NMR and Pattern Recognition Studies on the Time-Related Metabolic

Chem. Res. Toxicol. 2001, 14, 1401-1412

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NMR and Pattern Recognition Studies on the Time-Related Metabolic Effects of r-Naphthylisothiocyanate on Liver, Urine, and Plasma in the Rat: An Integrative Metabonomic Approach Nigel J. Waters,† Elaine Holmes,† Ann Williams,‡ Catherine J. Waterfield,‡ R. Duncan Farrant,§ and Jeremy K. Nicholson*,† Biological Chemistry, Division of Biomedical Sciences, Sir Alexander Fleming Building, Imperial College of Science, Technology and Medicine, London SW7 2AZ, U.K., and Preclinical Safety Evaluation, GlaxoSmithKline Research & Development, Ware, Herts., SG12 0DP, U.K., and Analytical Technologies, GlaxoSmithKline R & D, Stevenage, Herts. SG1 2NY, U.K. Received March 27, 2001

We present here a novel integrative metabonomic approach to probe toxic effects of drugs in experimental animals using R-naphthylisothiocyanate (ANIT) as a model hepatotoxicant. Male Han-Wistar rats were dosed with ANIT (150 mg/kg, n ) 25), and plasma and liver samples were collected for NMR and magic-angle spinning (MAS) NMR spectroscopy at 3, 7, 24, 31, and 168 h postdosing. Urine was collected continuously for 3 days prior to dosing and up to 168 h postdose. Histopathology and plasma clinical chemistry was also performed at all time points. Liver samples were analyzed either intact by 600 MHz 1H MAS NMR techniques or using high resolution (liquid state) 1H NMR of water-acetonitrile extracts. These data were related to sequential 1H NMR measurements in urine and plasma using pattern recognition methods. 1D 1H NMR spectra were data-reduced and analyzed using principal components analysis (PCA) to show the time-dependent biochemical variations induced by ANIT toxicity. From the eigenvector loadings of the PCA, those regions of the 1H NMR spectra and hence the combinations of endogenous metabolites marking the main phase of the toxic episode were identified. The ANIT-induced biochemical manifestations included a hepatic lipidosis associated with hyperlipidaemia; hyperglycaemia and glycosuria; increased urinary excretion of taurine and creatine; a shift in energy metabolism characterized by increased plasma ketone bodies with reduced urinary excretion of tricarboxylic acid cycle intermediates and raised hepatic bile acids leading to bile aciduria. The integration of metabolic data derived from several sources gives a holistic approach to the study of time-related toxic effects in the intact system and enables the characterization of key metabolic effects during the development and recovery from a toxic lesion.

Introduction We have previously reported that the use of proton NMR spectroscopy and pattern recognition (PR) studies of urine and magic angle spinning (MAS)1 NMR studies on tissues from animals treated with drugs and toxins can give insight into site and mechanisms of toxic damage (1-10). Here we have investigated the combined application of NMR and MAS NMR of urine, plasma, and tissue samples taken at various postdose time points in an integrated metabonomic (11) approach to analyze onset, progression, and recovery processes from a toxic lesion using ANIT as a model. ANIT is a model hepatotoxin that causes intrahepatic cholestasis in a reproducible and dose-dependent manner * To whom correspondence should be addressed. † Biological Chemistry. ‡ Preclinical Safety Evaluation. § Analytical Technologies. 1 Abbreviations: AMIX, analysis of mixtures; ANIT, R-naphthylisothiocyanate; CPMG, Carr-Purcell-Meiboom-Gill; D2O, 2H2O; FID, free induction decay; LDL, low-density lipoprotein; MAS, magic-angle spinning; PCA, principal components analysis; PR, pattern recognition; TMAO, trimethylamine-N-oxide; TSP, 3-(trimethylsilyl)[2,2,3,3-2H4] propionate; VLDL, very low-density lipoprotein.

in experimental animals (12), which is pathologically similar to drug-induced cholangiolitic hepatitis in man (13). Prominent features of acute ANIT hepatotoxicity include bile duct epithelial cell necrosis, cessation of bile flow, hepatic parenchymal cell injury and hyperbilirubinaemia (14). Periportal injury is characterized by cholangiolitic hepatitis, inflammation of the liver and biliary capillaries, mediated by portal oedema, and neutrophil infiltration. Bile duct obstruction produced by ANIT treatment leads to a proliferative lesion known as biliary epithelial cell hyperplasia (15). The mechanism(s) involved in ANIT toxicity have been shown to be neutrophil- and platelet-dependent (16, 17). ANIT causes hepatocellular damage in vitro by the release of neutrophil proteases, as demonstrated by an increase in activity of the serine proteases, cathepsin G, and elastase (18). ANIT accumulation in bile is glutathione dependent, and the resultant hepatotoxicity is dependent on glutathione levels (19). It is thought that a reversible glutathioneANIT conjugate (dithiocarbamyl-linked glutathione) is released from hepatic parenchymal cells and is directed into bile where it dissociates to reduced glutathione and

10.1021/tx010067f CCC: $20.00 © 2001 American Chemical Society Published on Web 10/15/2001

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ANIT. The bile duct is, therefore, exposed to a high concentration of ANIT (20). Previous studies have shown that ANIT treatment increases urinary excretion of bile acids, glucose, creatine, and taurine together and lowers the excretion of tricarboxylic acid cycle intermediates (6). For non-rigid solid materials or highly viscous liquids, high resolution 1H MAS NMR spectroscopy offers an approach whereby some of the major line broadening contributions related to restricted molecular motion are eliminated or considerably reduced (21). High resolution 1H NMR spectroscopic studies of excised tissues normally require relatively large amounts of tissue (>0.25 g) which must be extracted via protein precipitation methods to enable the collection of sharp line spectra. MAS NMR of tissues is non-destructive, requires no sample preparation, and additionally allows molecular compartmentation and dynamic interactions to be investigated (22). 1H MAS NMR spectroscopy has recently been applied to the study of whole biological tissue such as renal cortex and liver (23), adipose tissue (24), intact red blood cells (22), prostate (25), and brain (26). Procedures involved in this technique have been optimized to validate the information gained from such MAS NMR studies of toxicological episodes and disease processes (23). 1H MAS NMR spectroscopy of whole tissue has been applied to the study of cadmium-induced renal toxicity in the bank vole (2) and to the assessment of renal and mitochondrial toxicity of 2-bromoethanamine (4). We show here that the combination of 1H MAS NMR spectral data on tissues with 1H NMR-PR data from blood plasma, urine and liver tissue extracts, conventional clinical chemistry, and histopathological data can give a comprehensive overview of the effects of ANIT in vivo, gathering tissue specific and whole system biochemical information and its variation in time.

Materials and Methods Animal Handling Procedure and Tissue Preparation. Thirty-five male Han-Wistar rats (250 g, Charles River, Cambs., U.K.) were acclimatized for 13 days in plastic cages prior to group allocation and treatment. They were then housed individually in metabolism cages in a well-ventilated room at a temperature of 21 ( 2 °C and a relative humidity of 50 ( 10%, with a 12 h light/12 h dark cycle. Food [Rat and Mouse No. 1 Diet (Special Diet Services Ltd., Cambridge, U.K.)] and tap water were provided ad libitum. Body weights were measured daily. Each rat received either a single dose of ANIT in corn oil (p.o. 150 mg/kg, n ) 25) or corn oil only (p.o. 10 mL/kg, n ) 10). Animals were sacrificed (n ) 5) by exsanguination from the abdominal aorta under isoflurane anaesthesia at 3, 7, 24, 31, and 168 h after dosing with ANIT, and vehicle-dosed control animals at 24 and 31 h p.d. Triplicate samples of the left lateral lobe of the liver, weighing between 15 and 23 mg, were excised and immediately snap-frozen in liquid nitrogen. Another larger section was snap-frozen for tissue extraction. These samples were stored at -70 °C until NMR spectroscopic analysis. The remainder of the left lateral liver lobe, a kidney, and a testis were fixed in 10.5% phosphate buffered formalin, embedded in paraffin wax, sectioned (3-4 µm), and stained with haematoxylin and eosin for histopathological assessment. 1H MAS NMR Spectroscopic Analysis of Intact Tissues. Samples (15-23 mg) were rinsed in 0.9% saline D2O, placed in zirconia 4 mm diameter rotors (Bruker Analytische GmbH, Rheinstetten, Germany) and analyzed by 1H MAS NMR spectroscopy at 600.13 MHz using a Bruker DRX600 spectrometer at an MAS spin rate of 6000 Hz (23). MAS NMR spectra were acquired at 283 K, as measured by the thermocouple system and maintained by the cooling of the inlet gas pressures

Waters et al. responsible for sample spinning. The in situ temperature calculated using the chemical shifts of the water resonance (temperature sensitive) and the R-anomeric proton of glucose (temperature insensitive) averaged 288 K (27). For each sample, 128 transients were collected into 32 K data points using a water presaturation pulse sequence (D-90°-t1-90°-tm-90°-acquire FID). A secondary irradiation field is applied at the water frequency during the relaxation delay of 3 s and during the mixing period tm (100 ms), with t1 fixed at 3 µs. A spectral width of 12000 Hz and an acquisition time per scan of 1.36 s were used. The 1D Carr-Purcell-Meiboom-Gill (CPMG) spin-echo pulse sequence [90°-(τ-180°-τ)n acquisition] (τ ) 200 µs, n ) 200) with standard presaturation of the water resonance, using a fixed total spinspin relaxation delay of 80 ms was applied to measure spinecho 1H MAS NMR spectra on all samples. 1H NMR Spectroscopic Analysis of Liver Tissue Extracts. Samples of liver tissue (∼250 mg) taken from the left lateral lobe were homogenized in 2 mL of 50% acetonitrile in an ice/water bath. The homogenates were centrifuged at 5070g for 5 min at 4 °C. The supernatants were removed and lyophilized before being reconstituted in 1 mL of D2O. The reconstituted solutions were pipetted into 5 mm NMR tubes together with 0.1% 3-(trimethylsilyl)[2,2,3,3-2H4] propionate (TSP, an internal standard δ 0.0) and 1% sodium azide (bacteriostatic agent) in D2O. 1H NMR spectra were acquired on each sample at 600.13 MHz on a Bruker DRX600 spectrometer at ambient probe temperature (298 K). 1D single pulse experiments were carried out using the standard pulse sequence to achieve satisfactory water suppression (see above). For each sample, 128 transients were collected into 64 K data points with a relaxation delay of 2 s and a mixing period of 100 ms. A spectral width of 9600 Hz and an acquisition time per scan of 3.41 s were used. 1H NMR Spectroscopic Analysis of Blood Plasma. Aliquots (2.0 mL) of blood were collected into heparin containers from abdominal aortal exsanguination, and the plasma was separated by centrifugation and frozen prior to NMR measurement. For each sample, 250 µL of blood plasma was made up to a total volume of 600 µL by the addition of an appropriate volume of D2O. The samples were centrifuged at 4200g for 20 min at 4 °C. Aliquots (500 µL) were decanted into 5 mm NMR tubes and inverted several times to ensure thorough mixing. 1H NMR spectra were acquired on each sample at 600.13 MHz on a Bruker DRX600 spectrometer at ambient probe temperature (298 K). 1D single pulse experiments were carried out using the standard pulse sequence, as described for the acquisition of MAS NMR spectra. For each sample, 64 transients were collected into 64 K data points with a relaxation delay of 2 s and a mixing period of 100 ms. A spectral width of 9600 Hz and an acquisition time per scan of 3.41 s were used. The 1D CPMG spin-echo pulse sequence using a fixed total spin-spin relaxation delay of 80 ms was applied to measure spin-echo 1H NMR spectra on all samples. 1H NMR Spectroscopic Analysis of Urine. Urine was collected in 1% sodium azide during the following periods: predose (daytime collection 0-7 h period on day -5, -4, and -1), and 0-7, 7-24, 24-31, 31-48, 48-56, 56-72, 72-79, 7996, 144-151, and 151-168 h postdose. Aliquots (600 µL) of urine were mixed with phosphate buffer (300 µL of 0.1 M) and centrifuged at 2230g for 15 min. The supernatants (800 µL) were pipetted into sample vials together with 0.1% TSP/1% azide in D2O. NMR measurements were made on a Bruker AMX600 spectrometer, operating at 600.13 MHz 1H frequency, using a Bruker BEST flow-injection probe (Bruker Analyticshe GmbH, Rheinstetten, Germany). NMR spectra were acquired using the standard pulse sequence (as previously described). For each sample, 64 free induction decays (FIDs) were collected into 48 K data points using a spectral width of 12 000 Hz and an acquisition time per scan of 2.04 s. Automatic Data Reduction and Principal Components Analysis (PCA). Single pulse and CPMG (MAS-) NMR spectra were data reduced using the program AMIX (Bruker GmbH,

Metabonomic Investigations into Hepatotoxic Processes Germany). The spectral region δ 0.2-10.0 was segmented into regions of 0.04 ppm width giving a total of 256 integrated regions per NMR spectrum. The area for each segmented region was expressed as an integral value resulting in an intensity distribution description of the whole spectrum with 256 variables prior to PR analysis. The region of the spectrum, which included water (δ 4.8-5.4), was removed from the analysis for all groups to eliminate variation in water suppression efficiency. For urine spectra, the region containing urea (δ 5.4-6.0) was also excluded. For the aqueous liver extracts, the region of the spectrum containing a residual acetonitrile signal (δ 2.08) was removed from all spectra. All remaining spectral segments were scaled to the total integrated area of each spectrum (28). The single pulse and CPMG 1H NMR spectral data sets were imported into the SIMCA-P8.0 software package (Umetrics AB, Umea˚, Sweden) separately. These data were then scaled using centered scaling prior to PCA. With mean-centering, the average value of each variable is calculated and then subtracted from the data. PCA methods involve the calculation of linear combinations of the original descriptors, the PCs, such that each PC is orthogonal to all others with the first PC (PC1) containing the largest amount of variance with subsequent PCs containing progressively less variance (29). Thus, a plot of PC1 versus PC2 provides the most efficient 2D representation of the information contained in the data set. Much of the PCA data was presented as mean trajectory plots, where the magnitude and direction of each point represents the severity and extent of the lesion, and as an average of all individual samples in each group. Clinical Chemistry and Haematology. Clinical chemistry analysis was carried out on a Hitachi 917 analyzer using appropriate kits. Blood was collected at autopsy time points by abdominal aortal exsanguination. Blood (0.5 mL) was collected into EDTA anticoagulant. A Technicon H* 1 analyzer was used to determine a range of haematological parameters. Reticulocyte count was determined using a Sysmex R-1000 analyzer. In addition to this, 0.01 mL of blood was collected into thrombotest reagent and used to determine thrombotest time. Also, 1.8 mL of blood was collected into 0.2 mL of 0.106 M trisodium citrate. Prothrombin time, activated thromboplastin time and fibrinogen concentration were determined using a Sysmex CA-5000 analyzer. Unpaired Mann-Whitney tests were used to compare clinical chemical data between groups.

Results 1H MAS NMR Spectroscopic and Pattern Recognition Analysis of Intact Liver. 1H MAS NMR spectra of intact liver acquired at various time points after the administration of ANIT showed marked and consistent changes in the levels of endogenous metabolites from 7 h p.d. onward (Figure 1). The predominant changes identified in the PR analysis (Figure 2) included an increase in lipid, e.g., triglyceride at 24 and 31 h p.d., which fell to below control levels at 168 h p.d., a reduction in glucose and glycogen concentrations between 3 and 31 h p.d. before recovery at 168 h p.d. and elevated trimethylamine-N-oxide (TMAO), betaine, phosphocholine, and choline in all but one individual at 168 h p.d. On closer inspection of the 1H MAS NMR spectra, an increase in the bile acid C18 methyl signal strength (δ 0.7) at 24 and 31 h p.d. was observed and, in some individuals, an increase in the amino acids leucine and isoleucine signal intensities at 24-31 h p.d were observed. Figure 2 shows that, overall, the liver biochemistry perturbations had not recovered by 168 h. 1H NMR Spectroscopic and Pattern Recognition Analysis of Liver Extracts. The PCA analysis of aqueous 1H NMR spectra did show time-dependent alterations in the levels of endogenous metabolites (Figures 3 and 4). These included a subtle but progressive

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reduction in glycogen and glucose from 3 to 31 h p.d., which recovered to control levels by 168 h p.d., an increase in choline and phosphocholine concentrations from 3 to 168 h p.d., raised lactate at 24-31 h p.d. and the appearance of the C18 bile acid methyl signal at 2431 h p.d. Further examination of the spectra revealed augmented glutathione levels at 24 h p.d. The inherent differences between 1H NMR analysis of intact tissue and an aqueous extract of the same tissue led to the generation of different sets of metabolite information. Both 1H MAS NMR and solution-state 1H NMR spectroscopy identified the elevated bile acids and reduced glycogen and glucose together with changes in phosphocholine and choline in the liver. However, all perturbations of the lipid moieties were detected by 1H MAS NMR spectroscopy alone, while the 1H NMR analysis of aqueous extracts identified changes in lactate and glutathione. 1 H NMR Spectroscopic and Pattern Recognition Analysis of Blood Plasma. The 1H NMR spectra of blood plasma obtained at various time points after administration of ANIT exhibited an altered biochemical composition from 3 h p.d. onward (Figure 5 and 6). Spectra were assigned based on previous studies (30). The prominent changes in endogenous plasma metabolites, confirmed in the PR analysis (Figure 7), comprised an elevation in glucose at 7 h p.d., together with increased lipid, very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) concentrations at 31 h p.d. and a rise in phosphatidylcholine, choline, and creatine at 24 and 31 h p.d. In addition, subtle changes observed in the 1H NMR spectra over the entire time course included a drop in acetate concentration accompanied by the appearance of 3-D-hydroxybutyrate and acetoacetate signals. The 1H NMR spectra and pattern recognition analysis indicated recovery of the plasma profile by 168 h p.d. 1 H NMR Spectroscopic and Pattern Recognition Analysis of Urine. The 1H NMR spectra of urine showed consistent changes in endogenous metabolites from 7 h p.d. onward (Figure 8). The major changes identified in the PCA analysis (Figure 9) included the appearance of bile acids from 24 to 72 h p.d., an increase in succinate in the first 24 h p.d. before dropping below control levels until later time points, a decrease in 2-oxoglutarate and citrate from 7 to 144 h p.d., an increase in creatine from 48 to 96 h p.d., a rise in taurine at 72-96 h p.d., augmented levels of TMAO at 144 h p.d. onward and an elevation in glucose concentration at 7-96 h p.d. and later at 151-168 h p.d. Apart from the latter, these ANIT-induced perturbations of the 1H NMR urinary profile are in agreement with previous 1H NMR spectroscopic studies (6). The pattern recognition PCA analysis (Figure 9) showed maximum separation of treatment versus control at 24-48 h p.d. and this time point was characterized by low levels of succinate, 2-oxoglutarate and citrate, elevated glucose and bile aciduria. Later time points mapped toward the control and predose region indicating partial regeneration and recovery of the lesion. However, unlike plasma the metabolic trajectory did not return to control metabolic space by 151-168 h p.d. Clinical Chemistry Parameters. The activity of the plasma enzymes; alanine aminotransferase, aspartate aminotransferase, glutamate dehydrogenase, and sorbitol dehydrogenase all increased severalfold at 24-31 h p.d. (Table 1). The neutrophil count was elevated at 24, 31,

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Figure 1. Series of 600 MHz single pulse 1H MAS NMR spectra (δ 0.5-5.7) of the left lateral lobe of the liver at various time points following the administration of ANIT (150 mg/kg): (a) control, (b) 7 h, (c) 24 h, (d) 31 h, and (e) 168 h postdose. Lorentzian-Gaussian resolution-enhancement (LB ) -2, GB ) 0.3) was applied to all spectra. Key: Ala, alanine; Bet, betaine; Cho, choline; Glc, glucose; Gln, glutamine; GPC, glycerophosphorylcholine; GSH, glutathione; Ile, isoleucine; Lac, lactate; Leu, leucine; Lys, lysine; PCho, phosphocholine; Tau, taurine; Thr, threonine; TMAO, trimethylamine-N-oxide; Val, valine.

and 168 h p.d. A variety of metabolites in blood plasma also exhibited time-dependent alterations including elevated bile acids, cholesterol, and bilirubin at 24-31 h p.d., increased glucose at 7 h p.d. followed by a decline to below control at 168 h p.d. and raised blood lactate at 31 and 168 h p.d. In addition, Table 2 shows body weight data from treated animals over the study indicating a progressive loss postdose, followed by recovery to control values at day 7. Histopathological Studies. In liver 3 h p.d., a reduction in hepatocyte glycogen vacuolation resulting in increased hepatocyte eosinophilia was seen in four of five treated animals together with minimal bile duct epithelial hypertrophy. By 24 h p.d., slight to moderate bile duct degeneration and necrosis was seen in all

Figure 2. PCA metabolic trajectory plot mapping the average position of liver single pulse 1H MAS NMR spectra for each time point. Principal components 1 and 2 explain 95.9% of the data variance.

Metabonomic Investigations into Hepatotoxic Processes


Figure 3. Series of 600 MHz single pulse 1H NMR spectra (δ 0.5-5.7) of liver tissue aqueous extracts (left lateral lobe) at various time points following the administration of ANIT (150 mg/kg): (a) control, (b) 3 h, (c) 7 h, (d) 24 h, (e) 31 h and (f) 168 h postdose. Key: As for Figure 1 with the following additions, 3HB, 3-D-hydroxybutyrate; Ace, acetate; Asp, aspartate; Glu, glutamate; region A, glycerol; glucose and amino acid CH protons.

treated animals, accompanied by very slight to moderate peribiliary acute inflammation and oedema. Epithelial hypertrophy and bile duct hyperplasia was seen in four of five animals. Reduced hepatocyte glycogen vacuolation and increased hepatocyte eosinophilia was apparent in all animals at 24 h p.d. However, at 31 h p.d. the degree of peribiliary inflammation and oedema was less than that observed at 24 h. Four of five individuals also showed some evidence of parenchymal haemorrhagic necrosis. By 168 h p.d., the only hepatic change visible in treated animals was the presence of slight bile duct hyperplasia accompanied by marginal periportal fibrosis. There was a suggestion of an increase in periportal haematopoiesis. Oil-Red O stained liver sections showed lipid accumulation at 24 and 31 h p.d. No treatmentrelated changes were seen in the kidney at any time point. No treatment-related changes were seen in the testes of animals killed 3, 7, 24, or 31 h p.d. In animals killed 168 h p.d., slight or moderate sperm retention (at

Figure 4. PC map showing the mapping position of aqueous liver tissue extract 1H NMR spectra obtained from individuals following treatment with corn oil (+) or ANIT at various time points postdose: 3 h (2), 7 h (9), 24 h (O), 31 h ([), and 168 h (0). Principal components 1 and 2 explain 85.3% of the data variance.

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Figure 5. Series of 600 MHz single pulse 1H NMR spectra (δ 0.5-5.5) of blood plasma at various time points following the administration of ANIT (150 mg/kg): (a) control, (b) 3 h, (c) 7 h, (d) 24 h, (e) 31 h, and (f) 168 h postdose. Key: As for Figure 1 with the following additions, 3HB, 3-D-hydroxybutyrate; Aca, acetoacetate; d-Ala, deuterated alanine; Cit, citrate; Cre, creatine; Glu, glutamate; HDL, C18 methyl from cholesterol in high-density lipoprotein; L1, lipid CH2*CH2CO; L2, lipid CH2*CH2-CdC; L3, lipid CH2*-CdC; L4, lipid CH2*CO; L5, lipid CdC-CH2*-CdC; L6, albumin lysyl -CH2; L7, glyceryl of lipids CH2*OCOR; L8, unsaturated lipid CdCH*CH2CH2; LDL1, low-density lipoprotein terminal methyl; LDL2, low-density lipoprotein (CH2)n; NAG, N-acetyl glycoproteins; PC, phosphatidylcholine; Pyr, pyruvate; region A, glucose and amino acid CH resonances; VLDL1, very low-density lipoprotein terminal methyl; VLDL2, very low-density lipoprotein (CH2)n.

the lumen and base of late stage tubules) was seen in all animals (data not shown).

Discussion Changes in Lipid Metabolism and Storage. The elevated lipid levels observed in the 1H MAS NMR spectra at 24 and 31 h p.d. followed by the subsequent drop to below control levels at 168 h p.d. is in agreement with previous work conducted on mice (31). The increase in triglyceride levels represents drug-induced steatosis, which may be caused by a number of mechanisms. The accumulation of triglycerides and/or phospholipids is a common cellular response to toxic compounds, which is normally reversible. However, in severe cholestasis, the hepatocellular injury has been attributed to the direct

membrane-damaging action of bile acids (32, 33). ANIT is a cholestatic hepatotoxin, and an accumulation of bile acids within the bile ducts could lead to a “detergent effect” resulting in bile acid-mediated micellar solubilization of membrane lipid (32, 33). The phase transition from protein-rich membrane bilayer matrix to micelles, microscopic globular aggregates which permit solvation, may lead to the improvement in 1H NMR “visibility” of membrane lipid components. Another contributory factor to hepatic lipid accumulation could lie in the impaired apolipoprotein formation as a result of reduced protein synthesis, blocking the transport of lipid out of the cell as VLDL (34). Following the hepatic lipidosis at 24-31 h p.d., ANIT caused a marked rise in plasma lipids and lipoproteins



Figure 6. Series of 600 MHz CPMG 1H NMR spectra (δ 0.5-5.5) of blood plasma at various time points following the administration of ANIT (150 mg/kg): (a) control, (b) 3 h, (c) 7 h, (d) 24 h, (e) 31 h, and (f) 168 h postdose. Key: As for Figure 1 with the following additions, 3HB, 3-D-hydroxybutyrate; Aca, acetoacetate; Ace, acetate; Cit, citrate; Cre, creatine; d-Ala, deuterated alanine; DMG, dimethylglycine; Glu, glutamate; LDL1, low-density lipoprotein terminal methyl; LDL2, low-density lipoprotein (CH2)n; NAC1/NAC2, composite acetyl signals from R1-acid glycoprotein; PC, phosphatidylcholine; region A, signals from glycerol; glucose and amino acid CH protons; Suc, succinate.

measured at 31 h p.d. It has been shown that the onset of cholestasis is characterized by increased plasma levels of bile acids, bilirubin, medium chain length (C16-C20) phospholipids, free cholesterol, and enzymic markers of liver damage, such as alanine aminotransferase (35-37). In the present studies we observed increased plasma cholesterol at 24 and 31 h p.d. (Table 1), the likely cause being the inhibition of bile acid synthesis followed by incorporation into LDL (35), correlating well with the plasma 1H NMR-PR data. Density gradient ultracentrifugation of rat plasma following ANIT-treatment has demonstrated a distinct shift in lipoprotein density toward the low-density lipoprotein range (35), and the increase in VLDL and LDL levels identified in the plasma 1H NMR-PR data has provided further evidence of this pathophysiological response. Furthermore, both studies

Figure 7. PCA metabolic trajectory plot mapping the average position of plasma single pulse 1H NMR spectra for each time point. Principal components 1 and 2 explain 84.7% of the data variance.

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Figure 8. Series of 600 MHz single pulse 1H NMR spectra (δ 0.5-4.7) of urine at predose and various time points following the administration of ANIT (150 mg/kg). Key: as for Figure 1 with the following additions, 2-OG, 2-oxoglutarate; Ace, acetate; Cit, citrate; Cre, creatine; NAG, N-acetyl glycoproteins; Suc, succinate.

have highlighted the recovery to the control lipid pattern within 168 h p.d. The 1H MAS NMR-detected decrease in liver lipid at 168 h p.d. was accompanied by an increase in TMAO, betaine, phosphocholine, and choline. As such, this suggests the removal of the excess lipid by enzymatic conversion to intermediate species. Choline and phosphocholine are products of lipid catabolism and can be further metabolized to TMAO and betaine by an Noxidation step (38-40). The recovery phase of ANITinduced cholestasis was characterized by bile duct hyperplasia, and hence the time-related modulations in TMAO, betaine, phosphocholine, and choline levels may provide the basis of novel biomarkers for this proliferative response.

Figure 9. PCA metabolic trajectory plot mapping the average position of single pulse 1H NMR urine spectra for each time point. (4) 5 days, 4 days, and 1 day before dosing and vehicledosed control time points, (2) sequential time points after ANIT treatment. Principal components 1 and 2 explain 87.9% of the data variance.



Table 1. Effect of ANIT Treatment on Various Blood Plasma Parameters as Measured by Clinical Chemistry Assaysa plasma measurement

control

3h

7h

24 h

31 h

168 h

ALT (IU/L) AST (IU/L) GDH (IU/L) SDH (IU/L) neutrophils (×109/L) bile acids (µmol/L) bilirubin (µmol/L) cholesterol (mmol/L) glucose (mmol/L) lactate (mmol/L)

31.27 ( 4.25 53.7 ( 4.97 4.14 ( 0.7 15.64 ( 3.95 0.65 ( 0.29 27.6 ( 13.96 1.09 ( 0.12 1.48 ( 0.28 11.71 ( 1.37 2.99 ( 1.55

40.44 ( 10* 55.22 ( 1.52 3.14 ( 0.23* 18.12 ( 3.74 0.78 ( 0.22 13.2 ( 4.19 1.62 ( 0.58 1.75 ( 0.28 13.94 ( 1.41* 2.86 ( 1.04

32.78 ( 4.25 47.48 ( 5.33 2.84 ( 0.42** 18.42 ( 3.99 0.55 ( 0.3 14.52 ( 1.39 3.68 ( 1.18*** 1.91 ( 0.13** 17.64 ( 2.44*** 2.63 ( 2.26

109.18 ( 92.57 266.38 ( 218.67* 64.62 ( 54.11*** 78.74 ( 62.37** 1.85 ( 0.53** 729.18 ( 620.3*** 13.96 ( 10.09*** 2.1 ( 0.34** 11.82 ( 1.8 3.03 ( 1.76

228.74 ( 139.22*** 352.76 ( 168.24*** 94.22 ( 58.56*** 146.66 ( 79.61*** 1.81 ( 0.41*** 1037.44 ( 335.74*** 38.06 ( 14.26*** 2.84 ( 0.35*** 11.16 ( 1.78 5.26 ( 2.9

35.9 ( 3.94* 52.24 ( 2.64 3.66 ( 0.76 13.22 ( 2.36 1.34 ( 0.56* 37.12 ( 22.49 5.36 ( 1.86*** n.d. 8.49 ( 0.5*** 5.13 ( 1.96*

a Values expressed as mean ( SD. Statistics: (*) p < 0.05, (**) p < 0.01, and (***) p < 0.001 (n ) 5 for all groups, except control where n ) 10). Key: ALT, alanine aminotransferase; AST, aspartate aminotransferase; GDH, glutamate dehydrogenase; SDH, sorbitol dehydrogenase; n.d., no data available.

Table 2. Effect of ANIT Treatment on Body Weighta day

-5

-4

-1

0

1

2

3

4

5

6

7

body weight (g)

249.06 ( 9.7

253.06 ( 10.51

264.18 ( 12.45

267.19 ( 12.44

259.24 ( 13.03

248.50 ( 6.5

241.64 ( 9.41

242.12 ( 12.58

250.02 ( 11.21

252.42 ( 11.89

254.12 ( 11.37

a

Values expressed as mean ( SD.

Lesion-Specific Biomarkers of Hepatotoxicity. Clinical chemistry measurements revealed a severalfold increase in the activity of the plasma enzymes alanine aminotransferase, aspartate aminotransferase, glutamate dehydrogenase, and sorbitol dehydrogenase confirming liver damage, together with hyperbilirubinaemia and elevated plasma bile acids indicating liver cholestasis, at the 24 and 31 h p.d. time points. Raised numbers of circulating neutrophils at 24, 31, and 168 h p.d. suggested an inflammatory response to ANIT administration, centered on the liver. NMR analysis of urine presented bile-aciduria accompanied by glycosuria: lesion-specific biomarkers of cholestasis (6). The observed increase in urinary creatine (48-96 h p.d.) and taurine (72-96 h p.d.) levels are, in combination, well-documented biomarkers of hepatotoxicity (6, 41). Changes in taurine levels are closely related to changes in the pool of the precursor amino acid cysteine, which reflect both changes in protein synthesis and glutathione demand (42). It is thought that a reversible glutathione-ANIT conjugate (dithiocarbamyllinked glutathione) is released from hepatic parenchymal cells which is directed into bile where it dissociates to reduced glutathione and ANIT; a reaction which is necessary for hepatotoxicity to occur (19, 20). As the GSH-ANIT conjugate dissociates in the biliary system, an excess of free GSH will accumulate and this appears to be the case since 1H NMR spectral analysis of liver extracts showed an acute rise in glutathione at 24 h p.d. This would then cause a shift in the sulfur metabolite pathway, as less GSH production leads to the potentially toxic build-up of cysteine, which is bypassed by metabolism to taurine, prior to excretion into the urine. Bile Duct Obstruction and Damage. Histopathology confirmed the ANIT-induced lesion such that cholangiolitic hepatitis and bile duct degeneration were followed by bile duct hyperplasia, as is well-documented. In addition, conventional assay techniques identified raised bile acids and cholesterol levels in the plasma at the time points of maximum effect (24-31 h p.d.). The numbers of circulating neutrophils increased at maximum effect, also confirming an inflammatory response, characteristic of the ANIT-induced liver lesion (12). Under normal physiological conditions, bile salts secreted into the canalicular lumen aggregate phospholipid

(consisting principally of phosphatidylcholine) as micelles, which are transported in the bile prior to intestinal secretion (43). The hepatocyte canalicular membrane plays a crucial role in nascent bile formation and biliary secretion of bile salts, phosphatidylcholine, and cholesterol. The canalicular membrane is made up of mainly phosphatidylcholine, sphingomyelin, and cholesterol, where the two latter species provide decreased fluidity and increased resistance against the detergent effects of bile salts. However, cholestasis-induced hepatocyte necrosis and the subsequent membrane breakdown, will provide not only elevated bile acids, but also an increased available source of phosphatidylcholine for bile acidmediated micellar solubilization to take place. The bile duct obstruction would then lead to a reflux of these bile constituents into the plasma compartment (35), explaining the rise in phosphatidylcholine and bile acids observed in the plasma 1H NMR-PR and clinical chemistry analysis at 24 and 31 h p.d. As a consequence, abnormally high levels of bile acids would then be excreted into the urine. The increase in choline and phosphocholine over the entire time course in the 1H NMR spectra of aqueous liver tissue extracts was not observed in the 1H MAS NMR spectra. It is possible that the extraction procedure had released these species from a constrained motional environment within the cellular matrix, into solution where an isotropic motion had enabled their visualization by liquid state 1H NMR spectroscopy. The elevated levels of choline and phosphocholine maybe products of membrane breakdown and lipid catabolism as a result of the action of ANIT, which in the solid tissue contributed to the obstruction of the bile ducts resulting in a constrained molecular motion. The appearance of the bile acid methyl signal in liver extracts at 24-31 h p.d. is in agreement with the 1H MAS NMR-PR analysis and this is likely to be due to the bile duct obstruction leading to a cessation of bile flow. Effects on Glucose and Glycogen Metabolism. 1H NMR spectroscopic and clinical chemistry measurements on blood plasma highlighted a hyperglycaemic effect at 7 h p.d. This effect may also relate to the presence of plasma ketone bodies, 3-D-hydroxybutyrate and acetoacetate, from 3 h p.d. onward, verifying the onset of ketogenesis: β-oxidation of fatty acids and/or oxidation

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Figure 10. Chart illustrating the major fluctuations in metabolite profiles observed by 1H NMR spectroscopy over the experimental time course in the liver, blood plasma and urine. Key: v, above control levels; V, below control levels; LDL, low-density lipoprotein; PC, phosphatidylcholine; TMAO, trimethylamine-N-oxide; VLDL, very low-density lipoprotein. Metabolite changes in purple were observed in both extract and MAS NMR spectra. Note: L, P, and U refers to sampling of liver, blood plasma and urine, respectively.

of the ketogenic amino acids to form acetyl-CoA in the liver and the resultant transport of ketone bodies to peripheral tissues (44). The body weight data show a progressive loss over the first 4 days after dosing due to reduced food intake, hence causing a shift toward ketone body metabolism. The drop in the 1H (MAS)-NMR-detected hepatic concentrations of R-glucose, β-glucose and glycogen over the first 31 h p.d., together with the rise in liver and blood lactate concentration at 24-31 h p.d. implied an increased rate of glycogenolysis and glycolysis. The reduction in urinary levels of succinate, citrate, and 2-oxoglutarate also confirmed a general increase in energy

metabolism. Studies on isolated rat liver mitochondria suggest that toxicologically relevant concentrations of bile acids can alter mitochondrial function, initialized by mitochondrial membrane depolarization (45), and the subsequent perturbations in bioenergetics may lead to altered excretion of the tricarboxylic acid cycle intermediates. Integrated Metabonomics. Figure 10 summarizes the ANIT-induced perturbations of endogenous metabolites observed in the 1H NMR-PR analysis of intact liver, extracted liver, and biofluids, highlighting the correlations between time-dependent changes in each biological matrix.


A 1H MAS NMR spectroscopic and pattern recognition approach has enabled the detailed study of biochemical perturbations in intact liver tissue spectra following a hepatotoxic insult, thereby allowing a direct correlation with biofluid NMR spectra, histopathological data, and clinical chemistry parameters. The use of these NMRbased metabonomic techniques has allowed the visualization of key time periods in the development of a toxic injury, enabling the identification of lesion-specific, matrixspecific biomarkers, of cholestasis and hepatotoxicity. The variety and complexity of the biochemical changes arising from a single dose of the hepatotoxicant over time shows the importance of the use of multiparametric analytical approaches (such as NMR-PR) to the study of toxic episodes in order to relate biochemical changes to classically accepted pathological end-points.

References (1) Holmes, E., Nicholls, A. W., Lindon, J. C., Connor, S. C., Connelly, J. C., Haselden, J. N., Damment, S. J. P., Spraul, M., Neidig, P., and Nicholson, J. K. (2000) Chemometric models for toxicity classification based on NMR spectra of biofluids. Chem. Res. Toxicol. 13, 471-478. (2) Griffin, J. L., Walker, L. A., Troke, J., Osborn, D., Shore, R. F., and Nicholson, J. K. (2000) The initial pathogenesis of cadmium induced renal toxicity. FEBS Lett. 478, 147-150. (3) Nicholls, A. W., Nicholson, J. K., Haselden, J. N., and Waterfield, C. J. (2001) A metabonomic approach to the investigation of druginduced phospholipidosis: A NMR spectroscopy and pattern recognition study. Biomarkers 5, 410-423. (4) Garrod, S., Humpfer, E., Connor, S. C., Connelly, J. C., Spraul, M., Nicholson, J. K., and Holmes, E. (2000) High resolution 1H NMR and Magic Angle Spinning NMR spectroscopic investigation of the biochemical effects of 2-bromoethanamine in intact renal and hepatic tissue. Magn. Reson. Med. 45, 781-790. (5) Holmes, E., Nicholls, A. W., Lindon, J. C., Ramos, S., Spraul, M., Neidig, P., Connor, S. C., Connelly, J., Damment, S. J. P., Haselden, J., and Nicholson, J. K. (1998) Development of a model for classification of toxin-induced lesions using 1H NMR spectroscopy of urine combined with pattern recognition. NMR Biomed. 11, 235-244. (6) Beckwith-Hall, B. M., Nicholson, J. K., Nicholls, A. W., Foxall, P. J. D., Lindon, J. C., Connor, S. C., Abdi, M., Connelly, J., and Holmes, E. (1998) Nuclear magnetic resonance spectroscopic and principal components analysis investigations into biochemical effects of three model hepatotoxins. Chem. Res. Toxicol. 11, 260272. (7) Holmes, E., Bonner, F. W., and Nicholson, J. K. (1995) Comparative studies on the nephrotoxicity of 2-bromoethanamine hydrobromide in the Fischer 344 rat and the multimammate desert mouse (Mastomys natalensis). Arch. Toxicol. 70 (2), 89-95. (8) Anthony, M. L., Sweatman, B. C., Beddell, C. R., Lindon, J. C., and Nicholson, J. K. (1994) Pattern recognition classification of the site of nephrotoxicity based on metabolic data derived from proton nuclear magnetic resonance spectra of urine. Mol. Pharmacol. 46, 199-211. (9) Nicholson, J. K., Higham, D. P., Timbrell, J. A., and Sadler, P. J. (1989) Quantitative high resolution 1H NMR urinalysis studies on the biochemical effects of cadmium in the rat. Mol. Pharmacol. 36, 398-404. (10) Gartland, K. P. R., Bonner, F. W., and Nicholson, J. K. (1989) Investigations into the biochemical effects of region-specific nephrotoxins. Mol. Pharmacol. 35, 242-250. (11) Nicholson, J. K., Lindon, J. C., and Holmes, E. (1999) ‘Metabonomics’: understanding the metabolic responses of living systems to pathophysiological stimuli via multivariate statistical analysis of biological NMR spectroscopic data. Xenobiotica. 29, 1181-1189. (12) Goldfarb, S., Singer, E. J., and Popper, H. (1962) Experimental cholangitis due to alpha-naphthylisothiocyanate (ANIT). Am. J. Pathol. 40, 685-698. (13) Zimmerman, H. J. (1978) Drug-induced liver disease. Drugs 16, 25-45. (14) Plaa, G. L., and Priestly, B. G. (1976) Intrahepatic cholestasis induced by drugs and chemicals. Pharmacol. Rev. 28, 207-273. (15) Kosser, D. C., Meunier, P. C., Dulik, D. M., Leonard, T. B., and Goldstein, R. S. (1998) Bile duct obstruction is not a prerequisite for Type 1 biliary epithelial cell hyperplasia. Toxicol. Appl. Pharmacol. 152, 327-338.

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NMR and Pattern Recognition Studies on the Time-Related Metabolic

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