Metabonomic Evaluation of Melamine-Induced Acute Renal Toxicity in

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Metabonomic Evaluation of Melamine-Induced Acute Renal Toxicity in Rats Guoxiang Xie,†,‡,# Xiaojiao Zheng,§,# Xin Qi,§ Yu Cao,§ Yi Chi,§ Mingming Su,§ Yan Ni,§ Yunping Qiu,§ Yumin Liu,§ Houkai Li,§ Aihua Zhao,§ and Wei Jia*,† Department of Nutrition, University of North Carolina at Greensboro, North Carolina Research Campus, Kannapolis, North Carolina, 28081, and Shanghai Center for Systems Biomedicine and School of Pharmacy, Shanghai Jiao Tong University, Shanghai, 200240, P. R. China Received April 13, 2009

Abstract: The recent outbreak of renal failure in infants in China has been determined to be caused by melamine (Mel) and derivatives adulterated in the food. A metabonomic study was performed to evaluate the global biochemical alteration triggered by Mel ingestion in parallel with the acute renal toxicity in rats. Mel at 600, 300, and 100 mg/kg, cyanuric acid (Cya) at 100 mg/kg, and mixture of Mel and Cya (50 mg/kg each) were administered in five groups of Wistar rats, respectively, via oral gavage for 15 days. Urinary metabonomic profiles indicated that Mel perturbed urinary metabolism in a dose-dependent manner, with high-dose group showing the most significant impact. Metabonomic variations also suggest that the toxicity of low-dose (50 mg/kg) Mel was greatly elevated by the presence of Cya (at 50 mg/kg), which was able to induce a significant metabolic alteration to a level equivalent to that of 600 mg/kg Mel. Histological examination and serum biochemical analysis also indicated that the low-dose Mel-Cya mixture and high-dose Mel group resulted in the greatest renal toxicity. The high-dose Mel and low-dose Mel-Cya resulted in disrupted amino acid metabolism including tryptophan, polyamine, and tyrosine metabolism, and altered TCA and gut microflora structure. Keywords: metabonomics • melamine • cyanuric acid • urine • nephrotoxicity • ultraperformance liquid chromatography/time-of-flight mass spectrometry • multivariate statistical analysis

Introduction In 2004, the outbreak of renal failure in dogs linked to ingestion of specific commercial dog foods occurred in Asia.1 In 2007, there have been a number of dogs and cats which suffered renal failure2 in North America with identical histological and toxicological findings to those in 2004 after eating certain batches of wet pet food. These events were followed by an intensive recall of pet foods in North America 3 years * To whom correspondence should be addressed. E-mail: [email protected]. † University of North Carolina at Greensboro. ‡ Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University. § School of Pharmacy, Shanghai Jiao Tong University. # These authors contributed equally to this work. 10.1021/pr900333h

 2010 American Chemical Society

later. Investigations of pet foods involved revealed melamine (Mel)3 and one of its derivatives, Cyanuric acid (Cya), to have been present as an adulterant.4,5 Over the course of this investigation, it came to light that Mel was added intentionally to raise the apparent protein content of the pet food ingredients labeled wheat gluten or rice protein because of its high nitrogen content.6,7 In September 2008, renal crystals and associated renal failure were found among infants and children in China due to the consumption of the milk powder containing Mel, leading to nearly 53 000 sick individuals, with more than 12 800 hospitalizations and four infant deaths, by September 22.8,9 Mel (Supplemental Data Figure 1A), used for adulteration in foods, is an organic base and a trimer of cyanamide.10 The level of Mel found in pet foods was about 70 ppm and in dairy products from 0.09 to 2560 ppm (1 mg/kg).11 The compound is used to produce a wide range of brightly colored dishware, bowls and containers, and to create utensils and a resin coating for wood and textiles. It has also been used as fertilizer for crops during the 1950s and 1960s,7 and as nonprotein nitrogen (NPN) for cattle described in a 1958 patent.12 Mel is usually accompanied with several related compounds, all byproduct from the manufacturing of Mel, namely, ammeline (4,6-diamino5H-1,3,5-triazine-2-one), ammelide (6-amino-1H-1,3,5-triazine2,4-dione), and Cya (1,3,5-triazine-2,4,6-trione, Supplemental Data Figure 1B).4 These compounds are also in vivo microbial metabolites of Mel.13,14 The toxicity of Mel and Cya has been extensively evaluated1,3,15-17 and recent research showed that Mel and Cya yielded extensive gold-brown renal “stones” in radical spheres when administered together3 in fish and pigs. This type of “gold-brown” stone is consistent with crystals of Mel-Cya complex that can be prepared in the laboratory,1,3 which has also been observed in cats1,15 and other species when dosed with Mel or Mel-Cya mixutre.1,3 Reports from the nutrition literature indicated that feeding ammeline to sheep at a dose of approximately 100 mg/(kg/day) produced kidney stones after several weeks.18 The formation of inorganic crystals in kidney tubules has been believed as the mechanism by which Mel and related compounds caused kidney failure in infants and pets which triggered the large-scale pet food and baby formula recall. Metabonomics, an emerging -omics approach using nuclear magnetic resonance (NMR) spectroscopy or hyphenated gas chromatography/liquid chromatography-mass spectrometry (GC-MS or LC-MS) technologies, has been successfully used Journal of Proteome Research 2010, 9, 125–133 125 Published on Web 05/28/2009

technical notes

Xie et al. 19-21

for the evaluation of drug toxicity and nephrotoxicity induced by plant extracts.22,23 The technology provides quantitative information of holistic and time-dependent metabolic variation in response to xenobiotic interventions that is complementary to organ-specific biochemical and histological findings. The purpose of this study was to evaluate the global change of metabonome and, thus, understand the pathophysiological outcome in parallel to the acute renal toxicity in Wistar rats upon Mel ingestion at various doses.

Experimental Section Regents and Materials. Mel and Cya were obtained from Sigma-Aldrich (St. Louis, MO). Formaldehyde and sodium carboxymethycellulose (CMC-Na) were of analytical grade (China National Pharmaceutical Group, Shanghai, China). A 10% formalin solution was prepared from formaldehyde. CMCNa served as suspending agent to drug and was prepared in ultrapure water (Milli-Q SP water purification System, 18.2 MΩ, Millipore, Billerica, MA) at a concentration of 1%. Dosing and Sample Collection. The handling of all animals in this study was conformed to the national guidelines and performed at the Center for Laboratory Animals, Shanghai Jiao Tong University, Shanghai, China. A total of 42 male Wistar rats (80 ( 5 g), 4 weeks old, were purchased from the Shanghai Laboratory Animal Co. Ltd. (SLAC, Shanghai, China). After acclimation of 1 week in metabolic cages, these animals were randomly divided into six groups: the high dose Mel group (n ) 7), in which a daily dose of 600 mg/kg of body weight (equivalent to 1/5 of its reported LD5024) of Mel was administrated; middle dose Mel group (n ) 7), at 300 mg/kg; low-dose Mel group (n ) 7) at 100 mg/kg; low-dose Cya group (n ) 7) at 100 mg/kg; (Mel-Cya) group (n ) 7), dosed with a mixture of 50 mg/kg of Mel and 50 mg/kg of Cya; and the control group (n ) 7). Mel and Cya were administered to all treatment groups daily in 1% CMC-Na suspension via oral gavage for 15 consecutive days, and solution of 1% CMC-Na was given to control group. All experimental rats were fed with standard rat chow and received water ad libitum throughout the experiment, during which the food and water consumption was carefully recorded. The light cycle consisted of 12 h light and 12 h dark; the temperature was maintained at 20-22 °C, and the humidity between 45% and 65% throughout the study. An 18-h urine sample was collected from each rat between drug administration (at 20:00 p.m.) and 14:00 next day for a total of 19 days including four predose time points. A blood sample was drawn from the ocular orbit before all experimental rats were sacrificed at day 16. Sera obtained from blood samples were analyzed using a Hitachi 7600 automated analyzer to determine blood urea nitrogen (BUN), serum creatinine and serum uric acid. Urine samples were also evaluated for the presence of crystals. Histological Examination. Kidney tissues were obtained from the right kidney and fixed in neutral-buffered 10% formalin pending section and embedded in paraffin wax. A 3-µm histological section of the paraffin-embedded kidney tissue for each rat was stained with hematoxylin-eosin for histological analysis with Leica DMRE Microsystems equipment with SPOT FLEX Microscope Digital Camera at the Laboratory of Cell Biology and Tissue Pathology of Shanghai University of Traditional Chinese Medicine (Shanghai, China). Urine Sample Preparation. Rat urine samples were collected and immediately centrifuged at 13 000 rpm (15 700g) for 10 126

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min. The resulting supernatants were immediately stored at -80 °C pending metabonomic analysis. Analysis of Urine. Ultrapure water (500 µL) was added to urine sample (500 µL), after the sample was thawed and vortexed for 1 min, and then filtered through a syringe filter (0.22 µm) for analysis. A Waters ACQUITY UPLC system coupled with a tandem quadrupole-time-of-flight (Q-TOF) mass spectrometer equipped with a binary solvent delivery manager, a sample manager, and an electrospray interface (Waters Corporation, Milford, MA). Chromatographic separations were performed on a 2.1 × 100 mm 1.7 µm ACQUITY BEH C18 column. The column was maintained at 45 °C and eluted with a 1-99% acetonitrile (0.1% (v/v) formic acid)aqueous formic acid (0.1% (v/v) formic acid) gradient over 12 min at a flow rate of 0.40 mL/min. A 5-µL aliquot sample was injected into the column. UPLC-MS analysis was performed on a Micromass Q-TOF Premier (Waters, Manchester, U.K.) operating in positive ion electrospray. The mass accuracy analysis and detailed MS parameters were optimized according to our previously reported protocols.25 Nitrogen (N2) was used as the desolvation gas. The desolvation temperature was set to 350 °C at a flow rate of 600 L/h and source temperature of 120 °C. The capillary and cone voltages were set to 3200 and 45 V, respectively. The Q-TOF premier was operated in v mode with 10 000 mass resolving power. During metabolite profiling, centroid data were acquired for each sample from 50 to 1000 Da with a 0.10 s scan time and a 0.01 s interscan delay over a 12 min run time. Data Processing and Statistical Analysis. The UPLC-QTOFMS data of the urine samples were analyzed to identify potential discriminant variables. The ES positive (ES+) raw data were analyzed by the MarkerLynx applications manager version 4.1 (Waters, Manchester, U.K.) using parameters reported in our previous work.25 The parameters used were retention time (RT) range 0-12 min, mass range 50-1000 Da, mass tolerance 0.02 Da; internal standard detection parameters were deselected for peak retention time alignment; isotopic peaks were excluded for analysis; noise elimination level was set at 10.00; minimum intensity was set to 15% of base peak intensity; maximum masses per RT was set at 6 and, finally, RT tolerance was set at 0.01 min. The resulting 3-D matrix containing arbitrarily assigned peak index (retention time-m/z pairs), sample names (observations), and normalized peak area were exported to SIMCA-P software 11.0 (Umetrics, Umea˚, Sweden) for multivariate statistical analysis. The multivariate statistical analysis was performed using the method previously reported.25-27

Results and Discussion General Information about the Animal Experiment. As indicated in Supplemental Data Figure 2A-D, the body weight of the rats increased steadily during the experiment and there is no statistically significant difference among the six groups. Food consumption decreased slightly in the Mel-Cya group and the high-dose Mel group. The Mel-Cya, high-dose Mel, and middle-dose Mel groups consumed more water than the other groups, and as a result, the volume of urine excreted in these three groups was greater than that in the other groups. Blood tests showed an increase, although not statistically significant, in blood urea nitrogen (BUN) and serum creatinine levels in several treatment groups. The mean values of BUN and serum creatinine level of the Mel-Cya group (at a dose of 50 mg/kg Mel and 50 mg/kg of Cya) and high-dose Mel group (600 mg/kg) were the greatest among the treatment group,

technical notes

Metabonomic Evaluation of Mel-Induced Renal Toxicity in Rats Table 1. Effects of Mel on Kidney and Renal Function in Rats treatment

Control Low-dose Mel Middle-dose Mel High-dose Mel Low-dose Cya Low-dose Mel-Cya a

kidney weight (g) kidney/body weight (%) hemorrhage crystal BUN (mmol/L) serum creatinine (µmol/L) serum uric acid (µmol/L)

1.65 ( 0.14 1.69 ( 0.07 1.59 ( 0.09 1.90 ( 0.15 1.85 ( 0.17 1.80 ( 0.52

No hemorrhage was found.

b

0.86 ( 0.09 0.86 ( 0.02 0.81 ( 0.07 1.01 ( 0.09 0.92 ( 0.05 0.94 ( 0.31

–a –a mild severe slight severe

–b +c +c +c +c +c

4.81 ( 0.82 4.01 ( 0.56d 4.22 ( 0.42e 6.50 ( 2.92 4.23 ( 0.51 8.34 ( 5.07

No crystal deposition was found. c Crystal deposition was observed.

compared to the control group, suggesting the highest degree of renal impairment16,28 (Table 1) in these two groups. Serum uric acid levels were lower in most of the treatment groups compared to the control, which is different from the other reports of renal failure studies,29,30 presumably due to the increased urine excretion in the treatment groups (Supplemental Data Figure 2). The Mel-Cya group has the highest mean value of serum uric acid, which is greater than the control, suggesting that the renal impairment of this group is more severe than others despite of the fact that they excreted the highest volume of urine. At necropsy, the kidneys of the treatment groups were found to be edematous, with organ weight higher than those in the control group (Table 1). Histological examinations reveal that the Mel-Cya and high-dose Mel group showed extensive tubular dilatation in distal tubules and a small amount of hemorrhage, while the renal glomeruli were normal or not different from those of the control group. Crystal deposition in collecting ducts of renal papilla and inflammation in renal interstitium were also observed in these two groups. These observations were consistent with the previous reports;1 therefore, histological figures were not provided in this report. The hemorrhage was severe in the Mel-Cya group. Bleeding was found in the high-dose Mel group and crystals were found near the papilla of the renal tubule in all treatment groups but was more significant in high-dose Mel and Mel-Cya group (Table 1). These observations were in good agreement with blood test results. UPLC-QTOF-MS Analysis of Urine Samples. The UPLCQTOF-MS procedure in the ES+ mode was able to detect a significantly greater number of urinary metabolites than the ES negative mode, as revealed by the representative BPI spectra, due to higher degree of ion fragmentation. Therefore, the fullscan detection was set as positive ion mode in our analytical procedure. The desolvation gas flow, desolvation temperature and cone voltage were optimized for the molecular ions [M + H]+ on the mass spectrum. Figure 1 is the UPLC-QTOF-MS base peak intensity (BPI) chromatogram of urine samples of the control group (Figure 1A), the low (1B), middle (1C), high-dose Mel group (1D), the Cya group (1E) and the Mel-Cya group (1F). It appeared, through visual comparison of these chromatograms, the lowdose Mel group and the Cya group, both at 100 mg/kg, share very similar metabolic profiles, while the Mel-Cya group at a dose of (50 + 50) mg/kg, shares similar metabolic profile with the high dose (600 mg/kg) Mel group. This suggests that the toxicity of low-dose Mel is greatly increased by the presence/ addition of (low-dose) Cya. The typical Mel peak ion (m/z 127.0732, RT 0.7-0.8 min) can be found in the urine sample of all Mel groups and Mel-Cya group (Supplemental Data Figure 3). This is consistent with the previous report that Mel is not metabolized in vivo but excreted in the urine in its

13.45 ( 4.37 16.00 ( 0.82 13.46 ( 3.02 17.56 ( 9.31 15.71 ( 2.75 22.00 ( 12.66 d

143.65 ( 45.12 117.00 ( 26.72 115.89 ( 29.86d 112.79 ( 23.77d 106.08 ( 24.79 153.48 ( 70.93

p < 0.05. e p < 0.001.

original form when administrated to rats.31 To detect more treatment-related metabolic alterations, pattern recognition techniques were applied and the peak ion of Mel was removed from the spectrum in order to avoid the Mel peak interference. Principal component analysis (PCA) was performed to differentiate distinct urinary metabolite profiles of all groups (Figure 2 and Supplemental Data Figure 4). The trajectories of the metabolic profiles at different time points illustrated the time-dependent alteration of urine metabolites of the six groups (Figure 2 and Supplemental Data Figure 4), where all the treatment groups clustered together with Control group at the beginning and gradually moved apart. The trajectories of the two low-dose groups, Mel group and Cya group are the nearest to the Control group, suggesting the minimum metabolic changes occurred during the treatment, while Mel-Cya and high-dose Mel groups are the furthest, reflecting the greatest global metabolic alteration in the two groups during the treatment. The degrees of metabolomic alterations are in good agreement with histological status of kidney toxicity in these groups. PCA scores were plotted based on the urinary metabolites of the healthy control and high-dose Mel group on day 15 (Supplemental Data Figure 5A), which demonstrate a clear intergroup separation. A three-component partial least-squaresdiscriminant analysis (PLS-DA) model (Figure 3, top panel) (R2X ) 0.452, R2Y ) 0.999, Q2Y ) 0.896) was subsequently constructed to identify the differential metabolites contributing to the separation of the metabolic patterns of the control group and high-dose Mel treated group. The corresponding loadings plot (Figure 3, bottom panel) indicated increased urinary expression of 4-hydroxy-L-proline, nicotinate, spermidine, indole-3-acetaldehyde, urea, and tyramine, and decreased expression of indole-3-acetic acid, 5-hydroxytryptophan, N-acetyl-5-hydroxytryptamine, guanidoacetate, tyrosine and N-acetyl-L-tyrosine. The trajectories of the above metabolites at different time points from day 0 to day 15 are shown in Figure 4. A list of the most significant differential metabolites in high-dose Mel group and in Mel-Cya group (compared to the control group) is summarized in Table 2 and Supplemental Data Table 1 and 2. A group of differential metabolites (Supplemental Data Table 2) accountable for the separation of control group and Mel-Cya group was also identified through the corresponding loadings plot (figures were not given) derived from a three-component PLS-DA model (R2X ) 0.460, R2Y ) 0.998, Q2Y ) 0.671). Effect of High-Dose Mel Intake. Approximately 8000 peaks (defined by a pair of m/z value and RT) were obtained from each urine sample using the method reported in our previous work.25 Although only a fraction of these peaks are selected as differential metabolites, and subsequently identified, the information obtained from the metabonomic analysis is important to elucidate the pathophysiological changes as a result of high-dose Mel intervention. The unsupervised pattern recogniJournal of Proteome Research • Vol. 9, No. 1, 2010 127

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Figure 1. Comparison of UPLC-QTOF-MS base peak intensity (BPI) chromatograms of urine samples collected from (A) control group; (B) low-dose Mel group at 100 mg/kg; (C) middle-dose Mel group at 300 mg/kg; (D) high-dose Mel group at 600 mg/kg; (E) the Cya group at 100 mg/kg; and (F) the Mel-Cya group at (50 + 50) mg/kg.

tion method, PCA, was able to visualize the degree of metabolic alterations among different treatment groups. The PCA scores plot (Supplemental Data Figure 5A) could readily divide the Mel treatment groups into four clusters: healthy control, lowdose Mel, middle-dose Mel and high-dose Mel group. A threecomponent PLS-DA model (Figure 3, top panel) was used to identify the metabolites accountable for the differences between the control group and high-dose Mel group on day 15. A total of 44 differential metabolites were determined based on the mass of molecular and fragment ions, 20 of which were further validated using reference standards available in our laboratory. The metabolites most significantly perturbed in the 128

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Mel group are listed in Supplemental Data Table 1 along with the variable importance parameter (VIP) and p (Kruskal-Wallis) values. The significantly perturbed metabolic pathways mainly involve amino acid metabolism, such as tryptophan, polyamine, and tyrosine metabolism (Figure 5). Other metabolic changes observed involve TCA cycle and alteration of gut microbiota structure. Nicotinate, produced by tryptophan via the kynurenine pathway, significantly increased in the high-dose Mel group. The rate-limiting enzyme of kenurenine pathway, tryptophan 2,3-dioxygenase indole 2,3-dioxygenase (IDO),32 could be activated in different cell types during inflammation, leading

Metabonomic Evaluation of Mel-Induced Renal Toxicity in Rats

technical notes

Figure 2. Time-dependent trajectories of urinary metabolic profiles (scores plot of PC1 vs PC2, data were normalized) of control group, low-dose Mel group, middle-dose Mel group, high-dose Mel group, Mel-Cya group, and Cya group from day 0 to day 15.

Figure 3. The scores plot (top panel) and loading plot (bottom panel) of the PLS-DA model of the UPLC-QTOF-MS spectral data from the high-dose Mel group and control group on day 15.

to tryptophan depletion.33 The observed up-regulation of kynurenine pathway may be a consequence of cell injury in

the Mel treated rats, which was consistent with the observations of the animals dosed with Mel.1 Decreased urinary excretion Journal of Proteome Research • Vol. 9, No. 1, 2010 129

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Figure 4. Trajectories of six representative differential metabolites perturbed by the high-dose Mel (compared to control group) from day 0 to day 15.

of 5-hydroxytryptophan and N-acetyl-5-hydroxytryptamine were observed, indicating the down-regulated tryptophan metabolic pathway in the high-dose Mel group. Several important urinary metabolites that participate in polyamine metabolic pathway were found significantly altered in high-dose Mel group, including increased spermidine, 4-hydroxy-L-proline, and decreased 4-aminobutyraldehyde. Polyamines are commonly involved in many diverse cellular and physiological processes, such as cellular growth and differentiation, regulation of nucleic acid and protein synthesis. The disturbed polyamine metabolism indicated the abnormal tissue development associated with the Mel-induced toxicity.34 In addition, one product of arginine in urea cycle, guanidoacetate, was found decreased in our study. Urea cycle is highly related with polyamine metabolism; such disturbance could reflect the impaired renal function.35,36 130

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It was also observed that high-dose Mel altered the urinary excretion levels of phenylacetylglycine and trimethylamine oxide (TMAO), all of which are believed to be products of the gut microflora co-metabolization.37,38 This suggests that there is a significant involvement of gut flora in the Mel-induced metabolic alteration. Furthermore, the early elevation in urinary TMAO was suggested to be a marker of site-specific renal papillary injury in the rat.39 The important contribution of polyamines to a balanced gut function has been previously reported,40 in which polyamine-synthesis inhibitors could disrupt intestinal development in mice41,42 which supports our findings of the alteration gut microflora related metabolites. Comparison of Mel-Cya Mixture with High-Dose Mel. A total of 32 most significantly altered metabolites (Supplemental Data Table 2) in Mel-Cya group on day 15 were identified from a three-component PLS-DA model, determined based on the

technical notes

Metabonomic Evaluation of Mel-Induced Renal Toxicity in Rats

Table 2. Summary of the Differential Metabolites from VIP Values of Three Component PLS-DA Model in the High-Dose Mel Group and Low-Dose Mel-Cya Group at day 15 Compared to the Healthy Control Groupa metabolic pathway

metabolites identified

Tryptophan metabolism

2-Amino-5-hydroxybenzoatec 5-hydroxyindole-3-acetaldehydeb Indoleacetaldehydec 5-Hydroxytryptophanc N-acetyl-5-hydroxytryptamineb Nicotinateb Melatoninc 6-hydroxymelatoninc Nicotinamideb N-acetyl-L-tyrosineb Tyramineb Tyrosinec Dopab b L-arginine Ureab Spermidineb Guanidoacetateb Adeninec 4-aminobutyraldehydec 4-hydroxy-L-prolineb cis-Aconitatec R-Ketoglutaric acidb Citric acidb Phenylacetylglycinec Trimethylamine oxideb 3-Phenylpropionatec Cystinec Histidineb N-methyl-L-histidineb 2-Phosphoglyceratec Taurinec Homoserinec b L-isoleucine Dimethylglycineb Leukotriene E4c Dihydrothyminec Leukotriene A4c Pyridoxalb Pipecolic acidc 3-Methyluridinec 1-methylguaninec (S)-3-hydroxyisobutyratec Riboflavin (Vitamin B2)c Deoxy ADP (dADP)c Thymidineb 3-indolepropionic acidb Uracilb Adenosineb Hypoxanthineb Xanthineb Uric acidb S-(5′-adenosyl)-L-methionine p-toluenesulfonate saltb 3′,5′-cyclic adenosineb b D-(-)-Ribose b L-Threitol Pyridoxine (B6)b

Tyrosine metabolism

Arginine metabolism

TCA cycle Gut microflora Others

Mel-Cya group

V v V

high-dose Mel group

v V v V V v V V

V v V V V v V

v

v

v

V V v V v V v v V v V V v V v v V

V v V v v V v V v v v v V v v v v V v v v v V v V V v V V V v V v V v V

V V

a

b

The upward arrow (v) represents significantly elevated concentration, whereas the downward arrow (V) represents significantly lowered concentration. Metabolites identified by reference standards. c Metabolites identified by accurate mass measurement.

mass of molecular and fragment ions, among which 23 were further validated by reference standards available in our laboratory. These metabolites are listed in Supplemental Data Table 2 along with the variable importance parameter (VIP) and p (Kruskal-Wallis) values. A summary of 56 differential metabolites identified in the high-dose Mel group and Mel-Cya group at day 15 is provided in Table 2. These differential metabolites were generated by a multivariate statistical comparison between the dosed group and the healthy control group using a PLS-DA model. The two dosed groups, high-dose Mel group and Mel-Cya group, share 20 of the total of 56 differential metabolites, all of which have the same direction of perturbation (up- or down-regulation). All the differential

metabolites are mainly involved in several metabolic pathways, primarily, tryptophan, polyamine, and tyrosine metabolism and altered gut microflora structure. In conclusion, we applied an MS-based metabonomics approach to investigate the global biochemical alteration along with the Mel-induced acute renal toxicity in Wistar rats. Histological examinations of kidney specimen revealed acute renal lesion along with the formation of crystals occurring in all the treatment groups. The combined experimental results of metabolomics, serum biochemical analysis and histology indicated that Mel at all doses induced significant renal toxicity in a dose-dependent manner in rats, and that the mixture of Mel and cyanuric acid (50 mg + 50 mg/kg) and 600 mg of Mel/ Journal of Proteome Research • Vol. 9, No. 1, 2010 131

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Figure 5. The perturbed metabolic pathways in response to high dose Mel treatment: (A) Tryptophan-related metabolite changes; (B) Arginine-related metabolite changes; (C) Tyrosine-related metabolite changes. Dark square denotes an elevated concentration of metabolites present in the urine, whereas gray square denotes a decreased level of metabolites as compared to the healthy control.

kg resulted in the greatest renal toxicity and physiological alteration. The high-dose Mel (600 mg/kg) and low-dose MelCya perturbed the urinary expression of different metabolites, respectively, but impacted the same metabolic pathways, including tryptophan, polyamine, and tyrosine metabolism and alteration in the structure of gut microflora. Abbreviations: UPLC-QTOFMS, ultraperformance liquid chromatography/time-of-flightmassspectrometry;Mel,melamine; Cya, cyanuric acid; CMC-Na, sodium carboxymethycellulose; BUN, blood urea nitrogen; PCA, principal component analysis; PLS-DA, partial least-squares-discriminant analysis; VIP, variable importance parameter.

Acknowledgment. This work was financially supported by the International Collaborative Project of Chinese Ministry of Science and Technology (2006DFA02700) and Team of Research Excellence Award, the Science and Technology Commission of Shanghai Municipality (06DZ05906). Supporting Information Available: Summary of the differential metabolites from three component PLS-DA model in the high-dose Mel group at day 15, compared to the healthy control group; summary of the differential metabolites from three component PLS-DA model in the Mel-Cya group at day 15, compared to the healthy control group; structures of melamine and cyanuric acid; variations of the body weight, 132

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quantity of water consumption, quantity of food consumption, and the volume of urine excreted; urinary excretion of Mel (mean ( SD, in % of dose) in high-dose Mel group; timedependent trajectories of urinary metabolic profiles (scores plot of PC1 vs PC2 with each error bar, data were normalized) of control group, low-dose Mel group, middle-dose Mel group, high-dose Mel group, Mel-Cya group, and Cya group from day 0 to day 15; scores plot (t1 vs Num) generated from PLS-DA model of urinary metabolic profiles of control group, low-dose Mel group, middle-dose Mel group, and high-dose Mel group at day 15; trajectories of urinary metabolic profiles (PC1 vs PC2 scores plot, data normalized) of control group and high-dose Mel group from day 0 to day 15; scores plot (t1 vs Num) generated from PLS-DA model of urinary metabolic profiles of control group, low-dose Mel group, middle-dose Mel group, high-dose Mel, low-dose Mel-Cya group and low-dose Cya group at day 15; the permutation testing (1000 times) result of the PLS-DA model. This material is available free of charge via the Internet at http://pubs.acs.org.

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technical notes

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