An NMR-based metabonomic investigation of the subacute effects of

Mar 8, 2012 - The subacute toxic effects of 28 days of exposure to three dosages (250, 500, 1000 mg/kg/day) of melamine on Wistar rats were investigat...
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An NMR-based metabonomic investigation of the subacute effects of melamine in rats Ying-Jian Sun,†,‡,§ Hui-Ping Wang,‡,§ Yu-Jie Liang,‡,§ Lin Yang,‡ Wei Li,‡ and Yi-Jun Wu*,‡ †

Department of Veterinary Medicine and Animal Science, Beijing Agriculture College, Beijing 102206, P.R. China Laboratory of Molecular Toxicology, State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, 1-5 Beichenxi Road, Beijing 100101, P.R. China

‡

ABSTRACT: The subacute toxic effects of 28 days of exposure to three dosages (250, 500, 1000 mg/kg/day) of melamine on Wistar rats were investigated using nuclear magnetic resonance spectra, histopathological examination, and biochemical analysis. Rats treated with melamine developed adverse health effects compared to the controls, including decrease in body weight and kidney damage. Blood biochemical analysis showed that the blood urea nitrogen and creatinine increased distinctly compared to the control group. Urinary metabonomic analysis indicated that melamine caused an increase in succinate and citrate. Serum metabonomic analysis showed that the lowest dose led to an increase in dimethylglycine, N-acetylglycoprotein (NAC), accompanied by a decrease in taurine and glucose. Rats treated with the highest dose developed high levels of serum choline and 3hydroxybutyrate (3-HB) together with low lactate levels. Metabonomic analysis of liver tissue indicated that melamine caused an increase in NAC, choline, and creatine, accompanied by a decrease in lactate, trimethylamine-N-oxide, glutamate, and glucose. All three dosages resulted in an increase in glutamate, lactate, choline, glucose, and animo acids and a decrease in 3-HB and pyruvate in aqueous kidney extract. These results indicate that melamine not only caused renal disfunction but also disturbed the liver’s glucose, protein, and nitrogen metabolism. KEYWORDS: NMR, metabonomic, melamine, subacute effects

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INTRODUCTION Melamine, a synthetic nitrogenous product, is widely used in plastics, adhesives, glues, and laminated products such as plywood, cement, and cleansers. Recently, several cases of melamine being deliberately added to fertilizer, pet food, and even infant milk formula have come to light. Melamine is added to such products because its high nitrogen content artificially boosts apparent protein content. In 2007, an outbreak of nephrotoxic renal failure and death occurred in dogs and cats in the United States and other western countries following the consumption of melamine-contaminated pet food.1 More recently, an unprecedented epidemic of renal disease in children occurred in China after the consumption of melamine-tainted milk products.2−4 These incidents have prompted research on the diagnosis and treatment of melamine-related renal disease.5,6 Although some research on the toxicity of melamine to rats has been conducted,7−9 a metabonomic evaluation of subacute exposure to melamine in rats had not been completed. Metabonomics was initially defined as “the quantitative measurement of the dynamic multiparametric metabolic response of living systems to pathophysiological stimuli or genetic modification”.10 1H nuclear magnetic resonance © 2012 American Chemical Society

(NMR)-based metabonomics has proven a fast, effective, nondestructive method of obtaining good quality structural and quantitative information for investigating metabolic processes within whole organisms.11,12 This technique allows simultaneous detection of hundreds of low-molecular weight species within a biological matrix, generating an endogenous metabolic profile that is altered characteristically in response to external stimuli.13 Combining metabonomics with high resolution NMR and pattern recognition (PR) technology, such as principle components analysis (PCA), can facilitate visualization of inherent patterns in NMR data. This approach can also aid the identification of target sites of toxicity, assessment of the processes of toxic lesions, and the characterization of novel biomarkers.14,15 Metabonomics has had a significant impact on toxicology16−18 and is now widely used to identify target organ toxicity,19−21 in clinical diagnosis,22−24 to evaluate the safety of traditional Chinese medicine,25−27 and to assess the toxicity of candidate chemical agents.28,29 Received: December 16, 2011 Published: March 8, 2012 2544

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clean centrifuge tube, then dried and stored at −80 °C for NMR spectroscopic analysis.

We here report the results of a NMR-based metobonomic, biochemical, and histopathological investigation of the subacute multitoxic effects of melamine on experimental Wistar rats.

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Histopathology

The liver and kidney samples were fixed in 10% formalin, cut into 4 μm paraffin sections, and stained with hematoxylin and eosin for histopathological examination under a microscope.

MATERIALS AND METHODS

Chemicals

Blood Biochemical Analysis

Melamine (purity >99%) was obtained from the Hengye Zhongyuan Chemical Co. Ltd. (Beijing, China). All other reagents were obtained from commercial sources.

Standard spectrophotometric methods and an AutolabPM4000 Automatic Analyzer (AMS Co., Rome, Italy) were used to measure the following serum parameters; alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), albumin (ALB), blood urea nitrogen (BUN), and creatinine (CR). All parameters are expressed as mean ± SE.

Animals and Animal Husbandry

Six to eight week old Wistar rats were obtained from the Weitong Lihua Laboratory Animal Technology Company (Beijing, China). They were housed individually in cages and acclimatized for at least 1 week prior to the treatment. During the experiment, rats were kept at 22 ± 2 °C and 50−60% humidity under a 12 h light/dark cycle with free access to water and food. All animal procedures were performed in accordance with current Chinese legislation and approved by the CAS Institute of Zoology Animal and Medical Ethics Committee. Twenty male Wistar rats were randomly assigned to 4 groups, including a control group and three treatment groups so that each group was comprised of 5 rats. Based on the published oral half-lethal dose (LD50) of 3161 mg/kg for melamine in rats,30 we chose doses of 250, 500, and 1000 mg/ kg/day, hereafter described as low, medium, and high, for each of the treatment groups. Melamine was suspended in deionized water at a concentration of 1 mL/kg of the rat’s body weight and orally administrated for 28 consecutive days. Controls were treated with an equivalent volume of deionized water.

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H NMR Spectroscopic Measurement of Urine, Serum and Aqueous Liver and Kidney Extracts

Two-hundred microliters of buffer solution (0.2 M Na2HPO4 and 0.2 M NaH2PO4, pH 7.4) (BS) was mixed with 400 μL of urine to minimize variations in the pH of the urine. The sample was allowed to stand for 20 min prior to centrifugation at 3500× g for 5 min to remove any precipitates. Five-hundred microliters of the supernatant from each urine sample was added to 50 μL of 2,2′,3,3′,-deuterotrimethylsilylproprionic acid (TSP)-d4/D2O solution (1 mM, final concentration). The TSP acted as a chemical shift reference (δ0.0) and the D2O provided a lock signal. Water signals were suppressed by presaturation. 50 μL of BS and 50 μL of D2O were added to 400 μL of each serum sample. The water-suppressed Carr-Purcell-Meibom-Gill (CPMG) spinecho pulse sequence (RD-90°-(τ-180°-τ) nACQ) with a total spin−echo delay (2nτ) of 40 ms was used to attenuate broad signals from proteins and lipoproteins. Liver or kidney tissue aqueous extract was dissolved in 550 μL of D2O buffer solution (99.9% 0.1 M Na2HPO4/NaH2PO4, pH 7.4) containing 0.5 mM TSP. NMR spectra of these samples were recorded on a BrukerAv600 spectrometer at 298 K. Typically, 64 free induction decays (FIDs) were collected into 64 k data points over a spectral width of 8992.8 Hz with a relaxation delay of 5 s and an acquisition time of 0.91 s. The FIDs were weighted by an exponential function with a 0.3 Hz line-broadening factor prior to Fourier transformation. All spectra were referenced to the CH3 resonance of creatine at δ3.05.

Body Weight

The body weight of each rat was recorded daily during treatment periods. Collection of Urine and Serum

Individual urine samples of 24 h following the final dose of 28 days were collected by metabolic cage into ice-cold vessels containing 1% sodium azide.31,32 Supernatant liquor was obtained by centrifugation and then stored at −80 °C until required for NMR spectroscopic analysis. Animals were killed by injecting them with barbitalum natricum after the end of the 28-day experimental period. During the process, blood samples were collected. Serum samples were obtained from each blood sample by centrifugation and divided into two aliquots. One aliquot was used for biochemical analysis, and the other stored at −80 °C for NMR spectroscopic analysis.

Data Reduction and Principal Components Analysis of 1H NMR Spectra

Each 1H NMR spectrum of urine, serum, or aqueous tissue extract was segmented into regions of 0.04 ppm using MestRe-c 2.3 (http://qobrue.usc.es/jsgroup/MestRe-c). The area of each segmented region was calculated and the integral values used to create an intensity distribution of the whole spectrum. The δ5.2−4.2 region was excluded prior to statistical analysis to remove the variation in water suppression efficiency. For urine spectra, the region corresponding to urea (δ6.0−5.2) was also excluded to eliminate any cross-relaxation effects on the urea signal. All remaining regions of the spectra were then scaled to the total integrated area of the spectra to reduce any significant concentration differences. The values of all NMR data were mean-centered and paretoscaled prior to PCA using the SIMCA-P software package (Version 10, Umetrics AB, Umea, Sweden). Pareto scaling gives each variable a variance numerically equal to its standard deviation. Score plots based on NMR spectra were used to

Preparation of Kidney and Liver Samples

The kidneys and liver (left lobe) of each rat were removed immediately after death. Each liver or kidney sample was divided into two parts. One part was used for histopathological examination and the other as the source of aqueous extracts. To obtain the aqueous extracts, 100 mg of preweighed frozen liver or kidney tissue was homogenized in 400 μL of cold methanol and 85 μL of cold water in a homogenization tube. The homogenate was transferred to a 1.5 mL centrifuge tube, to which 400 μL of chloroform and 200 μL of cold water were added. The sample was vortexed for 60 s, then left on ice for 10 min to separate before finally being centrifuged for 5 min (2000× g, 4 °C) to remove precipitated protein and tissue debris. The upper polar layer was carefully transferred into a 2545

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visualize the separation between the experimental and control group. Loading plots identified the NMR spectral regions that contributed most to the separation of samples in the score plots. Each spectral region corresponded to a particular metabolite. An increase or decrease in the assigned spectra was therefore indicative of changes in endogenous metabolites. Statistical Analysis. One-way analysis of variance (ANOVA) was used to assess the statistical significance of differences in body weight and blood biochemical parameters among treatment groups. If significant effect was identified, posthoc multiple analysis was performed using the Dunnett’s test. A value of p < 0.05 was considered significant.

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RESULTS

Body Weight

The body weight of rats in all three treatment groups decreased but this was only significant (p < 0.05) in the low dose group (250 mg/kg) (Figure 1). It is interesting that rats in the higher

Figure 2. Photomicrographs of representative sections of the kidneys of rats orally treated with melamine at doses of (A) 0 mg/kg, (B) 250 mg/kg, (C) 500 mg/kg, and (D) 1000 mg/kg.

aqueous liver and kidney extracts are respectively shown in Figures 5 and 6. Representative 600 MHz 1H NMR spectra (δ4.5−0.5) of aqueous kidney extract are presented in Figure 6A. PCA results reveal that urine, serum, and aqueous liver and kidney extracts in all treatment groups are clearly distinct from those of the controls. This clear separation suggests that the metabolite profiles of the treated rats were altered by melamine. Data points of the low (250 mg/kg) and medium (500 mg/kg) treatment groups overlap in Figures 5 and 6, suggesting that these groups had similar metabolic profiles. The corresponding loading plots show that most of the separation of samples in the score plots is due to difference in metabolites. Urinary metabonomic analysis indicates that melamine caused an increase in succinate (2.41 ppm) and citrate (2.53, 2.57, 2.69 ppm). A rise in taurine (3.25 ppm) together with a drop in creatine (3.05 ppm) was observed in rats in the high treatment group (Figure 3). Figure 4 shows that rats in the low treatment group displayed increased serum dimethylglycine (2.78 ppm), N-acetylglycoprotein (NAC, 2.02 ppm), very lowdensity and low-density lipoprotein (VLDL/LDL, 0.86, 1.22, 1.26 ppm), accompanied by a decrease in taurine (3.25 ppm) and glucose (3.41, 3.45, 3.49, 3.73 ppm). Rats in the high treatment group developed high levels of serum choline (3.21 ppm) and 3-hydroxybutyrate (3-HB, 1.18 ppm) together with low levels of lactate (1.33, 4.12 ppm). Metabonomic analysis of aqueous liver extract indicated that choline (3.21 ppm) and creatine (3.05 ppm) increased in the low and medium treatment groups, and that this increase was accompanied by a decrease in lactate (1.33 ppm), glutamine (2.17 ppm), glutamate (2.53, 2.57 ppm) and glucose (3.49, 3.77 ppm). Increased NAC (2.13 ppm) together with decreased lactate and trimethylamine-N-oxide (TMAO, 3.25 ppm) was observed in the high treatment group. Melamine induced changes in endogenous metabolites in aqueous kidney extracts are shown in Figure 6. All three treatment groups developed elevated levels of glutamate (2.05, 2.09, 2.13, 2.45, 2.53, 2.65 ppm), lactate (1.33 ppm), choline (3.21 ppm), glucose and animo acids (3.41, 3.61, 3.69, 3.88, 3.96, 4.0, 4.04 ppm), and a decrease in 3-HB (1.18 ppm) and pyruvate (2.37 ppm) relative to the control. To sum up the above, urinary metabonomic profiles showed that melamine perturbed urinary metabolism in a dosedependent manner, with high-dose group showing the most

Figure 1. Body weights of rats in the control (0 mg/kg) group and those orally treated with 250, 500, or 1000 mg of melamine per kg of body weight.

treatment groups lost less weight than those in the lowest (Figure 1). We suspect that this was because melamine caused the dose-dependent deposition of calculus in the rats’ kidneys. Because calculus is relatively dense, it may have masked the loss of general body condition in the higher treatment groups. Histopathology

No obvious histological changes in the liver were observed in all treatment groups relative to the control although the volume of the kidneys of treated rats increased and yellow spots were apparent on their surface (data not shown). The normal microstructure of the kidneys of treated rats was, however, seriously damaged and an abundance of dark, green crystals had developed in the glomerulus (Figure 2). Blood Biochemical Analysis

Table 1 shows that levels of BUN and CR from rats in all three treatment groups increased distinctly compared to the control group (P < 0.05). No significant changes in other biochemical parameters were observed. 1

H NMR Spectroscopic and Pattern Recognition Analysis

Figures 3 and 4 show the results of metabonomic analysis of urine and serum, respectively, from each treatment group. PCA score plots (Figures 5B and 6B) and corresponding loading plots (Figures 5C and 6C) based on 1H NMR spectra of 2546

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Table 1. Selected Clinical Parameters of Rats Orally Treated with Melamine for 28 Daysa dosage (mg/kg/day) parameters

0

250

500

1000

AST (U/L) ALT (U/L) ALP (U/L) BUN (mg/dL) CR (μmol/L) ALB (g/L)

158.67 ± 9.57 62.67 ± 6.80 135.67 ± 20.04 7.68 ± 1.60 51.67 ± 5.25 33.63 ± 0.17

157.00 ± 10.98 53.00 ± 5.89 196.67 ± 32.50 47.61 ± 12.26* 204.00 ± 48.19* 33.93 ± 0.78

212.25 ± 58.20 78.75 ± 30.93 168.75 ± 35.67 43.53 ± 10.07* 148.00 ± 25.67* 31.65 ± 2.62

162.50 ± 18.31 60.00 ± 11.90 172.50 ± 34.51 28.27 ± 2.94* 136.25 ± 24.20* 34.75 ± 0.99

Rats were treated with three doses of melamine for 28 consecutive days. Data were presented as mean ± SD of n = 5 animals per groups. Statistical analysis was performed by one-way ANOVA. *p < 0.05 indicates statistically significant differences relative to the control group. Abbreviations: ALB, albumin; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, urea nitrogen; CR, creatinine.

a

Figure 4. PCA score plots (B) and corresponding loading plots (C) based on 1H NMR spectra (δ4.5−0.5) (A) of serum from rats orally treated with melamine at doses of (a) 0 mg/kg (▲), (b) 250 mg/kg (◇), (c) 500 mg/kg (●), and (d) 1000 mg/kg (*).

Figure 3. PCA score plots (B) and corresponding loading plots (C) based on 1H NMR spectra (δ4.5−0.5) (A) of the urine of rats orally treated with melamine at doses of (a) 0 mg/kg (▲), (b) 250 mg/kg (◇), (c) 500 mg/kg (●), and (d) 1000 mg/kg (*).

melamine and uric acid comprised 61−81% of stone weight.34 Damaged kidney structure, including dark, green crystals in the glomerulus, is evidence that melamine is a nephrotoxin. Histological examinations of kidney specimen revealed the formation of numerous crystals occurring in all the treatment groups, which indicated that melamine at all doses induced significant renal toxicity. BUN is the product of animo acids and CR is the ultimate product of creatine metabolism in skeletal muscle. Concentrations of BUN and CR are important indicators of renal function. High levels of BUN and CR in treated rats are further evidence that melamine causes renal dysfunction. However, the nephrotoxicity of melamine is probably the result of the combined effect of melamine and its byproduct cyanuric acid. It was recently reported that cyanuric acid given alone did not result in any renal dysfunction; however, coadministration of melamine and cyanuric acid together has been proved to develop crystal

significant perturbation. For serum and liver tissues, some significantly perturbed metabolites were detected in the low and middle dose group, but some other metabolites that was perturbed significantly were also detected in the high dose group; these metabolites changes are inconsistent performance in the three different dose levels. For kidney extracts, all of the three treatment groups developed a similar level of the metabolites elevated or decreased, suggesting the possibility that the low dose of melamine could sufficiently induce the metabolic changes in kidney tissue.

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DISCUSSION The observed decrease in body weight in all treatment groups suggests that melamine caused a general loss of body condition in the treated rats. Melamine has low acute oral toxicity but excessive exposure causes renal stones.33 Chemical analysis of the calculi in treated rats revealed that equimolar amounts of 2547

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Figure 5. PCA score plots (B) and corresponding loading plots (C) based on 1H NMR spectra (δ4.5−0.5) (A) of aqueous liver extract from rats orally treated with melamine at doses of (a) 0 mg/kg (▲), (b) 250 mg/kg (◇), (c) 500 mg/kg (●), and (d) 1000 mg/kg (*).

Figure 6. PCA score plots (B) and corresponding loading plots (C) based on 1H NMR spectra (δ4.5−0.5) (A) of aqueous kidney extract from rats orally treated with melamine at doses of (a) 0 mg/kg (▲), (b) 250 mg/kg (◇), (c) 500 mg/kg (●), and (d) 1000 mg/kg (*).

deposition in distal renal tubules, which was the main cause of renal impairment.35 Urine is the most easily obtained biofluid. Components and concentrations of urinary metabolites, which are directly affected by the functions of different body systems, are therefore good indicators of metabolic status. Serum is a complex mixture of a large number of constituents; 1H NMR spectra of serum from animals under similar physiological conditions are highly reproducible. Animal tissue extracts provide an instant “snapshot” of the cellular metabolic processes taking place when these were collected. Highresolution 1H NMR spectra of tissue extracts aids the interpretation of metabolic profiles from cells, tissues, or organs. Each peak in the 1H NMR spectra of urine, serum, or tissue extracts accurately corresponded to the hydrogen atom of different metabolic chemicals. The relative signal intensity of each peak reflected the relative contents of each component in detected samples relative to the control. Comparison of metabolite profiles between treatment groups and the control allows changes in the function of related organs to be inferred. Figure 3 shows changes in some endogenous urinary metabolites, such as citrate, succinate, taurine, and creatinine, in each treatment group. Citrate and succinate are the intermediates in the tricarboxylic acid (TCA) cycle process which is localized mainly in liver mitochondria. Although the influence of other factors cannot be excluded, the increased amount of citrate and succinate in the urine of melaminetreated rats suggests that melamine affected the activity of mitochondrial enzymes involved in the TCA cycle, resulting in the disruption of energy metabolism in the liver. These observations were consistent with the previous report that melamine at dose of 600 mg/kg could alter metabolic changes

involved in TCA cycle.9 Elevated urinary taurine and creatine has been found to be a biomarker of liver damage.14,36,37 Taurinuria, albeit without concomitant creatinuria, has been reported to occur following the administration of hydrazine, which can cause marked steatosis in the liver and inhibition of protein and/or glutathione synthesis.38 This suggests that the observed increase in taurine in the urine of melamine-treated rats is indicative of liver damage. The creatine-phosphocreatine system is crucial for cellular energy transportation. The decreased levels of creatine observed in the urine of rats in the high treatment group are therefore consistent with disruption of the energy metabolism. Serum metabonomic analysis revealed that many metabolites changed after exposure to the lowest (250 mg/kg) dose of melamine, such as NAC, VLDL/LDL and glucose. An increase in serum VLDL/LDL may reflect alteration of the carbohydrate metabolism.39 High levels of NAC in serum and aqueous liver extract could imply that melamine affected the activities of key enzymes involved in the protein metabolism of the liver.40 Rats in the highest treatment group displayed an increase in serum choline and 3-HB, together with a decrease in serum lactate. Choline is a constituent of cell membranes and lipoprotein phospholipids which play an important role in the integrity of cell membranes and the lipid metabolism. Increased choline in serum and aqueous liver and kidney extracts suggests that membrane fluidity was disrupted by melamine.41 3-HB is indicative of enhanced β-oxidation and can be utilized as an alternative energy source when glucose is limited.42 Levels of ketone bodies can increase when acetyl-CoA derived from βoxidation of free fatty acid exceeds the capacity of the TCA 2548

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cycle.43 Elevation of serum 3-HB indicates that melamine affected the activities of the key enzymes involved in the metabolism of ketone bodies in the liver and induced a shift in energy metabolism toward fatty acid β-oxidation and ketogenesis. A decrease in serum lactate and glucose was related to increased utilization of pyruvate in the TCA cycle and a decrease in anaerobic cell respiration, which is one type of energy metabolism.44 Elevated creatine in both blood and tissue has been reported to be a sign of renal insufficiency.45 Lower rates of glomerular filtration, which might be expected due to perturbation of the rennin-angiotension system, can cause a reduction in renalcortical blood flow. Increased creatine in aqueous liver extracts indicates that melamine disrupted renal glomerular filtration. Glutamate and glutamine are amino suppliers important to amino acid synthesis.46 We speculate that the observed decreases in glutamate and glutamine in rats’ aqueous liver extract may reflect disturbance of the nitrogen metabolism, a view supported by the associated drop in TMAO. In addition, reduced TMAO suggests that melamine blocks the N-oxidation pathway of choline and glycerophosphorylcholine in the kidney.44 A decrease in hepatic glucose suggests an increase in the rate of glycogenolysis and glycolysis.47 High levels of glutamate, lactate, glucose, and animo acids in the aqueous kidney extract of melamine-treated rats may therefore indicate that renal tubular reabsorption was disrupted by melamine.

REFERENCES

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CONCLUSIONS Our results indicate that melamine causes renal insufficiency in rats and disturbances to the glucose, protein, and nitrogen metabolism of rats’ livers. Our approach demonstrates the benefits of metabonomic analysis in assessing the comprehensive effects of melamine on animal and human health.

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AUTHOR INFORMATION

Corresponding Author

*Yi-Jun Wu, Institute of Zoology, CAS, 1-5 Beichenxi Road, Beijing 100101, China. E-mail: [email protected]. Tel: 86-1064807251. Fax: 86-10-64807099. Author Contributions §

These authors contributed equally to this work

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the grants from the National Basic Research Program of China (No. 2012CB114103) and the State Key Laboratory of Integrated Management of Pest Insects and Rodents (No. ChineseIPM1005).

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ABBREVIATIONS ALB, albumin; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; CR, creatinine; FIDs, free induction decays; 3-HB, 3-hydroxybutyrate; 1H NMR, 1H nuclear magnetic resonance; LDL/VLDL, low- and very low-density lipoprotein; NAC, N-acetylglycoprotein; PCA, principal components analysis; SIMCA, soft independent modeling of class analogy; PR, pattern recognition; TCA, tricarboxylic acid; TSP, 2,2′,3,3′-deuterotrimethylsilylpropionic acid. 2549

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dx.doi.org/10.1021/pr2012329 | J. Proteome Res. 2012, 11, 2544−2550