Metabolic Influence of Acute Cyadox Exposure on Kunming Mice

Article pubs.acs.org/jpr

Metabolic Influence of Acute Cyadox Exposure on Kunming Mice Chongyang Huang,†,‡ Hehua Lei,† Xiuju Zhao,†,§ Huiru Tang,*,† and Yulan Wang*,† †

Key Laboratory of Magnetic Resonance in Biological Systems and State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Centre for Magnetic Resonance, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan 430071, Peopleʼs Republic of China ‡ University of Chinese Academy of Sciences, Beijing 100049, Peopleʼs Republic of China S Supporting Information *

ABSTRACT: Cyadox is an antibiotic drug and has the potential to be used as a feedstuff additive in promoting the growth of animals. However, the toxicity of cyadox should be fully assessed before application, and this has prompted the current investigation on the metabolic responses of mice to cyadox exposure, using a metabonomic technique. Three groups of Kunming mice were respectively given a single dose of cyadox at three different concentrations (100, 650, and 4000 mg/kg body weight) via gavage. We present here the metabolic alterations of urine, plasma, liver, and renal medulla extracts induced by cyadox exposure. The metabolic alterations induced by cyadox exposure are dose-dependent, and metabolic recovery is achieved only for low and moderate levels of cyadox exposure during the experimental period. Cyadox exposure resulted in a disturbance of gut microbiota, which is manifested in depleted levels of urinary hippurate, trimethylamine-N-oxide (TMAO), dimethylamine (DMA), and trimethylamine (TMA). In addition, mice exposed to cyadox at high levels caused accumulations of amino acids and depletions of nucleotides in the liver. Furthermore, marked elevations of nucleotides and a range of organic osmolytes, such as myo-inositol, choline, and glycerophosphocholine (GPC), and decreased levels of amino acids are observed in the renal medulla of cyadoxexposed mice. These results suggest that cyadox exposure causes inhibition of amino acid metabolism in the liver and disturbance of gut microbiota community, influencing osmolytic homeostasis and nucleic acids synthesis in both the liver and the kidney. Our work provides a comprehensive view of the toxicological effects of cyadox, which is important in animal and human food safety. KEYWORDS: cyadox, NMR-based metabonomics, multivariate statistical analysis

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INTRODUCTION Cyadox (Figure 1), a typical antibiotic drug belonging to the quinoxaline family, has potential as a growth-promoting additive

found in pigs fed with 200 ppm (mg/kg diet) cyadox for 6 weeks.3 However, no significant increase in body weight was found in female Wistar rats when they were fed on a diet containing 50 or 150 mg/kg cyadox.5 A large body of research has focused attention on the toxicity of the cyadox and showed that cyadox is safe in many aspects. For example, cyadox was not toxic or mutagenic to the reproduction system,6−9 and no carcinogenic effects were noted in animals dosed with cyadox.10 However, mild and reversible phototoxicity was found for cyadox when it was tested on female BALB/c mice.11 In addition, short and unstable intracellular reactive oxygen species (ROS) were released from cyadox, causing mitochondrial disruption and subsequent oxidative stress.12 Furthermore, traditional toxicological investigations into the effects of cyadox showed that the threshold level at which no observable adverse effects are seen on the reproductive and development systems was 400 mg/kg diet13 and 150 mg/kg diet,14 respectively. Understanding the effects of cyadox on

Figure 1. Chemical structure of cyadox.

in the feedstuff for piglets, chicken, and fish. Cyadox elicits antibacterial activity against some pathogenic bacteria such as Staphylococcus hyicus, Pasterella multocide, and Escherichia coli1 and can affect the levels of anaerobic rumen fungi metabolites, such as lactate and acetate.2 Nabuurs et al.3 showed that cyadox played a role in promoting the growth of piglets by increasing the food percent conversion and improving lean meat percentage, which was later confirmed by Wang et al.4 in a study into the effect of cyadox on growth and nutrient digestibility in weanling piglets. Furhtermore, a significant increase in body weight was © 2012 American Chemical Society

Received: November 2, 2012 Published: December 12, 2012 537

dx.doi.org/10.1021/pr301033a | J. Proteome Res. 2013, 12, 537−545

Journal of Proteome Research

Article

collected. Renal cortex and renal medulla were separated immediately. All samples were snap-frozen in liquid nitrogen and transferred to −80 °C for storage until NMR analysis.

global endogenous metabolism could provide additional information on the action of cyadox, which is important for assessment of the safety of cyadox. The specific goal of the present study is to investigate the endogenous biological effects of cyadox on mice. The metabonomic approach employing 1H nuclear magnetic resonance (NMR) spectroscopy or mass spectrometry with interpretation by multivariate statistical analysis methods provides a suitable tool to achieve such an objective. Metabonomics is defined as “the quantitative measurement of the dynamic multiparametric metabolic response of living systems to pathophysiological stimuli or genetic modification”.15 It can provide complementary information for exploring “wholeorganism functional integrity over time after drug exposure”.16 NMR is a nondestructive detection technique,16 and together with multivariate statistical analysis, could be utilized to investigate endogenous metabolic changes induced by xenobiotic intake. Principal component analysis (PCA),17 projection to latent structures discriminant analysis (PLS-DA),18 and orthogonal projection to latent structures discriminant analysis (O-PLS-DA)18 are the three major multivariate statistical analysis methods commonly used in metabonomics. This methodology has been successfully applied to investigate nephrotoxic lesions induced by HgCl2 and 2-bromoethanamine,19 hepatic lesions induced by hepatotoxins including αnaphthyl isothiocyanate, D-(+)-galactosamine and butylated hydroxytoluene,20 time-related toxic effects induced by αnaphthylisothiocyanate,21 and biochemical consequences and hepatotoxicity induced by aflatoxin B122 and by perfluorocarboxylic acids.23 In this study, we employ an NMR metabonomics profiling technique to evaluate the metabolic changes induced by cyadox exposure in a mouse model. We present dynamic metabolic profiles of urine and profiles of plasma, liver, and kidney extracts obtained from mice after acute exposure to cyadox at different concentrations. It is anticipated that cyadox is a potential drug for farm industry; however, testing the drug directly on farm animals would involve much larger animal numbers due to larger interanimal variations generated from the less well controlled environment in a farm. In order to comply with 3R rules, a mouse model is employed here. We hope the results generated from the mouse model will provide knowledge on the toxic effects of cyadox from a holistic point of view and offer an important reference for the safety assessment of cyadox, which could be taken into consideration when setting guidelines for the safe use of cyadox.

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Clinical Biochemistry and Histopathology Analysis

Clinical biochemistry assays were used to analyze the levels of glucose (Glc), total cholesterol (CHOL), creatinine (CREA), blood urea nitrogen (BUN), triglyceride (TG), albumin (ALB), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and total protein (TP) in sera. Liver and kidney tissues were fixed in neutral-buffered 10% formalin, sectioned, and stained with hematoxylin and eosin. Sections were reviewed under a light microscope. Sample Preparation and NMR Spectroscopy

A total of 100 μL of urine sample was mixed with 400 μL of 50% D2O and 50 μL of phosphate buffer (1.5 M, K2HPO4:NaH2PO4 ≈ 4:1, pD ≈ 7.4), containing 100% D2O as a field lock signal and 0.05% TSP (sodium 3-trimethylsilyl-2,2,3,3-tetradeuteropropionate) as a chemical shift reference. The mixture was centrifuged (16090g, 4 °C, 10 min), and 500 μL of supernatant was transferred into a 5 mm NMR tube for NMR spectral acquisition. Liver and renal cortex and medulla tissues were weighed and extracted three times in a 2:1 ratio of methanol and water by use of a homogenizer (Qiagen Tissue-Lyser, Retsch GmBH, Germany). The supernatants were combined and freeze-dried after removal of methanol. The extracts were reconstituted in 600 μL of phosphate buffer (0.1 M, K2HPO4:NaH2PO4 ≈ 4:1 pD ≈ 7.4, 50% D2O, 0.001% TSP), and 550 μL of supernatant was piped into the 5 mm NMR tube after centrifugation (16090g, 4 °C, 10 min). The plasma sample was prepared by mixing 30 μL of plasma with 30 μL of phosphate saline solution (0.045 M, K2HPO4:NaH2PO4 ≈ 4:1, pD ≈ 7.4, 0.9% NaCl), containing 50% D2O and transferred into a 1.7 mm micro-NMR tube. One-dimensional 1H NMR spectra were acquired for urine, liver, renal cortex, and renal medulla extracts at 298 K on a Bruker Avance III 600 MHz NMR spectrometer (Bruker BioSpin, Germany) equipped with 5 mm TCI cryogenic inverse detection probe, by use of the first increment of nuclear Overhauser effect spectroscopy (NOESY) pulse sequence [recycle delay (RD)− 90°−t1−90°−tm−90°−acquisition] with water presaturation.24 A recycle delay time of 2 s, t1 of 3 μs, and mixing time (tm) of 80 ms were set. A total of 64 and 128 scans for urine and extracts of liver and kidney, respectively, were collected into 32K data points with spectral width of 20 ppm. Two 1H NMR spectra were acquired for each plasma sample, namely, a spin−spin relaxation (T2) edited spectrum by use of a Carr−Purcell−Meiboom− Gill25 [CPMG, RD−90°−(τ−180°−τ)n acquisition] sequence with a fixed total spin−spin relaxation delay of 80 ms (2nτ) and a water-presaturated one-dimensional 1H NMR spectrum, with a broadband inverse detection probe on a Bruker AV II 500 NMR spectrometer operating at 500.13 MHz for proton frequency (Bruker, BioSpin, Germany). A total of 256 scans are collected into 32K data points with a spectral width of 20 ppm. For assignment purposes, five two-dimensional (2D) NMR spectra including 1H−1H J-resolved spectroscopy (J-Res), 1H−1H correlation spectroscopy (COSY), 1H−1H total correlation spectroscopy (TOCSY), 1H−13C heteronuclear single quantum coherence spectroscopy (HSQC), and 1H−13C heteronuclear multiple bond correlation spectroscopy (HMBC) were acquired for selected samples of urine, plasma, liver, and kidney extracts.22,23

EXPERIMENTAL DESIGN

Animal Experiment and Sample Collection

A total of 48 female Kunming mice aged 6 weeks old were purchased from The Center for Disease Control and Prevention (CDC) in Hubei province. After adaptation to experimental conditions in an SPF animal facility at Wuhan Institute of Virology for 2 weeks, the mice were randomly divided into four groups of 12 mice: control group (C), low-dose group [L, 100 mg/kg body weight (bw)], moderate-dose group (M, 650 mg/kg bw), and high-dose group (H, 4000 mg/kg bw). Different dose levels of cyadox were mixed with olive oil and administrated into mice via gavage. Urine samples were collected 1 day before dosage and at 8 and 16 h and then on a daily basis until 7 days postdose. The mice were sacrificed at 8 days postdose, and plasma and organ tissues including liver and kidney were 538



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Figure 2. Typical 1H NMR spectra from urine, extracts of liver and renal medulla. (U1, L1, and K1) urine, liver extract, and renal medulla extract from a mouse dosed with high levels of cyadox; (U2, L2, and K2) urine, liver extract, and renal medulla extract from a control mouse. The aromatic regions of urine and renal medulla spectra are magnified 4 times compared to those of corresponding aliphatic regions. The aromatic regions of liver spectra are magnified 8 times compared to the aliphatic regions. Keys: 1, isoleucine; 2, leucine; 3, valine; 4, threonine; 5, lactic acid; 6, alanine; 7, lysine; 8, acetic acid; 9, glutamate; 10, glutamine; 11, aspartic acid; 12, methionine; 13, β-hydroxybutyrate; 14, succinate; 15, methanol; 16, oxidized glutathione; 17, glucose; 18, uracil; 19, uridine; 20, fumaric acid; 21, tyrosine; 22, phenylalanine; 23, histidine; 24, hypoxanthine; 25, inosine; 26, nicotinate; 27, formate; 28, tryptophan; 29, AMP; 30, trimethylamine (TMA); 31, glycogen; 32, glycine; 33, trimethylamine N-oxide (TMAO); 34, pantothenic acid; 35, uridine 5′monophosphate (UMP); 36, α-ketoisocaproic acid; 37, 4-hydroxyhydratropate; 38, oxoglutaric acid; 39, citrate; 40, dimethylamine (DMA); 41, 3methyl-2-oxovaleric acid; 42, α-ketoisovalerate; 43, 3-ureidopropionic acid; 44, creatine; 45, creatinine; 46, taurine; 47, hippurate; 48, benzoic acid; 49, trigonelline; 50, phenylacetylglycine (PAG); 51, methylamine; 52, m-HPPA sulfate; 53, cis-aconitate; 54, dimethyl sulfone; 55, sarcosine; 56, choline; 57, phosphocholine (PC); 58, glycerophosphocholine (GPC); 59, myo-inositol; 60, ethanolamine; 61, N-methylnicotinamide; 62, indoxyl sulfate; 63, hypotaurine; 64, dimethylglycine; 65, butyric acid; 66, mannose; 67, IMP; 68, IDP; 69, ITP; 70, pinitol; 71, trans-2-hydroxycinnamate; 72, 2,5dihydroxybenzoate.

NMR Spectral Processes and Analysis

and the drug metabolite (δ 7.89−8.24 and 9.30−10.00) were excluded from urine spectra. A probabilistic quotient normalization method26 was performed for the spectra of plasma and urine, while normalization on weight of tissues was conducted for liver and kidney extracts before multivariate analysis on the data set scaled to unit variance by use of Simca-P 11.0 software (Umetrics, Sweden). Principal component analysis (PCA) was performed on the data set to generate an overview of the metabolic effects of cyadox exposure. Orthogonal projection to latent structures discriminant analysis (O-PLS-DA) was subsequently performed on the data set, and the validity of the model was certified by a 7-fold cross-validation method27 and permutation test method.28 Interpretation of the data was aided

The free induction decays were multiplied by an exponential window function with a 1 Hz line-broadening factor prior to Fourier transformation. NMR spectra were manually corrected for phase and baseline distortions by use of Topspin 2.0 (Bruker, BioSpin, Germany). TSP with a chemical shift at δ 0.00 was used as a spectral reference for urine, liver, and kidney extracts, whereas the NMR spectra of the plasma were referenced to the anomeric proton of α-glucose at δ 5.233. All the 1H NMR spectra were bucketed with spectral width of 0.002 ppm by use of AMIX software (version 3.9.2, Bruker, BioSpin, Germany). The regions containing water (δ 4.5−5.15) were removed to avoid the effects of imperfect water suppression. Additionally, signals from urea 539



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by cross-validated scores plots and coefficient plot that was generated from the back-transformed loadings incorporated with color-coded coefficients of the loadings plotted in MATLAB (The Mathworks Inc., Natwick, MA, version 7.1).29 The coefficients of the loadings represent weights of metabolites in contributing to the separation between classes; the red color denotes significance in the differentiation between classes, while the blue color indicates no significance. Here, based on the number of samples included in each dosage group, correlation coefficients higher than 0.553 were regarded as significant in the discrimination based on a 95% confidence limit.

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RESULTS

Clinical Biochemistry and Histopathology Analysis

ALT levels decreased significantly in the sera of all dosed mice, whereas the levels of creatinine are markedly elevated in the sera of the high-dose group (Table 1 in Supporting Information). Histopathology examination shows no abnormality in the kidney obtained from mice of all dosed groups, whereas livers obtained from mice dosed with moderate and high levels of cyadox display hydropic degeneration around the central vein. Additional spotty necrosis is also observed in the hepatic lobule in mice dosed with a high level of cyadox (Figure 1 in Supporting Information).

Figure 3. PCA trajectories generated from urinary profiles obtained from control (C), low-dose (L), moderate-dose (M), and high-dose (H) groups during the entire experimental time.

dosed mice. Metabolites such as hippurate, methylamine (MA), dimethylamine (DMA), trimethylamine (TMA), trimethylamine N-oxide (TMAO), 2-(4-hydroxyphenyl)propanoic acid (2HPPA), phenylacetylglycine (PAG), indoxyl sulfate, dimethyl sulfone, 2,5-dihydroxybenzoate, 2-hydroxycinnamate, and benzoic acid decreased continuously with time, and the levels of metabolites such as creatine, creatinine, hypotaurine, taurine, pinitol, and trigonelline are increased. These alterations are more persistent in the high-dose group than in the low- and moderatedose groups. The levels of butyric acid are decreased while the levels of formate are increased at some time points. The levels of sulfo-conjugated m-hydroxyphenylpropionic acid (m-HPPA sulfate) decrease at the early time points and increase at later time points in the moderate- and high-dose groups. The same O-PLS-DA strategy is performed for the NMR spectral data obtained from liver and renal medulla extracts (Figure 6), and parameters indicating model quality and crossvalidation results are also listed in Table 3 in Supporting Information. The results suggest that a single dose at a low level of cyadox exposure had no effect on the liver metabonome after 7 day of exposure. The moderate- and high-dose exposures induce similar changes in the liver metabonome, which includes increased levels of organic acids (formate, acetate, pantothenic acid) and a range of amino acids (including isoleucine, leucine, valine, lysine, glutamate, aspartic acid, glycine, tyrosine, phenylalanine, and histidine), as well as TMAO, uracil, hypoxanthine, and inosine (only in the high-dose group). However, the levels of glutamine, glycerophosphocholine (GPC), and a range of nucleotides including adenosine monophosphate (AMP), inosine triphosphate (ITP), inosine diphosphate (IDP), inosine monophosphate (IMP), and uridine monophosphate (UMP) are decreased in the livers obtained from moderate- and high-dose cyadox-exposed mice (Figure 6, LM and LH). The metabonome of the renal cortex is not affected by cyadox exposure; however, cyadox exposure causes dose-dependent alterations in the metabonome of renal medulla (Figure 6, KL, KM, and KH). For the low-dose group, marked depleted levels of acetic acid, glutamate, glucose, mannose, and fumaric acid and elevated levels of TMAO are found in the renal medulla. Elevated levels of TMAO and creatine and depleted levels of alanine, glutamate, glutamine, glycine, acetic acid, citrate, glucose, and mannose are associated with the renal medulla of moderate-dose mice.

Metabolite Assignments for 1H NMR Spectroscopy

Typical 1H NMR spectra of urine, liver, and renal medulla extracts obtained from control mice and mice dosed with high levels of cyadox are shown in Figure 2. Metabolite assignments are carried out as described in the literature30−32 and confirmed by two-dimensional NMR spectra. Urinary spectra show signals from metabolites in the glycolytic pathway, tricarboxylic acid cycle intermediates, amino acid synthesis and degradation pathways, and circulation of bile acid, as well as cometabolites with microbial metabolism. The spectra of liver and renal medulla extracts display signals from amino acids, nucleotides, and a range of organic acids. Data analysis of 1H NMR spectra of plasma and extracts of renal cortex suggests no marked differences between the metabolic profiles of control and dosed mice; hence the spectra of plasma and extracts of renal cortex are not included here. The NMR resonance assignments together with proton chemical shifts and their multiplicity were listed in Table 2 in Supporting Information. Multivariate Data Analysis

To generate an overview of the metabolic response of mice to cyadox exposure, the PCA trajectories of urinary profiles are constructed (Figure 3). The urinary metabolic profiles of mice dosed with cyadox deviated from controls initially and recovered after day 2 postdosage for the low-dose group. Those in the moderate-dose group are recovered after day 6 postdosage, and no recovery is achieved for the urinary metabolic profiles of the high-dose group over the entire experimental period. In order to discover altered metabolites associated with different levels of cyadox dosage, pairwise O-PLS-DA comparisons between urinary metabolic profiles of dosed mice and those of control mice are constructed for the different time points. Examples of cross-validated scores plots and corresponding coefficient plots are displayed in Figure 4. A summary of altered key urinary metabolites associated with duration and dose levels is shown in Figure 5, and parameters indicating model quality and crossvalidation results are listed in Table 3 in Supporting Information. Here, red color corresponds to an increase and blue color corresponds to a decrease in the levels of metabolites in the 540



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Figure 4. OPLS-DA scores plots (left) and corresponding color-coded correlation coefficient loadings plots (right) generated by comparisons between urinary spectra of control mice (black boxes) and those from mice dosed with low (red dots), moderate (green diamonds), and high (violet stars) levels of cyadox at day 1 postdosage.

Figure 5. Dynamic alterations of key urinary metabolites in response to high (C-H), moderate (C-M), and low (C-L) levels of cyadox exposure. The color indicates a correlation coefficient as scaled on the right-hand side. Red denotes an increase in the levels of metabolite in the dosed mice compared to the control group, whereas blue indicates a decrease.

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Marked metabolic effects are also observed in the renal medulla of high-dose mice. For example, the levels of a range of amino acids, including isoleucine, leucine, valine, alanine, lysine, glutamate, glutamine, glycine, tyrosine, phenyalanine, and histidine, and the levels of acetic acid, ethanolamine, glucose, uridine, cis-aconitate, and mannose are decreased markedly, whereas the levels of choline (Cho), GPC, myo-inositol, TMAO, creatine, IMP, and AMP are increased after cyadox exposure at high level.

DISCUSSION

Cyadox has been reported to be of low toxicity, which is supported by our observation of unchanged metabolic profiles of plasma, which in turn could be understood in terms of minimum disturbance of homeostasis by the drug. Nevertheless, a holistic investigation into the toxic effects of cyadox has not been extensively performed; we therefore employed a metabonomics strategy and studied the alterations of metabonomes of multiple 541



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Figure 6. OPLS-DA scores plots (left) and correlation coefficient loadings plots (right) derived from NMR spectra of extracts of liver (L) and renal medulla (K) of mice, indicating a discrimination between control mice (black boxes) and mice dosed with low (red dots), moderate (green diamonds), and high (violet stars) levels of cyadox. (LM, LH) Liver extracts of control mice compared with mice dosed with moderate and high levels of cyadox; (KL, KM, and KH) Extracts of control mice compared with those from mice dosed with low, moderate, and high levels of cyadox.

which indicates a disturbance of gut microbiota. This observation is consistent with the antimicrobial activity of cyadox. Other cometabolites between gut microbiota and host include indoxyl sulfate, 2-(4-hydroxyphenyl)propanoic acid (2-HPPA), 2,5dihydroxybenzoate, 2-hydroxycinnamate, PAG, MA, DMA, TMA, and TMAO,40 which are all reduced in urine of cyadoxdosed mice, particularly those dosed with moderate and high levels. In addition, the levels of urinary short-chain fatty acid (SCFA), such as butyric acid, produced by gut microbiota,41 are decreased in mice exposed to all levels of cyadox, which could also have a contribution from alterations of the intestinal microbes. The recovery of these cometabolites varies depends on the nature of cometabolites and dose levels. For example, the levels of hippurate recover to normal at day 2 postdose for the

biological matrices of mice, including urine, plasma, liver, and kidney, induced by single exposure of cyadox. Cyadox is an antibiotic drug, and hence it is anticipated that cyadox ingestion would cause a disturbance in the gut microbial community and subsequent cometabolism between the gut microbial community and host. Hippurate is one of the cometabolites between gut microbiota and host and is a product of conjugation of glycine and benzoic acid,33 which takes place in the mitochondria34 of the liver and kidney.35 Benzoic acid is a product of the intestinal microbial degradation of aromatic compounds in food.36−38 Therefore, hippurate is regarded as an indicator for balance in the gut microbial community.39 In the present investigation, we found a marked reduction in the levels of hippurate in the urine of mice dosed with cyadox (Figure 5), 542



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Figure 7. Metabolic pathways altered by acute cyadox exposure. Key: Red-colored metabolite shows the markedly increased metabolites and bluecolored metabolite is for decreased metabolites; metabolites in black indicate unchanged metabolites.

suggests that cyadox exposure prohibits nucleotide synthesis, which in turn could affect nucleic acid synthesis; this is another important line of evidence supporting the notion that cyadox exposure causes a dysfunction of liver functions (Figure 7). Furthermore, the observation of elevated levels of Nmethylnicotinamide (NMN) and trigonelline implies that cyadox exposure promotes antioxidation activity of the host. This is because trigonelline and N-methylnicotinamide are the respective methylated metabolites of niacin (vitamin B3) and nicontiamide, which can be generated during the conversion of Sadenosylmethionine to S-adenosylhomocysteine during biosynthesis of cysteine, an essential amino acid of glutathione synthesis.49 The kidney is another organ responsible for the detoxification of xenobiotic drugs. Although no pathological changes in the kidney were observed in the histopathology, there were metabolic alterations in the extracts of renal medulla, in particular from mice dosed with high levels of cyadox. The most prominent metabolic effect of cyadox on renal medulla is the elevated levels of a range of organic osmolytes, such as choline, GPC, TMAO, taurine, and myo-inositol. This observation suggests that cyadox exposure causes disturbances in kidney osmolarity,50,51 which ultimately compromise kidney function. We observed an elevation in the levels of plasma creatinine, which indicates that the function of the kidney in blood creatinine clearance is compromised upon dosing with cyadox at high levels (Table 1 in Supporting Information). In addition, we observe elevations in the levels of nucleotides, such as AMP and IMP, and depletions in the levels of uridine in the renal medulla obtained from highlevel cyadox-dosed mice, which suggests that cyadox exposure causes disruption in nucleic acid synthesis (Figure 7). Furthermore, tricarboxylic acid cycle intermediates were depleted, indicating alterations in energy metabolism. Cyadox exposure causing metabolic disruptions in mice is manifested further in the depletion of a range of amino acids, which occurs in a dose-dependent fashion (Figure 6, KL, KM, and KH). This observation implies that cyadox exposure could also result in the inhibition of protein synthesis. Our view is particularly supported by the depleted levels of tyrosine, since tyrosine is known to be used only in protein synthesis.52

low-dose group and at day 7 for the moderate-dose group. However, the levels of hippurate, benzoate, and indoxyl sulfate do not recover in the high-dose group throughout the entire experimental period, which suggest that recovery of gut microbiota is a long process. In this study, we also observed a reduction in the levels of m-HPPA sulfate at an early stage postdose and elevation in its level at a later stage postdose. The level of m-HPPA sulfate is closely related to the activity of sulfotransferase.42 Sulfotransferases catalyzes the transfer of a sulfonate group to target xenobiotic drugs, aiding their metabolism.43 Therefore it is plausible to assume that the availability of the sulfate group might be limited at the early stage of cyadox exposure and become available at a later stage of exposure, leading to the observed fluctuation in the levels of mHPPA sulfate. Liver is the primary organ for the detoxification of xenobiotic drugs; hence the function of the liver could be compromised by cyadox exposure, which is what we found in the present investigation. For example, hydropic degeneration is observed in the liver of mice dosed with moderate and high levels of cyadox, which suggests imbalance of ion and fluid homeostasis.44 The observed alterations in the levels of TMAO and GPC are highly likely to be consistent with this observation since these metabolites are known to act as osmolytes, ameliorating the imbalance of fluid homeostasis.45,46 Dysfunction of the liver is also manifested in the elevated levels of amino acids in the liver of cyadox-dosed mice (Figure 6, LM and LH). It is known that most amino acids are mainly metabolized in the liver.47 Hence, accumulation of amino acids in the liver indicates that cyadox causes inhibition of amino acid metabolism. This notion is also consistent with the observed reduction in the ALT level, which ultimately leads to accumulation of glutamate in the liver of cyadox-dosed mice. A decreased level of ALT was also previously observed in rats chronically dosed with cyadox, further supporting that cyadox exposure causes liver dysfunction.13 A study on the toxicity of microcysin-LR, a protein phosphatase inhibitor, has also shown decreased ALT synthesis in hepatocytes.48 In addition, we observed a marked reduction in the levels of nucleotides, such as uridine 5′-monophosphate, AMP, ITP, IDP, and IMP, with a concurrent increase in the levels of nucleoside and nucleobase, such as inosine, uracil, and hypoxanthine, in the liver of cyadox-exposed mice. Since the liver is the primary organ for nucleotide synthesis, our observation 543



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CONCLUSION How time and dose levels affect the metabolic responses of Kunming mice to cyadox exposure was revealed by multivariate data analysis of NMR spectroscopic profiles of multiple biological matrices. Low and moderate doses of cyadox induce reversible metabolic changes, whereas irreversible metabolic changes are observed when mice are exposed at high doses (4000 mg/kg bw). A high dose of cyadox induces disturbance of cometabolism between mice and gut microbiota, as well as inhibition of amino acid metabolism and antioxidative stress of liver. In addition, cyadox exposure causes an imbalance of osmolytic homeostasis and affects nucleic acid synthesis in both the liver and kidney. The metabonomic approach used to investigate the toxicity of cyadox provides evidence for metabolic disturbances induced by cyadox exposure and offers a holistic view on the toxicity of the drug. Our results suggest that cyadox is of low toxicity and has a potential to be used as a feedstuff additive, provided the toxicity of long-term exposure in model animals is assessed, which is currently ongoing. This information is vital in evaluating the safety of cyadox in the farming industry.

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PAG, phenylacetylglycine; m-HPPA sulfate, m-hydroxyphenylpropionic acid; Cho, choline; GPC, glycerophosphocholine; AMP, adenosine monophosphate; ITP, inosine triphosphate; IDP, inosine diphosphate; IMP, inosine monophosphate; UMP, uridine monophosphate; NMN, N-methylnicotinamide; SCFA, short-chain fatty acid

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ASSOCIATED CONTENT

S Supporting Information *

Three tables listing clinical biochemistry test, NMR assignment of metabolites, and statistics of the model; and one figure showing histopathological results of liver from mice in groups C, L, M, and H. This material is available free of charge via the Internet at http://pubs.acs.org.”

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected], fax +86-27-87199291, tel +8627-87197143(Y. W.); e-mail [email protected], fax +8627-87199291, tel +86-27-87198430 (H.T.). Present Address §

School of Biology and Pharmaceutical Engineering, Wuhan Polytechnic University, P. R. China. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by grants from the Ministry of Science and Technology of China (2009CB118804, 2012CB934004) and the National Nature Science Foundation of China (20825520, 21221064). We would also like to acknowledge S.X. Tang for English correction.

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ABBREVIATIONS NMR, nuclear magnetic resonance; PCA, principal component analysis; PLS-DA, projection to latent structures discriminant analysis; O-PLS-DA, orthogonal projection to latent structures discriminant analysis; CPMG, Carr−Purcell−Meiboom−Gill; Ile, isoleucine; leu, leucine; Val, valine; Lys, lysine; Asp, aspartic acid; Gly, glycine; Glu, glutamate; Gln, glutamine; His, histidine; Phe, phenylalanine; Tyr, tyrosine; Trp, tryptophan; Glc, glucose; CHOL, total cholesterol; CREA, creatinine; BUN, blood urea nitrogen; TG, triglyceride; ALB, albumin; AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase; TP, total protein; MA, methylamine; DMA, dimethylamine; TMA, trimethylamine; TMAO, trimethylamine-N-oxide; 2HPPA, 2-(4-hydroxyphenyl)propanoic acid; 544



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Metabolic Influence of Acute Cyadox Exposure on Kunming Mice

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