Comparative NMR-Based Metabonomic Investigation of the Metabolic

Article pubs.acs.org/crt

Comparative NMR-Based Metabonomic Investigation of the Metabolic Phenotype Associated with Tienilic Acid and Tienilic Acid Isomer Muireann Coen,*,† Peter M. Rademacher,‡ Wei Zou,§ Michael Scott,∥ Patricia E. Ganey,⊥ Robert Roth,⊥ and Sidney D. Nelson‡ †

Biomolecular Medicine, Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, London SW7 2AZ, United Kingdom ‡ Department of Medicinal Chemistry, University of Washington, 1959 NE Pacific Street, Health Sciences Building, Seattle, Washington 98195-7610, United States § Department of Microbiology and Molecular Genetics, 2215 Biomedical Physical Sciences, Michigan State University, East Lansing, Michigan 48824-1302, United States ∥ Department of Pathobiology and Diagnostic Investigation, G-347 Veterinary Medical Center, Michigan State University, East Lansing, Michigan 48824-1314, United States ⊥ Department of Pharmacology and Toxicology, 221 Food Safety and Toxicology Building, Michigan State University, East Lansing, Michigan 48824-1302, United States S Supporting Information *

ABSTRACT: An NMR-based metabonomic approach was applied to study the systems level metabolic effects of two closely related thiophene compounds, tienilic acid (TA) and tienilic acid isomer (TAI). The metabonomic data were anchored with traditional clinical chemistry and histopathologic analyses. TA was removed from the market as a result of suspected immune-mediated hepatotoxicity, whereas TAI is an intrinsic hepatotoxin. Equimolar doses of TA and TAI were administered to Sprague− Dawley rats, and sampling was conducted at 2, 6, and 24 h post-treatment. Histopathologic analyses revealed development of a significant hepatic lesion 24 h post-TAI treatment with a parallel increase in plasma alanine aminotransferase (ALT) activity. In contrast, TA was not associated with the development of a hepatic lesion or an increase in plasma ALT activity. High-resolution NMR spectral metabolic profiles were generated for liver extracts, plasma, and urine at multiple time points. Multivariate statistical tools were applied to model the metabolic profiles and identify discriminatory metabolites that reflected both the adaptation to TA administration and the onset and progression of TAI-induced hepatotoxicity. TAI was shown to induce marked metabolic effects on the metabolome at all time points, with dramatic metabolic perturbations at 24 h post-treatment correlating with the histopathologic and clinical chemistry evidence of a hepatic lesion. The TAI-induced metabolic perturbations provided evidence for the generation of electrophilic reactive metabolites and a significant impairment of bioenergetic metabolic pathways. TA induced early metabolic perturbations that were largely resolved by 24 h post-treatment, suggesting the reestablishment of metabolic homeostasis and the ability to adapt to the intervention, with hepatic hypotaurine potentially representing a means of assessment of hepatic adaptation. This comparative metabonomic approach enabled the discrimination of metabolic perturbations that were common to both treatments and were interpreted as nontoxic thiophene-induced perturbations. Importantly, this approach enabled the identification of temporal metabolic perturbations that were unique to TAI or TA treatment and hence were of relevance to the development of toxicity or the ability to adapt. This approach is applicable to the future study of pharmacologically and structurally similar compounds and represents a refined means of identification of biomarkers of toxicity.

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INTRODUCTION

retic hypotensive drug, was withdrawn from the market shortly after its introduction due to numerous reported cases (>500) implicating it as the cause of clinical hepatotoxicity.1 The mechanism for this idiosyncratic drug-induced liver injury

Drug-induced liver injury (DILI) represents a significant challenge in both the preclinical and the clinical setting, and there is a pressing need to further understand the mechanistic basis for DILI and to define biomarkers of diagnostic and prognostic value. Tienilic acid [TA, ticrynafen, 2,3-dichloro-4(thiophene-2-carbonyl)phenoxyacetic acid], a uricosuric diu© 2012 American Chemical Society

Received: June 20, 2012 Published: September 26, 2012 2412

dx.doi.org/10.1021/tx3002803 | Chem. Res. Toxicol. 2012, 25, 2412−2422

Chemical Research in Toxicology

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and assessment of clinical chemistry markers of liver injury together with histopathologic assessment. TAI was shown to induce a significant hepatic lesion at 24 h post-treatment, in comparison to TA, which did not induce a hepatotoxic response. This result enabled phenotypic anchoring of wellestablished toxicological end points to NMR spectral profiling of plasma, urine, and liver extracts at time points of 2, 6, and 24 h following TA and TAI treatment. This approach was capable of discriminating the TA-induced metabolic signature of adaptation from the TAI-induced metabolic signature reflecting the onset, development, and progression of hepatotoxicity.

(IDILI) remains unclear. It is believed that an immunemediated toxic mechanism may play a significant role given the observation of antibodies (anti-liver and -kidney microsomal type 2 autoantibodies) directed against CYP2C9, for which TA is a suicide substrate.2,3 However, a significant proportion of TA-treated patients who develop hypersensitivity and hepatotoxicity often lack the typical characteristics of immunemediated toxic reactions and do not have detectable antibodies towards either TA or CYP2C9.4,5 This presents the question as to whether TA can also induce toxic effects as a direct, intrinsic hepatotoxicant. In a study of the effect of TA in phenobarbital-induced isolated, perfused rat liver, reduced bile flow and sulfobromophthalein clearance were observed together with a minimal increase in the release of aspartate aminotransferase (AST) into the perfusate.6 The administration of relatively large doses of TA (480 mg/kg in male Sprague−Dawley rats, orally) for 28 days resulted in increased liver weight, a marginal increase in alanine aminotransferase (ALT) activity, and evidence for unicellular hepatic necrosis.7 It has also been demonstrated in rats depleted of glutathione that TA (doses up to 1000 mg/kg) caused a significant elevation in serum ALT activity and extensive centrilobular hepatocellular necrosis together with induction of biomarkers of oxidative stress.4,8 The 3-thenoyl regioisomer of tienilic acid [TAI, 2,3-dichloro-4-(thiophene-3carbonyl)phenoxyacetic acid] has been reported to be an intrinsic hepatotoxicant in rats.9−11 In contrast to TA, TAI does not act as a suicide substrate for CYP2C912 and displays a greater degree of nonspecific covalent binding to liver microsomal proteins than TA.9,10,13 Both TA and TAI are believed to form reactive electrophilic metabolites following oxidation of the thiophene ring, with covalent binding of TA epoxide to CYP2C9 (or CYP2C11 in rats) resulting in suicide inactivation. In contrast, TAI sulfoxide is released from the active site of CYP2C9 and reacts with nucleophiles such as glutathione and nonspecifically with numerous liver proteins once glutathione is depleted.9,10,13−15 Metabonomics involves the application of advanced analytical platforms, typically nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography coupled with mass spectrometry (LC-MS), to profile the small molecule complement of biofluids and tissues.16 The complex spectral profiles are typically modeled using multivariate statistical approaches to extract the biological information related to the phenotype of interest.17,18 Metabonomic applications in preclinical studies have proved of great value for simultaneous determination of the metabolic fate of a drug in addition to the associated endogenous metabolic alterations.19−23 A particular strength of metabonomic applications in toxicology has been the identification and study of the temporal metabolic trajectory of response to toxic insult enabling the onset, progression, and recovery of toxicity to be assessed for compounds with diverse modes of action.24 In addition, metabonomic approaches have led to the identification of unique metabolic phenotypes relevant to variability in toxic response. This approach has also demonstrated the potential to elucidate the underlying mechanistic bases for variability in toxic response and to identify preintervention signatures that were predictive of differential response.25−27 Herein, we report the application of NMR-based metabonomics to study the differential metabolic response induced by TA and TAI administration in a rodent model. The model involved administration of an equimolar dose of TA and TAI

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EXPERIMENTAL PROCEDURES

Animal Husbandry. Male Sprague−Dawley [Crl:CD(SD)IGS BR] rats (250−300 g) were obtained from Charles River Laboratories (Portage, MI). Animals were housed in temperature (294−300 K) and humidity (30−70% RH) -controlled rooms with a 12 h light/dark cycle throughout the study. They were acclimated to the facility for 1 week prior to the start of the study. Animals were provided food (Standard Rodent Chow/Tek 8640; Harlan Teklad, Madison, WI) and drinking water ad libitum apart from an overnight fasting period of 15 h prior to the administration phase. TA and TAI Administration. TA and TAI (Figure 1a) were synthesized in the laboratory of Professor Nelson as previously

Figure 1. (A) Chemical structure of TA and its isomer (TAI). (B) Plasma ALT activity (U/L) for vehicle control (Ctrl) and TA-treated and TAI-treated animals at 2, 6, and 24 h post-treatment. The table shows the mean, standard deviation, standard error of the mean, and the range for each cohort. (**) p < 0.01, calculated from an unpaired Mann−Whitney test between controls and TAI-treated at 24 h postdose. described.15 The purity of TA and TAI was 99% as determined by LCUV and LC-MS with structural characterization conducted using 1H NMR spectroscopy. TA and TAI were prepared in Trizma base solutions (0.25 M) at a concentration of 75 mg/mL, such that the molar ratio of TA or TAI to Trizma base was 1:1.1 (pH 7.4). Each drug was suspended in solution by vortexing, sonicating, and vortexing again. In a preliminary study, rats intravenously administered 250 mg/ kg TAI developed significant hepatotoxicity at 24 h post-treatment (plasma ALT activity of 600 U/L), whereas this dose of TA resulted in minimal toxicity (ALT activity below 200 U/L). Accordingly, this dose level and dosing route were chosen for the metabonomic study as it represented a model of TAI-induced hepatotoxicity and enabled comparative administration of an equimolar, non-hepatotoxic dose of TA. Animals were fasted overnight for a period of 15 h prior to 9:00 a.m. administration intravenously (rate of approximately 1.5 mL/min) with 250 mg/kg TA (n = 5 per time point, total of n = 15), 250 mg/kg 2413



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TAI (n = 5 for the 2 and 6 h time points, n = 6 for the 24 h time point, total of n = 16), or vehicle (Trizma base in HPLC-grade water, n = 5 per time point, total of n = 15). Food was returned once the administration was complete. Clinical observation was carried out during the administration phase and at 2, 6, and 24 h post-treatment. Sample Collection. Animals were euthanized with isoflurane at 2, 6, or 24 h post-treatment. Rats were housed singly in metabolism cages with urine collection funnels cooled with dry ice. For the group euthanized at 24 h, urine samples were collected from each rat over the following time periods: 0−2, 2−6, and 6−24 h. Urine was not collected from the cohorts euthanized at 2 and 6 h post-treatment. Collected urine samples were frozen immediately at −80 °C for further analysis. The total urine volume obtained from each rat for each collection period was recorded; only one sample was obtained from the 2 h control cohort given the short duration of this early sampling period; hence, the data (urine volume, spectral data) were grouped for all controls at 2 and 6 h (total n = 6). Blood was collected from the vena cava into heparin-containing Vacutainer tubes for centrifugation to collect plasma. Necropsy was performed immediately after euthanasia with a section of the left lateral lobe of the liver being immediately snap-frozen in liquid nitrogen and stored at −70 °C until analyzed by NMR spectroscopy. Determination of TA and TAI Plasma Concentration. TA and TAI plasma concentrations were determined using liquid chromatography coupled to a Quattro Micro Triple quadrupole mass spectrometer (Waters) coupled to a Schimadzu SC10A binary pump via an electrospray ionization interface. A Phenomenex (Torrance, CA) Synergi Polar-RP column (2.1 mm × 75 mm, 5 μM particle size) was used for separation with a flow rate of 0.3 mL/min. LC conditions consisted of two solvents, solvent A (99.9% H2O containing 0.1% formic acid) and solvent B (99.9% acetonitrile containing 0.1% formic acid). The gradient method used was as follows: solvent B was held at 25% for 3 min followed by a linear gradient to 95% B over 9 min. Compounds were quantified by multiple reaction monitoring (MRM) in the positive ion mode for the transition 331 → 247 for TA and TAI and 325 → 247 for the internal standard 2-(4-benzoyl-2,3dichlorophenoxy) acetic acid. For MS analysis, the cone voltage was 30 eV, and the collision energy was 20 eV. The source temperature was 120 °C, and the desolvation temperature was 350 °C. TA and TAI ionize with nearly identical efficiencies. Standard curves were generated by dissolving known quantities of TA into plasma. The standard curve samples and 50 μL of plasma from rats at 2, 6 and 24 h post-dose (250 mg/kg IV) were then spiked with internal standard. Plasma proteins were precipitated with ACN and centrifuged at 10000 g for 15 min and analyzed (50 μL aliquots). The lower limit of quantitation was 1 ng/mL (3 nM) and the assay was linear up to 10000 ng/mL (30 μM). Clinical Chemistry and Histopathology. The plasma ALT activity was determined using an Infinity-ALT kit (Thermo Corporation, Waltham, MA). Three slices (ca. 3 mm thick) from a section of the left lateral lobe of the liver were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Sections of all liver slices for each rat were assessed light microscopically by a pathologist (MAS) who was masked to time and treatment other than knowing which samples were from vehicle control rats. All abnormalities were characterized, and the degree of vacuolar change, cell death, presence of glycogen, and inflammation were graded on a 0−3 scale with 0 representing no lesion and grades 1, 2, and 3 representing mild, moderate, and marked changes, respectively. 1 H NMR Spectroscopy of Urine. Urine samples were thawed, vortexed, and allowed to stand for 10 min prior to mixing aliquots (400 μL) with phosphate buffer [200 μL, 0.2 M containing 99% deuterium oxide (D2O), 1 mM 3-(trimethylsilyl)-[2,2,3,3-2H4]propionic acid sodium salt (TSP), and 3 mM sodium azide] and centrifuged at 15900g for 10 min. Supernatants (550 μL) were transferred into NMR tubes (507-HP-7, Norell, Landisville, NJ). The D2O provided a field frequency lock, and TSP provided a chemical shift reference (1H, δ 0). 1H NMR spectra were acquired on a Bruker Avance-600 spectrometer, operating at 600.13 MHz 1H frequency and

a temperature of 300 K, using a Bruker TXI probe (Bruker Biopsin, Rheinstetten, Germany) and an automated sample handling carousel (Bruker). NMR spectra were acquired using the standard onedimensional solvent suppression pulse sequence (relaxation delay, 90° pulse, 4 μs delay, 90° pulse, mixing time, 90° pulse, acquire FID).28 For each sample, 512 transients were collected into 64K data points using a spectral width of 12000 Hz with a relaxation delay of 4 s, a mixing time of 100 ms, and an acquisition time of 2.73 s. A linebroadening function of 0.3 Hz was applied to all spectra prior to Fourier transformation (FT). 1 H NMR Spectroscopy of Plasma. Plasma samples were thawed, vortexed, and allowed to stand for 10 min prior to mixing aliquots (200 μL) with 0.9% saline containing 20% D2O (400 μL). Samples were spun at 15900g for 10 min, the supernatant fluid (500 μL) was placed in NMR tubes (507-HP-7, Norell), and NMR spectra were acquired at a 1H observation frequency of 600.13 MHz and temperature of 300 K. Chemical shifts were referenced to that of αglucose (1H, δ 5.23), and D2O provided a field-frequency lock. The Carr−Purcell−Meiboom−Gill (CPMG) spin−echo pulse sequence with a fixed spin−spin relaxation delay, 2nτ of 64 ms (n = 160, τ = 200 μs), was applied to acquire 1H NMR spectra of all plasma samples.28 For each sample, 384 transients were collected into 64K data points using a spectral width of 12000 Hz with a relaxation delay of 4 s and an acquisition time of 2.73 s. A line-broadening function of 0.3 Hz was applied to all spectra prior to FT. 1 H NMR Spectroscopy of Aqueous-Soluble Liver Extracts. Liver tissue samples (median sample weight of 100 mg) were added to ice cold acetonitrile/water (1 mL, 50% ACN:50% H2O) and homogenized for 8 min using a ball-bearing tissue lyser (QiagenTissueLyser, Retsch GmBH, Haan, Germany). The homogenized samples were spun at 15900g for 10 min, and the supernatant was removed and lyophilized overnight prior to reconstitution in phosphate buffer (600 μL, 0.2 M containing 99% D2O, 3 mM TSP, and 3 mM sodium azide). The CPMG spin−echo pulse sequence with a fixed spin−spin relaxation delay, 2nτ of 64 ms (n = 160, τ = 200 μs), was applied to acquire 1H NMR spectra of all samples. For each sample, 512 transients were collected into 64K data points using a spectral width of 12000 Hz with a relaxation delay of 4 s and an acquisition time of 2.73 s. A line-broadening function of 0.3 Hz was applied to all spectra prior to FT. Assignment of NMR Spectral Resonances. Two-dimensional hetero- and homonuclear NMR spectroscopic experiments were carried out on representative samples for metabolite identification purposes, together with use of metabonomic literature and spectral databases (BMRB, Colmar, HMDB, Bruker S-Base). In addition, experiments where standard chemicals were “spiked” into biofluid, and extract samples were conducted to confirm spectral resonance assignments. Statistical Analysis of NMR Spectral Data. Full-resolution NMR data were imported into MATLAB (R2009b, Mathworks Inc., 2009). The regions corresponding to water/HDO (δ 4.7−4.9) and TSP (δ −0.2−0.2) were removed from all spectra. In addition, the urea resonance region was removed from urinary spectra (δ 5.6−6). The urinary spectral data were normalized using the probabilistic quotient method29 to account for concentration differences between samples as a result of the diuretic effect of TA and TAI. The liver aqueous extract spectral data were also normalized using the probabilistic quotient method, whereas plasma spectra were not normalized. The urinary spectra were aligned using the recursive segment-wise peak alignment algorithm (RSPA30) incorporating a guide tree.31 Principal components analysis (PCA) was initially carried out on unit-variance scaled, full-resolution spectral data to reduce the spectral dimensionality and identify inherent clustering of samples and potential outliers (data not shown). Pairwise, partial-least-squares-discriminant analysis (PLS-DA) and orthogonal projection on latent structures discriminant analysis (O-PLS-DA) models were computed to maximize multivariate separation of control and treated classes and of TA- and TAI-treated cohorts.18,32 PLS is a supervised pattern recognition algorithm that models the linear relationship between a spectral matrix (X) and a descriptor such 2414



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as class membership (Y dummy matrix of zero and one) for discriminant analysis (PLS-DA) or a continuous end point such as a clinical chemistry parameter in the case of regression modeling. OPLS-DA is an extension of this algorithm that prefilters orthogonal and hence classification-irrelevant variation from the spectral data matrix and thereby greatly improves interpretability of spectral variation between classes. The prefiltered, structured noise in a data set, which will confound interpretation in PLS-derived models, is modeled separately from the variation related to class membership and can also be further interpreted via the loading matrices. The scores plot provides an interpretation of the inherent clustering of samples in multivariate space. In this manuscript, we have presented the crossvalidated predictive scores (Tcv) from discriminant analysis models that applied a class membership (Y), of control and treated. The loadings coefficients enable the NMR spectral resonances that are responsible for separation between classes in the scores plot to be identified. The loadings coefficient plot is constructed from a “backscaled” model: the data are autoscaled by division of each variable by its standard deviation, and the variable weights are also incorporated.33 The loadings coefficient plot of the full resolution spectral data is represented with the weight of the variables color-coded; hence, the highly discriminatory variables are clearly identified in the plot as those with high correlation value colors, typically red/orange. As fullresolution NMR data are used for modeling, the spectral structure is retained, and the plot can be visually interpreted as a pseudo-NMR spectrum to determine the significant metabolites that vary in response to the treatment of interest. To prevent overfitting of spectral data, the 7-fold cross-validation method was used, and the cross-validation parameter Q2 was calculated to assess the statistical robustness and predictive ability of models. In addition, permutation tests were performed to test the robustness and validity of the models, where the Y vector was randomly permuted 1000 times and the permutation p value was obtained by ranking the actual Q2Y with the permuted Q2Y. In addition, integration of the discriminatory NMR spectral resonances was carried out with subsequent calculation of Mann− Whitney test p values to assess statistical significance [(*) p < 0.05, (**) p < 0.01]. The median log2 fold change of the integrals for the statistically significant discriminatory hepatic metabolites was calculated for each treatment group and time point relative to the median of the controls and displayed as a heat map, which enabled simplified visualization and comparison of the scale of metabolic change between each treatment and time point together with assessment of the degree of interanimal variability. The statistical significance of differences in plasma ALT activity was assessed from a Mann−Whitney test p value [(*) p < 0.05, (**) p < 0.01].

Clinical Chemistry and Histopathology. The plasma ALT activity was measured for vehicle control and TA and TAI treatment groups (Figure 1b). At 2 h postdose, no increase in ALT was seen for TA- or TAI-treated groups relative to vehicle controls. An increased, albeit highly variable rise in ALT was seen at 6 h post-TAI treatment [688 ± 946 U/L mean ± standard deviation (SD)]; two animals had dramatically elevated ALT activity with values greater than 800 U/L. A uniform and statistically significant rise in ALT activity was observed at 24 h post-TAI treatment (336 ± 267 U/L, p < 0.01 relative to controls), whereas TA or vehicle caused no such elevation. The vehicle groups showed no statistically significant difference in plasma ALT activity at 2, 6, and 24 h posttreatment. Masked histopathologic analysis revealed a lesion that was specific for the 24 h TAI-treated group, and that corresponded with the significantly increased plasma ALT activities of this group. The three 24 h TAI-treated rats with the highest plasma ALT activities had multifocal regions of centrilobular to bridging hepatocyte vacuolation, degeneration, and necrosis that were associated with intralesional aggregates of macrophages and lymphocytes and few scattered neutrophils (Figure 2). In contrast, hepatocyte vacuolar change and necrosis were not detected in any of the vehicle control or TA-treated rats at 24 h post-treatment. Every vehicle- and TA/TAI-treated rat at 2 and 6 h postdosing had few small, centrilobular to random foci of macrophagic and lymphocytic inflammation, most of which were accompanied by individual hepatocyte death. These were considered minor background lesions unrelated to drug treatment. Similar lesions with less hepatocyte death were present in three of the five 24 h TA-treated rats, but these lesions were absent from the 24 h control rats, and they were not detected in the 24 h TAI-treated rats where they may have been masked by much more substantial lesions. The plasma ALT activity and hepatic extract metabolic profiles of the 2, 6, and 24 h time point control cohorts were stable, which suggested that the presence of this background lesion did not affect the validity of results. Indistinct cytoplasmic vacuolation consistent with glycogen stores increased with time in the control rats: it was mild in two of five control rats at 2 h, mild to moderate in all control rats at 6 h, and marked in all control rats at 24 h. Similarly, glycogen appeared to increase over time in the TA-treated rats; it was absent from all TA-treated rats at 2 h after dosing, mild to moderate in four of five TA-treated rats at 6 h, and moderate to marked in all five TA-treated rats at 24 h. TAI-treated rats also lacked evident glycogen stores at 2 h, but increases over time were less pronounced; at 6 h postdosing, glycogen was limited to only moderate stores in the two rats with the lowest 6 h plasma ALT activities, and at 24 h, glycogen stores reached a moderate degree in only one rat, which had the lowest plasma ALT activity. Mild to moderate midzonal to periportal or diffuse lipidlike hepatocyte vacuolation was present in all TA- and TAI-treated rats 2 h after treatment, in three out of five of each at 6 h, and in none at 24 h postdosing. In contrast, very mild lipidlike vacuolation was present in two of five control rats at 2 h but in no control rats at 6 or 24 h postdosing. Thus, there was less evidence of lipid in hepatocytes of control rats as compared to treated rats, and it was progressively less apparent over time. Metabonomic Results. The application of a nontargeted metabonomic approach to the study of the systems wide response to TA and TAI enabled recovery of information on numerous endogenous metabolic consequences. Pairwise O-

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RESULTS Plasma Concentration of TA and TAI and Urinary Output. The mean 2 h post-treatment plasma concentration of TA was 20.1 ± 2.9 μM and for TAI was 23.4 ± 3.7 μM, following an iv bolus dose of 250 mg/kg. At 6 h post-treatment, the plasma concentration of TA was 1.0 ± 1.2 μM and for TAI was 0.6 ± 0.2 μM. At 24 h post-treatment, the plasma concentration of both compounds was below the limit of detection (3 nM). The mean 2 h post-treatment urine output for TA was 2.1 ± 0.9 mL/h and for TAI was 2.7 ± 1.2 mL/h, which were both statistically significantly different (p < 0.05) relative to control output (0.29 ± 0.12 mL/h as determined from the grouped 2 and 6 h time point collections). There was no statistically significant difference in urine volume output at 6 h posttreatment for TA- and TAI-treated animals in comparison to controls. At 24 h post-treatment, there was a statistically significant (p < 0.05) increase in the urinary output of the TAItreated cohort relative to controls, whereas no difference was seen for TA-treated animals. 2415



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Figure 3. O-PLS-DA model discriminating control and TAI aqueous soluble liver extract NMR spectral profiles 2 h post-treatment (Y-Ctrl TAI represents a dummy matrix of zero for Ctrl and one for TAI). (A) Cross-validated scores (Tcv) for control (Ctrl, red, n = 5) and TAI (blue, n = 5) cohorts, (B) loadings coefficient plot for the aliphatic spectral region, and (C) loadings coefficient plot for the aromatic spectral region. The height of spectral peaks represents the covariance, and the color code corresponds to the coefficient of determination, r2. Model statistics: R2Y = 0.99, Q2Y = 0.88, and permutation p value ** < 0.01. Key: Leu, leucine; Isoleu, isoleucine; Val, valine; D-3-HB, D-3hydroxybutyrate; GSSG, oxidized glutathione; and GSH, reduced glutathione. * represents resonances arising from both GSSG and GSH.

Figure 2. TAI-induced hepatic lesion 24 h post-treatment. (A) Multifocal centrilobular to bridging hepatocyte degeneration and necrosis with multifocal intralesional inflammatory aggregates and inapparent glycogen stores; bar = 100 μm. (B) Greater magnification of the severe perivenular vacuolar degeneration and necrosis shown in the upper portion of panel A; vacuolated hepatocytes with small, condensed nuclei are intermixed with shrunken hypereosinophilic necrotic hepatocytes and multifocal to coalescing aggregates of macrophages and lymphocytes; bar = 50 μm. Hematoxylin and eosin.

hepatic metabolites differentiating the control cohort from TAItreated at the early 2 h post-treatment time. TAI treatment was associated with a depletion of glycogen and glucose, glutathione (representing both oxidized and reduced forms), lactate, and alanine together with an increase in the levels of tyrosine, inosine, D-3-hydroxybutyrate (D-3-HB), creatine, glutamate, valine (Val), isoleucine (Isoleu), and leucine (Leu). A comparable model that differentiated controls from TA-treated at 2 h post-treatment revealed a similar effect on many of these metabolites; glycogen, glucose, glutathione (oxidized and reduced), lactate, and alanine were markedly depleted, and tyrosine, inosine, Val, Isoleu, Leu, and D-3-HB were significantly elevated. A unique and statistically significant change observed in response to TA treatment was an increase in hepatic hypotaurine levels. An O-PLS-DA model discriminating TA-treated from TAI-treated cohorts enabled determination of the metabolic changes induced at 2 h post-treatment that were unique to either TA or TAI treatment (Figure 4; R2Y = 0.99, Q2Y = 0.62, permutation p value < 0.01). The model differentiating TA-treated from TAI-treated animals showed

PLS-DA models of full-resolution NMR spectral data were computed to identify metabolites that discriminated between the control and the treated classes. Comparisons were made between the vehicle control group and the TA- or TAI-treated groups and also between the TA- and the TAI-treated groups at each time point (2, 6, or 24 h) to identify both common and unique metabolic markers of TA and TAI treatment. Endogenous Hepatic Metabolic Profiles Reflective of TA and TAI Treatment. Marked metabolic alterations were identified from O-PLS-DA pairwise models of the hepatic aqueous extract NMR spectral profiles as a result of TA and TAI administration relative to control profiles. An O-PLS-DA model of the hepatic aqueous extract NMR profiles that discriminated TAI treatment from vehicle controls 2 h postdosing is shown in Figure 3. The cross-validated scores (Tcv) of control and TAI-treated samples are very clearly separated for this robust model (Figure 3A; R2Y = 0.99, Q2Y = 0.88, permutation p value < 0.01). The loadings coefficient plot (Figure 3B,C) highlights the significant number of endogenous 2416



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variability in the TAI-treated cohort suggestive of a less uniform recovery in comparison to the TA-treated cohort. At 24 h post-treatment (Table 1A in the Supporting Information and Figure 5A), depletion of betaine and increased levels of glutamate and Choline/PCho were seen following both TA and TAI treatment, although the scale of these metabolic changes was greater for TAI treatment. Metabolic changes that were unique to TAI treatment included increased D-3-HB, creatine, and glycine and depleted levels of glycogen and glucose. A complete recovery to control hepatic levels was observed for hepatic glycogen, glucose, creatine, and D-3-HB following TA treatment in comparison to TAI treatment. Hepatic hypotaurine was depleted following TA treatment at 24 h. The log fold change plot captures the unique metabolic alterations induced by TA and TAI at 24 h post-treatment, with evidence of far greater metabolic perturbation in the TAItreated cohort in comparison to the TA-treated cohort (Figure 5A). An O-PLS regression model of the hepatic metabolic profiles 24 h post-treatment with the histopathologic glycogen score was computed to explore the pattern of hepatic metabolites that correlated with a traditional toxicological end point. The crossvalidated scores (Tcv) plot (Figure 6a) showed clear discrimination of control and TA-treated samples representing marked and moderate glycogen scores from the TAI-treated cohort that predominantly represented mild glycogen scores. The O-PLS regression model loadings coefficient plot showed a positive correlation of the histopathologic glycogen score with hepatic glycogen, glucose, and betaine together with a negative correlation with creatine (Figure 6B). Endogenous Plasma Metabolic Profiles Reflective of TA and TAI Treatment. At the 2 h post-treatment time point (Table 1B in the Supporting Information and Figure 5B), significant increases in plasma tyrosine and creatine were seen with concomitant reduction of alanine, acetate, and formate for both TA and TAI treatment groups relative to controls. Discriminatory metabolites that were unique to TAI treatment included increased plasma levels of citrate and a reduction in resonances arising from lipid moieties. At 2 h post-treatment, a statistically significant increase in the levels of phenylalanine and lactate was observed following TA treatment alone. At 6 h post-treatment (Table 1B in the Supporting Information and Figure 5B), an increase in creatine and phenylalanine was observed for both TA and TAI treatments. Metabolic alterations that were unique to TAI treatment included a continued increase in tyrosine and citrate, whereas an increase in formate was observed following TA treatment. At 24 h post-treatment (Table 1B in the Supporting Information and Figure 5B), the plasma metabolic profiles revealed dramatic alterations in metabolites for TAI treatment relative to controls or TA treatment. Metabolic changes that were common to both treatments included increased levels of phenylalanine, acetate, and formate, although the scale of the change in these metabolites was greater for TAI treatment (Table 1B in the Supporting Information and Figure 5B). Metabolic changes that were unique to TAI treatment included depleted levels of lactate and lipid moieties and increased levels of tyrosine, citrate, creatine and creatinine. Endogenous Urinary Metabolic Profiles Reflective of TA and TAI Treatment. Metabolic alterations in response to TA and TAI administration were evident from statistical modeling of the urinary NMR spectral profiles from 2 h posttreatment onward. The urinary spectral cohort represented

Figure 4. O-PLS-DA model discriminating TA and TAI aqueous soluble liver extract NMR spectral profiles 2 h post-treatment (Y-TA TAI represents a dummy matrix of zero for TAI and one for TA). (A) Cross-validated scores (Tcv) for TA (red, n = 5) and TAI (blue, n = 5) 2 h samples and (B) loadings coefficient plot for the aliphatic spectral region. The height of spectral peaks represents the covariance, and the color code corresponds to the coefficient of determination, r2. Model statistics: R2Y = 0.99, Q2Y = 0.62, and permutation p value ** < 0.01.

very clear separation of the scores (Figure 4A) for the TA- and TAI-treated groups representing the unique treatment-related metabolic profiles. The loadings coefficient plot (Figure 4B) showed that the TAI-treated animals had increased hepatic levels of creatine and glutamate, relative to TA treatment, whereas the TA-treated cohort had increased levels of hypotaurine relative to TAI. A summary of these discriminatory hepatic metabolic alterations and the associated statistical significance for each individual metabolite following TA or TAI treatment relative to controls at each time point post-treatment is given in Table 1A in the Supporting Information. A heatmap of the median log fold change of the discriminatory hepatic metabolites following TA and TAI treatment is provided in Figure 5A. The metabolic changes that were common to both treatments at 6 h post-treatment (Table 1A in the Supporting Information and Figure 5A) included increased hepatic levels of D-3-HB and creatine and depleted levels of glycogen relative to controls. The metabolic changes that were unique to TAI treatment included increased levels of choline/phosphocholine (Choline/PCho) and glutamate and depleted levels of glucose, lactate, betaine, and alanine. A metabolic change that was unique to TA treatment at 6 h post-treatment was a statistically significant depletion of the hepatic level of hypotaurine. Hepatic glutathione (oxidized and reduced) levels were seen to have recovered to control levels following both TA and TAI treatment at 6 h, albeit with a degree of interindividual 2417



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Figure 5. Log fold change heat maps for the discriminatory metabolites identified in (A) liver, (B) plasma, and (C) urinary 1H NMR spectral profiles of TA-treated and TAI-treated cohorts relative to control at 2, 6, and 24 h post-treatment. Each pixel represents the log-2 fold change of a metabolite integral (columns) in a single spectrum (rows, representing each animal) relative to the median peak integral in the time-matched control spectra. The metabolic changes that were insignificant based on an unpaired Mann−Whitney test p value have been set to zero. Key: Choline/PCho, choline/phosphocholine − NMe3 resonance; plasma lipid moieties, integrals of −(CH2)n, CH3, and CHCH resonances. Glutathione represents the integral of the resonance arising from both oxidized and reduced forms (δ 2.99).

treatments as evidenced on inspection of the log fold change heat map (Figure 5C). A statistically significant decrease in hippurate and NMNA was observed for both treatments, although the scale and uniformity of the change were far greater with respect to TAI treatment (Table 1C in the Supporting Information and Figure 5C). A significant recovery to control levels was observed for 2-OG, lactate, citrate, glucose, D-3-HB, and fumarate in the TA treated cohort. Urinary levels of 2-OG, citrate, and fumarate remained depleted in TAI-treated animals, with a unique observation of depleted levels of D-3-HB. At 24 h post-treatment, further unique TAI-induced changes included a dramatic increase in branched chain amino acids, namely, Val and alanine, and an increase in lactate. Multivariate statistical modeling also revealed significantly increased levels of Isoleu and Leu following TAI treatment at 24 h post-treatment.

temporal collection (0−2, 2−6, and 6−24 h) from the cohort of animals that were necropsied at 24 h post-treatment. At the 2 h post-treatment time point, increased levels of D-3HB and lactate and depleted levels of citrate, 2-oxoglutarate (2OG), fumarate, hippurate, and N-methylnicotinic acid (NMNA) were seen for both TA and TAI (Table 1C in the Supporting Information and Figure 5C). At 6 h post-treatment, a continued decrease in citrate, 2-OG, fumarate, hippurate, and NMNA together with a continued increase in lactate were seen for both TA and TAI treatment groups (Table 1C in the Supporting Information and Figure 5C). The scale and uniformity of the increase in lactate were greater following TA treatment in comparison to TAI treatment at 6 h (Figure 5C). An additional unique TA-induced change at 6 h posttreatment was the observation of increased urinary glucose and D-3-HB (Table 1C in the Supporting Information and Figure 5C). The urinary profiles reflecting the 24 h post-treatment cohort displayed marked differences for the respective TA and TAI

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DISCUSSION Comparative exposure and clearance of TA and TAI were determined from similar plasma concentrations for TA and TAI 2418



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metabonomics to generate urine, plasma, and liver metabolic profiles enabled the temporal metabolic response to both TA and TAI treatments to be elucidated. Unique TAI-induced metabolic changes were identified in urine, plasma, and liver, and these changes both preceded and coincided with the 24 h post-treatment plasma ALT activity increase and observation of a histopathologic lesion. With respect to multivariate modeling of metabolic profiles of hepatic tissue extracts, several endogenous metabolic alterations were identified following TA and TAI treatment. We observed depleted hepatic reserves of glycogen, glucose, and lactate and a concomitant increase in D-3-HB for TAtreated and TAI-treated animals at 2 h. However, with respect to TA-treatment, glucose and lactate had recovered to control levels by 6 h, and D-3-HB and glycogen had recovered to control levels at 24 h postdose. In contrast, hepatic glycogen, lactate, and glucose remained markedly depleted and D-3-HB elevated following TAI treatment at 6 and 24 h post-treatment, suggesting the progression of toxic insult rather than recovery. The continued and marked TAI-induced depletion of hepatic glycogen and glucose reserves suggested a sustained effect on hepatic energy metabolism and an increased demand on glycolysis and a compensatory shift in energy metabolism to lipolysis. The disturbance of hepatic metabolic homeostasis induced by TAI treatment was far greater than the comparative TA treatment as evidenced from the log fold change heatmap plots. The modeling of temporal data enabled the trajectory of metabolic response to be followed for both TA and TAI treatments relative to controls. With respect to TA treatment, the early hepatic metabolic consequences identified at 2 and 6 h post-treatment were found to return to control levels by 24 h post-treatment, reflecting recovery of metabolic homeostasis. In contrast, the hepatic metabolic effects of TAI treatment remained marked at 24 h post-treatment, which correlated with increased plasma ALT activity and histopathologic evidence of necrosis and inflammation. It must be noted that food intake and body weights were not recorded in this study, and future work that incorporates these data and includes a pair-feeding regimen would be optimal to determine if TA and TAI treatment alter dietary consumption. However, there is no evidence in the literature for a TA or TAI-induced effect on body weight, and given the comparative approach and identification of unique metabolic markers for each treatment, it is unlikely that dietary effects represent a confounding factor in data interpretation. An O-PLS regression model of the histopathologic glycogen score and the hepatic metabolic profiles at 24 h post-treatment enabled the metabolic signature of relevance to this particular histopathologic end point to be determined. This was of relevance and interest given the unique TAI-induced depletion of glycogen at 24 h post-treatment, which was not observed in the control or TA-treated cohorts. The differentiation of the TAI-treated cohort from the control and TA-treated cohort showed a positive correlation of the histopathologic glycogen score with hepatic levels of glycogen, glucose, and betaine. In addition, a negative correlation between the histopathologic glycogen score and creatine was identified. This approach highlights the ability to successfully integrate metabonomic data with a single histopathologic end point, in this instance to discriminate a specific panel of metabolites that were correlated with hepatic glycogen depletion. A metabolic change that was unique to TA treatment was the early 2 h increase in hepatic hypotaurine and subsequent

Figure 6. O-PLS regression model of 24 h post-treatment hepatic 1H NMR spectral profiles and histopathologic glycogen scores (zero representing absence; 1, mild; 2, moderate; and 3, marked hepatic glycogen stores). (A) Cross-validated scores (Tcv) of controls (red, n = 5), TA-treated (blue, n = 5), and TAI-treated (green, n = 6) cohorts and (B) loadings coefficient plot for the aliphatic spectral region. The height of spectral peaks represents the covariance, and the color code corresponds to the coefficient of determination, r2. Model statistics: R2Y = 0.98, and Q2Y = 0.83.

at 2 and 6 h post-treatment and the absence of TA and TAI at 24 h post-treatment. The maximal TA plasma concentration was lower than that reported in the literature following TA administration in man (single 250 mg oral dose, concentration range of ca. 15−25 μg/mL or 45−75 μM34−36). Indeed, the exposure to TA in rats has been shown to equate to 1/16th of a comparative dose in humans when administered orally, which is believed to result from lower absorption in rats.4,36 The diuretic action of both TA and TAI was also comparable with a 7- and 9-fold increase in urinary output, respectively, at 2 h post-treatment relative to controls, which supports the common pharmacological effect of these isomers. Clinical chemistry analyses found that TAI treatment resulted in a statistically significant increase in plasma ALT activity at 24 h post-treatment. This TAI-induced increase in plasma ALT activity paralleled the 24 h post-treatment histopathologic findings of TAI-induced hepatic necrosis and inflammation. No significant increase in plasma ALT activity was observed for the TA-treated or control cohorts, all of which lacked the TAI-induced histopathologic lesion. The clinical chemistry and histopathologic analyses were complemented through application of a nontargeted, NMRbased metabonomic approach. The application of NMR-based 2419



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depletion of hypotaurine at 6 and 24 h post-treatment. Increased hypotaurine levels have previously been identified following partial hepatectomy in the rat, in both liver37,38 and urine.37,39 The increase in hepatic hypotaurine following partial hepatectomy has been suggested to reflect an increased import of amino acids necessary for protein synthesis in the regenerating liver37 and potentially a reduction in the hepatic capacity for oxidation of hypotaurine to taurine.40 Hepatic hypotaurine levels were also increased following acute ethanol administration, which correlated with decreased hepatic glutathione, suggesting that cysteine was preferentially metabolized to hypotaurine in response to oxidative stress injury.41 Further evidence to support this is the observation that N-acetylcysteine administration favors formation of hypotaurine rather than glutathione in the liver of mice.42 Hypotaurine has also been shown to protect in vitro cellular systems from oxidative stress.43 The early increase in hepatic hypotaurine with TA treatment may represent a metabolic marker of hepatic metabolic adaptation, as its presence at 2 h post-TA administration was followed by a multivariate metabolic trajectory that suggested recovery of metabolic homeostasis. Indeed, TA has previously been shown to induce changes in genes involved in liver regeneration in a preclinical rodent model (100 mg/kg).44 The ability to detect a temporal metabolic phenotype that is suggestive of adaptation is of potential relevance with respect to clinical translation of this approach for differentiation of individuals who tolerate or adapt to drug-related homeostatic alterations from those who fail to adapt and progress to clinically significant liver injury from a particular drug treatment.45 It is possible that the absence of increased hepatic hypotaurine at 2 h post-treatment in the TAItreated cohort reflects an inability to adapt or the absence of this potentially protective response. This observation correlates with the marked and sustained effect of TAI on energy metabolism and the progression of hepatotoxicity. Further experiments are necessary to elucidate the mechanistic basis for the TA-induced increase in hepatic hypotaurine at 2 h posttreatment and to understand the time course of change and relevance to hepatic function. The increased plasma levels of the aromatic amino acid, tyrosine, following TAI administration at 2, 6, and 24 h, and the increased hepatic level at 2 h suggested impairment in hepatic protein synthesis and subsequent inhibition of tyrosine aminotransferase. Hypertyrosinemia has previously been seen following challenge with diversely acting hepatotoxicants such as galactosamine, thioacetaminde, ethionine, and isoniazid.26,46 The observation of decreased glutathione levels, representing total glutathione (both oxidized and reduced forms), in the aqueous hepatic extract profiles following both TA and TAI administration at the early time point (2 h post-treatment) supports the generation of electrophilic reactive metabolites of both TA and TAI. A recovery of hepatic total glutathione to control levels was observed following TA treatment at 6 h posttreatment. A recovery towards control levels was also observed following TAI treatment at 6 h; however, a degree of interanimal variability was observed, with complete recovery observed at the later 24 h post-treatment time point. This observation agrees with published literature showing a TAinduced decrease in hepatic reduced glutathione concentration at 3−6 h postdose and a compensatory increase at 24 h postdose.4 TA has also been shown to induce changes in genes involved in the response to oxidative stress4,44 and to covalently bind to diverse cellular proteins. 14

An increase in plasma and hepatic levels of creatine was observed following TAI treatment, particularly at the later time point of 24 h post-treatment. Increased plasma levels of creatine and hypercreatinuria have previously been identified for numerous hepatotoxicants, and increases have been proposed to be a useful marker of liver injury that may result from leakage from damaged hepatocytes.47 The marked depletion of plasma lipids observed following TAI treatment at 24 h post-treatment further suggests a significant effect of TAI on energy homeostasis and might have resulted from impaired hepatic lipogenesis and secretion of hepatic lipoproteins. Depletion of TCA cycle intermediates, 2-OG, fumarate, and citrate, was observed in the urine of TA- and TAI-treated cohorts at 2 and 6 h postdose. The urinary levels of these metabolites recovered to control levels for TA treatment at 24 h in comparison to TAI treatment, further suggestive of homeostatic recovery with TA treatment. The depletion of TCA cycle metabolites is commonly observed in urinary metabolic profiles following treatment with a wide range of toxicants and, although not agent-specific, suggests a disturbance of mitochondrial metabolism. The observation of increased plasma citrate was unique to TAI administration at 24 h, further suggestive of mitochondrial toxicity, which supports the evidence for impairment of hepatic energy metabolism following TAI treatment. The observation of increased urinary excretion of amino acids, namely, Val, Isoleu, Leu, and alanine, together with increased plasma levels of the ketogenic amino acids phenylalanine and tyrosine following TAI treatment at 24 h complement the increase in levels of the ketone body, D-3HB, and together further support perturbation of energy metabolism. The increased urinary excretion of these amino acids and lactate together with increased urinary output following TAI treatment at 24 h may also reflect renal tubular dysfunction; however, no histopathologic data were available for the kidney to support this possibility. In summary, the comparison of systems-wide metabolic profiles reflecting exposure to TA and TAI enabled unique metabolic changes to be identified for TAI treatment, which preceded or coincided with histopathologic evidence of a significant necrotic lesion. This approach proved powerful, as it enabled comparison with the metabolic effects of TA treatment, which were not associated with a necrotic lesion. In contrast to TAI, the TA-induced treatment effects on the endogenous metabolome were found to be largely reversible, and the unique observation of increased hepatic hypotaurine at 2 h posttreatment may represent a marker to assess the onset of a regenerative and adaptive hepatic response. The metabolic changes that were common to both agents were interpreted as thiophene-related effects; hence, this approach presented a means to delineate metabolic changes that were of significance with respect to the progression to overt hepatotoxicity. This comparative metabonomic study of closely related thiophene compounds represents a novel approach for rapidly identifying metabolic markers that are of relevance to the development of toxicity and for generating mechanistic hypotheses. This approach is of relevance for future application to study drugs with closely related structures and pharmacological activities to refine identification of mechanisms and biomarkers of toxicity. 2420



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(8) Nishiya, T., Kato, M., Suzuki, T., Maru, C., Kataoka, H., Hattori, C., Mori, K., Jindo, T., Tanaka, Y., and Manabe, S. (2008) Involvement of cytochrome P450-mediated metabolism in tienilic acid hepatotoxicity in rats. Toxicol. Lett. 183 (1−3), 81−89. (9) Bonierbale, E., Valadon, P., Pons, C., Desfosses, B., Dansette, P. M., and Mansuy, D. (1999) Opposite behaviors of reactive metabolites of tienilic acid and its isomer toward liver proteins: Use of specific antitienilic acid-protein adduct antibodies and the possible relationship with different hepatotoxic effects of the two compounds. Chem. Res. Toxicol. 12 (3), 286−296. (10) Dansette, P. M., Amar, C., Valadon, P., Pons, C., Beaune, P. H., and Mansuy, D. (1991) Hydroxylation and formation of electrophilic metabolites of tienilic acid and its isomer by human liver microsomes. Catalysis by a cytochrome P450 IIC different from that responsible for mephenytoin hydroxylation. Biochem. Pharmacol. 41 (4), 553−560. (11) Mansuy, D. (1997) Molecular structure and hepatotoxicity: Compared data about two closely related thiophene compounds. J. Hepatol. 26 (Suppl. 2), 22−25. (12) Lopez-Garcia, M. P., Dansette, P. M., and Coloma, J. (2005) Kinetics of tienilic acid bioactivation and functional generation of drug-protein adducts in intact rat hepatocytes. Biochem. Pharmacol. 70 (12), 1870−1882. (13) Dansette, P. M., Amar, C., Smith, C., Pons, C., and Mansuy, D. (1990) Oxidative activation of the thiophene ring by hepatic enzymes. Hydroxylation and formation of electrophilic metabolites during metabolism of tienilic acid and its isomer by rat liver microsomes. Biochem. Pharmacol. 39 (5), 911−918. (14) Koen, Y. M., Sarma, D., Williams, T. D., Galeva, N. A., Obach, R. S., and Hanzlik, R. P. (2012) Identification of Protein Targets of Reactive Metabolites of Tienilic Acid in Human Hepatocytes. Chem. Res. Toxicol. 25 (5), 1145−1154. (15) Rademacher, P. M., Woods, C. M., Huang, Q., Szklarz, G. D., and Nelson, S. D. (2012) Differential Oxidation of Two ThiopheneContaining Regioisomers to Reactive Metabolites by Cytochrome P450 2C9. Chem. Res. Toxicol. 25 (4), 895−903. (16) Nicholson, J. K., Holmes, E., Lindon, J. C., and Wilson, I. D. (2004) The challenges of modeling mammalian biocomplexity. Nat. Biotechnol. 22 (10), 1268−1274. (17) Eliasson, M., Rannar, S., and Trygg, J. (2011) From data processing to multivariate validationEssential steps in extracting interpretable information from metabolomics data. Curr. Pharm. Biotechnol. 12 (7), 996−1004. (18) Trygg, J., Holmes, E., and Lundstedt, T. (2007) Chemometrics in metabonomics. J. Proteome Res. 6 (2), 469−479. (19) Coen, M., Holmes, E., Lindon, J. C., and Nicholson, J. K. (2008) NMR-based metabolic profiling and metabonomic approaches to problems in molecular toxicology. Chem. Res. Toxicol. 21 (1), 9−27. (20) Lindon, J. C., Holmes, E., and Nicholson, J. K. (2006) Metabonomics techniques and applications to pharmaceutical research & development. Pharm. Res. 23 (6), 1075−1088. (21) Nicholson, J. K., Connelly, J., Lindon, J. C., and Holmes, E. (2002) Metabonomics: A platform for studying drug toxicity and gene function. Nat. Rev. Drug Discovery 1 (2), 153−161. (22) Patterson, A. D., Gonzalez, F. J., and Idle, J. R. (2010) Xenobiotic metabolism: A view through the metabolometer. Chem. Res. Toxicol. 23 (5), 851−860. (23) Robertson, D. G., Watkins, P. B., and Reily, M. D. (2011) Metabolomics in toxicology: Preclinical and clinical applications. Toxicol. Sci. 120 (Suppl. 1), S146−S170. (24) Holmes, E., Bonner, F. W., Sweatman, B. C., Lindon, J. C., Beddell, C. R., Rahr, E., and Nicholson, J. K. (1992) Nuclear magnetic resonance spectroscopy and pattern recognition analysis of the biochemical processes associated with the progression of and recovery from nephrotoxic lesions in the rat induced by mercury(II) chloride and 2-bromoethanamine. Mol. Pharmacol. 42 (5), 922−930. (25) Coen, M., Goldfain-Blanc, F., Rolland-Valognes, G., Walther, B., Robertson, D. G., Holmes, E., Lindon, J. C., and Nicholson, J. K. (2012) Pharmacometabonomic investigation of dynamic metabolic

ASSOCIATED CONTENT

S Supporting Information *

Table of metabolic changes identified in liver extracts, plasma, and urine following administration of TA and TAI. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Funding

The MRC Integrative Toxicology Training Partnership (ITTP) is gratefully acknowledged for financial support to M.C. through a Career Development Fellowship. The NIH is gratefully acknowledged for financial support to S.D.N. (F33 GM089052) and to R.R. (R01 DK087886). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Professors Jeremy Nicholson and Elaine Holmes, Biomolecular Medicine, Department of Surgery and Cancer, Imperial College London, are acknowledged for hosting Professor Sidney Nelson's NIH Fellowship and for access to high-field NMR spectroscopic facilities. Mr Steven Robinette, Biomolecular Medicine, is acknowledged for assistance with in-house software and figure design.

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ABBREVIATIONS Ctrl, control; TA, tienilic acid; TAI, tienilic acid isomer; NMR, nuclear magnetic resonance; D-3-HB, D-3-hydroxybutyrate; 2OG, 2-oxoglutarate; NMNA, N-methylnicotinic acid; ALT, alanine aminotransferase; GSSG, oxidized gluathione; Choline/ PCho, choline/phosphocholine; Isoleu, isoleucine; Leu, leucine; Val, valine; CPMG, Carr−Purcell−Meiboom−Gill; FT, Fourier transformation; PCA, principal components analysis; PLS-DA, partial-least-squares-discriminant analysis; O-PLS-DA, orthogonal projection on latent structures discriminant analysis; SEM, standard error of mean

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Comparative NMR-Based Metabonomic Investigation of the Metabolic

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