2B). The significant metabolites contributing to the differentiations between T-2 toxin-exposed and control groups in plasma profiles of broiler chickens were further examined using PLS-DA and OPLS-DA strategies. The results suggested that the metabolic disturbance occurred in broiler chickens at 0.5 h post-dose. For the analysis of piglet plasma, PLS-DA and OPLS-DA were applied to compare the 1H NMR data obtained at 1, 1.5, 2, 4, 6, 12, 24, 36, 48, 60, and 72 h post-dose to those obtained at 0.5 h post-dose. This was performed because plasma samples from control piglets were not available.

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RESULTS AND DISCUSSION H NMR Spectra of Plasma and Tissue Extract Samples. A total of 56 metabolites were identified from 1H NMR spectra of tissue extracts of broiler chickens and from plasma of both broiler chickens and piglets (Figure 1). The identification was achieved according to literature data32−34 and unambiguously confirmed by various 2D NMR spectra. The spectra of plasma contained signals from lipid, glucose, choline, organic acids (e.g., lactate, formate, and 3-hydroxybutyrate), amino acids (e.g., valine, isoleucine, tyrosine, and phenylalanine), and tricarboxylic acid (TCA) intermediates (citrate, fumarate, and succinate). The spectra of tissue extracts contained resonances from amino acids, carbohydrates (glucose 1

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Figure 4. Dynamic metabolic alterations in the plasma of broiler chickens and piglets induced by T-2 toxin injection. (A) Significantly changed metabolites in the plasma of broiler chickens and piglets after T-2 toxin exposure at different time points. (B and C) Time dependence of changed amino acids in the plasma of T-2 toxin-treated (B) broiler chickens and (C) piglets.

increased concentrations of tryptophan and histidine were only observed in T-2 toxin-treated broiler chickens, while elevations in the levels of glutamate and glutamine were only observed in T-2 toxin-treated piglets. Interestingly, we observed a higher level of energy-related metabolites (citrate and fumarate) at the early time points in T-2 toxin-treated broiler chickens, while we only observed higher levels of lactate at the early time points and lower levels of citrate, fumarate, and pyruvate at the later time points in T-2 toxin-exposed piglets. Furthermore, the level of glycerophosphocholine (GPC) and formate decreased, while the level of choline increased in the T-2 toxin-exposed broiler chickens. Increased levels of creatine, urea, and formate and decreased levels of N-acetyl-glycoproteins were observed in the plasma of T-2 toxin-exposed piglets. The changes for all the significantly changed amino acids over time were displayed in panels B and C of Figure 4. The results showed that T-2 toxin injection caused a prompt increase in amino acids, reaching maximum levels at 2 h post-dose in broiler chickens (Figure 4B). In piglets (Figure 4C), the levels of amino acids were increased, reaching maximum levels at 4 h post-dose, and then

However, the fact that metabolic changes were only observed in piglets after 2 h post-dose and no differences in metabolic profiles were observed in prior samples compared to those collected at 0.5 h post-dose justified the use of samples collected at 0.5 h as appropriate control samples. Examples of cross-validated scores plots and corresponding coefficient plots were displayed in Figure 3. A summary of the metabolites significantly changed in T-2 toxin-treated broiler chickens and piglets over all time points was shown as a heat map (Figure 4A). In the heat map, metabolites highlighted in red color correspond to those showing an increase in concentrations after T-2 toxin exposure, while those in blue color showed decreased concentrations. On the one hand, the heat map indicated that T-2 toxin induced similar metabolic perturbations in the plasma of broiler chickens and piglets. For example, the T-2 toxintreated animals contained a lower level of glucose and lipid but a higher level of amino acids (isoleucine, leucine, valine, alanine, lysine, threonine, methionine, tyrosine, and phenylalanine) at early exposure time points. On the other hand, species differences were revealed in the metabolic responses of broiler chickens and piglets to T-2 toxin injection. For instance, 718


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Figure 5. OPLS-DA scores (left) and coefficient plots (right) showing T-2 toxin-induced metabolic alterations in (A) kidney, (B) liver, and (C) spleen of broiler chickens.

gradually reduced to minimum levels at 24 h post-dose and finally back to normal levels thereafter. Every three broiler chickens in the T-2 toxin-treated group were sacrificed at 1, 2, 3, and 4 h post-dose; hence, there were three samples for each tissue type at each time point. The number of samples obtained from each time point was not sufficient for multivariate statistical analysis; therefore, tissue samples taken from different time points were combined as the T-2 toxin-treated group. The metabolic changes in the broiler liver, kidney, and spleen extracts were analyzed using the OPLS-DA strategy. The correlation coefficient plots (Figure 5) showed the metabolites significantly discriminating organs of T2 toxin-exposed broiler chickens from those of control broiler chickens (the significantly changed metabolites were summarized in Table 1). T-2 toxin injection significantly elevated the levels of amino acids in kidney, liver, and spleen. Moreover, the levels of lactate, creatine, uracil, uridine diphosphate (UDP), and xanthine were increased, while the levels of inosine and αglucose were reduced in the kidney of T-2 toxin-treated broiler chickens. In the liver, significant increases in the levels of GSSG, creatine, choline, uridine, UDP, xanthine, and formate together with decreases in the levels of malate, succinate, fumarate, α-glucose, trimethylamine (TMA), taurine, and inosine were associated with T-2 toxin exposure. Furthermore, increased levels of lactate, succinate, choline, uridine, UDP, uracil, xanthine, hypoxanthine, and inosine and decreased levels of adenosine monophosphate (AMP), inosine monophosphate (IMP), taurine, and nicotinamide were found in the spleen of

T-2 toxin-treated broiler chickens. The dynamic alterations of amino acids and nucleotide metabolites in liver, kidney, and spleen of broiler chickens were displayed in Figure 6. After T-2 toxin injection, amino acids in liver and spleen (panels A and C of Figure 6) exhibited similar trends, with a marked increase at the early time points (1−2 h), and remained in plateaus at later time points (2−4 h). In the kidney (Figure 6B), the levels of amino acids were also elevated at early time points and then displayed a trend toward returning to initial levels. It is worth noting that the peak concentrations of amino acids were reached at 2 h post-dose in all observed tissues and tyrosine showed the most outstanding changes. These findings coincided with the data obtained from plasma samples (Figure 4B). The alteration of nucleotide metabolites reached a maximum in the liver, kidney, and spleen of broiler chickens at 4 h post-dose (panels D−F of Figure 6). T-2 Toxin Altered Energy Metabolism and Induced Oxidative Stress. Intravenous injection is the fastest way to deliver and distribute a drug to all parts of the body through the bloodstream. It was anticipated that the intravenous injection of T-2 toxin would cause various quick metabolic responses. We noted that energy metabolism was altered after T-2 injection (Figure 7). The significantly reduced levels of glucose in plasma, liver, and kidney of T-2 toxin-treated broiler chickens implied that glycolysis is stimulated to produce more energy to reduce or repair the damage associated with T-2 toxin and maintain homeostasis. The accumulation of lactate, a product of anaerobic glycolysis, in the kidney and spleen of dosed broiler 719


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following acute T-2 toxin exposure.13 The difference in sensitivity and metabolic responses of various organs to T-2 toxin may be explained by the fact that organs possess different levels of detoxifying enzymes or cellular receptors to T-2 toxin.10 Moreover, a noticeable increase in creatine levels was observed in the liver and kidney of T-2 toxin-treated broiler chickens. Creatine can interconvert with phosphocreatine through a reversible reaction catalyzed by creatine kinase. Acting as an energy reservoir, phosphocreatine can be rapidly converted to creatine and release adenosine triphosphate (ATP) in response to a high energy demand. The suppression of the TCA cycle caused by T-2 toxin exposure will inevitably result in the insufficient production of energy. Hence, the accumulated creatine in the liver and kidney of T-2 toxinexposed broiler chickens may be the result of the accelerated conversion of phosphocreatine to creatine to meet this energy demand (Figure 7). Our data revealed that the level of GSSG in liver tissues displayed a distinct increase following T-2 toxin exposure (Figure 5). GSSG is the oxidized form of glutathione, a reduced form of metabolite that can react with reactive oxygen species and xenobiotics to protect critical cellular macromolecules, such as nucleic acids and proteins, from oxidative damage. Hence, the increased levels of GSSG suggested that T-2 toxin injection triggered oxidative stress (Figure 7). Previous reports also highlighted that T-2 toxin induces oxidative stress, leading to increased levels of hepatic malondialdehyde and reduced levels of GSH in broiler chicks following 7 days of T-2 toxin exposure.11 Furthermore, Hoehler et al.12 observed lipid peroxidation in response to T-2 toxin treatment and proposed that this occurs through the generation of hydroxyl radicals. T-2 Toxin Impacts on Amino Acid and Nucleic Acid Metabolisms. In our current investigation, we noted the increased levels of a range of amino acids in the plasma and organs of T-2 toxin-dosed broiler chickens and plasma of piglets (Figure 4A and Table 1), which has been previously observed in rat36,37 and guinea pigs.38 As a lipophilic molecular, T-2 toxin can interact with lipid or protein components of cellular membranes, disrupting the membrane function to facilitate its rapid distribution to different organs and organelles. This facilitates the fast metabolizing of T-2 toxin through deacetylation, hydroxylation, glucuronide conjugation, and deepoxidation, causing systemic disruptions.39 T-2 toxin is known to inhibit protein synthesis in the liver and other tissues through its strong affinity for the 60S ribosomal subunit,7 leading to accumulated amino acids in a wide range of biological compartments. The concentrations of amino acids in the plasma (Figure 4B), liver, kidney, and spleen tissues (panels A−C of Figure 6) of T-2 toxin-treated broiler chickens reached peak values at the same time points (2 h after dosing), which supports the view of rapid toxic effects of T-2 toxin. Of note, an increase in tryptophan levels (Figure 4A) was induced by T-2 toxin, which has been previously observed in rats,36 sometimes together with that of its metabolites, serotonin and 5hydroxyindole acetic acid, in the brain.40 This observed increase in the levels of tryptophan could accelerate the synthesis of serotonin and, subsequently, fire the serotonergic neurons. This may, in turn, explain a series of symptoms of T-2 toxicity, such as feed refusal and loss of muscle coordination. In the current investigation, the levels of tryptophan were not increased in piglets, which suggest that promotion of the tryptophan level by T-2 toxin is species-dependent. Indeed, the toxicity of T-2 toxin in animals is strongly dependent upon

Table 1. Significantly Changed Metabolites in the Kidney, Liver, and Spleen of T-2 Toxin-Treated Broiler Chickens coefficienta key

metabolite

kidney

liver

spleen

1 2 3 5 6 7 8 10 11 13 14 15 16 17 18 19 20 25 27 30 31 33 34 35 36 37 38 39 40 41 42 43 44 49 52 54

isoleucine leucine valine lactate threonine alanine lysine glutamate glutamine GSSG succinate malate aspartate asparagine TMA creatine choline taurine α-glucose glycine uridine UDP inosine uracil fumarate tyrosine phenylalanine xanthine hypoxanthine formate AMP IMP nicotinamide tryptophan histidine β-alanine

0.71 0.71 0.86 0.84 − 0.69 0.70 − − − − − − − − 0.61 − − −0.89 − − 0.76 −0.72 0.62 − 0.94 0.75 0.71 − − − − − − 0.75 0.81

− − 0.83 − 0.87 0.73 0.77 − − 0.82 −0.82 −0.88 − − −0.85 0.78 0.82 −0.96 −0.96 − 0.84 0.86 −0.62 − −0.88 0.91 − 0.75 − 0.76 − − − 0.79 − 0.92

0.95 0.94 0.96 0.84 0.92 0.84 0.94 0.65 0.85 − 0.75 − 0.72 0.93 − − 0.77 −0.86 − 0.83 0.88 0.83 0.65 0.81 − 0.98 0.92 0.85 0.65 − −0.70 −0.63 −0.78 0.95 0.92 −

“−” indicates metabolites that show no significant changes in the concentration after T-2 toxin treatment. Metabolites with positive coefficient values indicate a significant increase, whereas metabolites with negative values indicate a decrease after T-2 toxin treatment. a

chickens supported the notion of stimulated glycolysis. Reduced levels of glucose and the accumulation of lactate were observed in the plasma of piglets after T-2 toxin exposure, which further demonstrated this notion. The level of tricarboxylic acid cycle intermediates, such as succinate, fumarate, and malate, concurrently decreased in the liver of T-2 toxin-treated broiler chickens, which suggested that T-2 toxin injection suppressed the TCA cycle (Figure 7). This observation can be explained by previous reports of T-2 toxin decreasing mitochondrial respiration35 through the inhibition of the activity of mitochondrial succinate dehydrogenase and the upregulation of the activity of reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase.17 We observed increased levels of succinate in the spleen of T-2 toxin-exposed broiler chickens, which indicated that the adverse impacts of T2 toxin on energy metabolism are organ-specific. These organspecific effects on energy metabolism were also found in rats 720


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Figure 6. (A−C) Time dependence of significantly changed amino acids and (D−F) metabolites related to nucleotide metabolism in the liver, kidney, and spleen of T-2 toxin-treated broiler chickens.

Figure 7. Summary of altered metabolic pathways induced by T-2 toxin exposure. Metabolite with a red or blue color signifies a significant increase or decrease in T-2 toxin-treated broiler chickens as compared to that in control broiler chickens. Metabolite with a black color signifies no marked change.

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experimental species. For instance, the LD50 for intravenous exposure of T-2 toxin in mice is 4.2 mg/kg of body weight,41 while that for rats42 and pigs43 is 0.9 and 1.21 mg/kg of body weight, respectively. Similarly, the lowest adverse effect level (LOAEL) is 29 μg/kg of body weight per day in pig, 40−48 μg/kg of body weight per day in poultry, and 300 μg/kg of body weight per day in young ruminants.4 One of the prominent findings in our study is that T-2 toxin caused marked metabolic alterations in the levels of nucleotides, nucleosides, and nucleobases (Table 1), which is consistent with the known toxicity of T-2 toxin, e.g., its ability to induce DNA damage44−47 and inhibit DNA synthesis.9,10 The alteration of nucleic acid metabolism (panels D−F of Figure 6) occurred within 4 h of T-2 toxin treatment, which lags behind that of amino acids (panels A−C of Figure 6). This observation supports the notion that the impacts of T-2 toxin on DNA are a secondary result of its effects on protein synthesis.9,48 In summary, we found that T-2 toxin-induced systemic metabolic changes in exposed animals (Figure 7). The primary metabolic disturbance associated with T-2 toxin exposure included stimulation of anaerobic glycolysis, suppression of the TCA cycle, and perturbation of amino acid and nucleotide metabolism. Moreover, T-2 toxin induced oxidative stress to animals. We also noted that disturbance of nucleic acid metabolism is a secondary result of its effects on protein synthesis. Although the metabolic responses of broiler chickens and piglets to T-2 toxin show considerable similarities, the alteration of tryptophan levels only occurred in broiler chickens, indicating differences in the toxicity of T-2 toxin between species. Our study provides a comprehensive overview of the metabolic consequences of T-2 toxin exposure in broiler chickens and piglets. This information is relevant to the assessment of the safety of consuming meat from exposed animals.

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REFERENCES

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b05076. NMR resonance assignments of metabolites in the plasma, liver, kidney, and spleen samples (Table S1) (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Telephone: +86-27-87197143. Fax: +86-27-87199291. E-mail: [email protected]. Funding

This work is supported by grants from the Ministry of Science an d T e ch n o l o g y o f Ch i n a ( 20 09 CB 11 88 04 a n d 2012CB934004) and the National Natural Science Foundation of China (21375144). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED PCA, principal component analysis; PLS-DA, projection to latent structure discriminant analysis; OPLS-DA, orthogonal projection to latent structure discriminant analysis 722


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