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Articles Integrated Metabonomic Analysis of the Multiorgan Effects of Hydrazine Toxicity in the Rat Sarah Garrod,† Mary E. Bollard,† Andrew W. Nicholls,†,‡ Susan C. Connor,‡ John Connelly,‡,§ Jeremy K. Nicholson,† and Elaine Holmes*,† Imperial College of Science, Technology and Medicine, Exhibition Road, London SW7 2AZ, United Kingdom, and Investigative Preclinical Toxicology, GlaxoSmithKline Pharmaceuticals, Park Road, Ware, Herts SG12 0DP, United Kingdom Received April 19, 2004
Hydrazine is a model toxin that induces both hepatotoxic and neurotoxic effects in experimental animals. The direct biochemical effects of hydrazine in kidney, liver, and brain tissue were assessed in male Sprague-Dawley rats using magic angle spinning nuclear magnetic resonance (NMR) spectroscopy. A single dose of hydrazine (90 mg/kg) resulted in changes to the biochemical composition of the liver after 24 h including an increase in triglycerides and β-alanine, together with a decrease in hepatic glycogen, glucose, choline, taurine, and trimethylamine-N-oxide (TMAO). From histopathology measurements of liver tissue, minimal to mild hepatocyte alteration was observed in all animals at 24 h. The NMR spectra of the renal cortex at 24 h after dosing were dominated by a marked increase in the tissue concentration of 2-aminoadipate (2-AA) and β-alanine, concomitant with depletions in TMAO, myo-inositol, choline, taurine, glutamate, and lysine. No alteration to the NMR spectral profile of the substantia nigra was observed after hydrazine administration, but perturbations to the relative concentrations of creatine, aspartate, myo-inositol, and N-acetyl aspartate were apparent in the hippocampus of hydrazine-treated animals at 24 h postdose. No overt signs of histopathological toxicity were observed in either the kidney or the brain regions examined. Elevated alanine levels were observed in all tissues indicative of a general inhibition of alanine transaminase activity. By 168 h postdose, NMR spectral profiles of treated rats appeared similar to those of matched controls for all tissue types indicative of recovery from toxic insult.
Introduction Hydrazine has been reported as a hepatotoxic metabolite of the antituberculosis agent isoniazid and the antihypertensive drug hydralazine (1-3) and has been extensively studied as a toxic model of steatosis (4, 5). Although widely described as a hepatotoxin, hydrazine has also been shown to have neurotoxic (6), carcinogenic, and mutagenic effects (7, 8). Accidental hydrazine poisoning in man has been reported to cause vomiting, longterm effects on the central nervous system (CNS),1 severe irritation of the respiratory tract, and hepatotoxicity (9). Hydrazine-induced steatosis has been extensively studied in animal models, but to date, the understanding of * To whom correspondence should be addressed. Tel: 00-44-20-75943230. Fax: 0044-20-7594-3226. E-mail:
[email protected]. † Imperial College of Science, Technology and Medicine. ‡ GlaxoSmithKline Pharmaceuticals. § Present address: Apotec Inc., 4100 Weston Road, Ontario M9L 2Y6, Canada. 1 Abbreviations: 2-AA, 2-aminoadipic acid; 2-AAT, 2-aminoadipate aminotransferase; 1D, one-dimensional; 2D, two-dimensional; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CNS, central nervous system; CPMG, Carr-Purcell-Meiboom-Gill; GABA, γ-aminobutyric acid; MAS, magic angle spinning; PLP, pyridoxal 5′-phosphate; TMAO, trimethylamine-Noxide; TOCSY, total correlation spectroscopy.
the definitive mechanism of toxicity remains incomplete. Electron microscopy of the livers from hydrazine-dosed rats has shown lipid droplets and swollen mitochondria in the midzonal hepatocytes 30 min after dosing (10), with subsequent changes at 2 h postdose (p.d.) including fatty vacuolation in scattered hepatocytes, which then extended to the periportal and midzonal regions by 4 h. By 24 h p.d., fatty vacuolation was more prominent, with diffuse rather than zonal distribution (10). Other histopathological studies have shown a transient depletion of hepatic glycogen, with a concomitant accumulation of fat in the periportal and midzonal hepatocytes by 24 h p.d. No abnormal structures were observed at 72 h p.d. inferring that hepatic recovery had occurred by this time (11). Previous studies have reported that the biochemical effects of hydrazine include inhibition of hypotaurine dehydrogenase, γ-aminobutryate (GABA) aminotransferase, ornithine-oxo-acid aminotransferase, tyrosine aminotransferase, alanine-oxoacid aminotransferase, and 2-aminoadipic acid aminotransferase (3). It has also been reported that the appearance of hepatic lesions following hydrazine dosing was accompanied by an increase in plasma alanine aminotransferase (ALT) activity, al-
10.1021/tx0498915 CCC: $30.25 © 2005 American Chemical Society Published on Web 01/25/2005
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though little difference in the activity of plasma aspartate aminotransferase (AST) and alkaline phosphatase (ALP) activity was detected (5). The neurotoxic effects of hydrazine, which have been shown to lead to disturbances in the CNS and seizures shortly after dosing, have been proposed to arise from alterations in the cerebral concentrations of the excitatory amino acid GABA (3). An alternative hypothesis for the CNS effect is the presence of increased levels of 2-aminoadipic acid (2-AA) in the brain (12). Previous studies have shown 2-AA to be a glial toxin causing limbic seizures and convulsions in animal models (13, 14). Several time-related biomarkers of hydrazine-induced toxicity in the rat have been elucidated using 1H NMR spectroscopic analysis of urine samples. Biochemical perturbations in the 1H NMR spectral urinary profiles included decreases in 2-oxoglutarate, citrate, succinate, hippurate, trimethylamine-N-oxide (TMAO), and glucose and increases in alanine, citrulline, N-acetyl citrulline, arginosuccinate, and taurine (5). Increased urinary levels of taurine and creatine are often observed when experimental animals are dosed with hepatotoxins, and it has been hypothesized that these concomitant events are linked through a defensive process whereby there is an increased synthesis of taurine (15). The synthesis of taurine requires cysteine as a precursor, but the synthesis of cysteine from methionine also produces creatine as a byproduct (15). In the present study, rats were dosed with a single oral dose of hydrazine (90 mg/kg) to directly probe biochemical changes in the liver, kidney, and brain (hippocampus and substantia nigra) using 1H magic angle spinning (MAS) NMR spectroscopy. The hippocampus was examined as it was the subject of previous studies due to its susceptibility to 2-AA toxicity (16). Similarities between the destruction of the dopaminergic cells in the substantia nigra, characteristic of Parkinsons disease (17, 18), and the lesions observed in rats after administration of hydrazine have also been reported; therefore, this region was also studied. 2-Aminoadipateaminotransferase, the enzyme that catalyzes the reaction between 2-AA and 2-oxoglutarate to give 2-oxoadipate and L-glutamate, is expressed in the kidney (19); therefore, the renal cortex was examined to assess the presence of 2-AA in this tissue. The direct biochemical effects of hydrazine on liver tissue were also investigated to identify and confirm biochemical variations of the commonly observed steatosis (11). 1 H NMR spectroscopy-detected perturbations in biofluids, such as urine and plasma, can give rise to surrogate markers of tissue specific toxicity (20, 21). However, because urine contains components from metabolic processes occurring throughout the body, direct analysis of tissues affords the ability to correlate characteristic alterations in biofluid composition with histopathological evidence. Recent technological advances in high-resolution 1H MAS NMR spectroscopy have provided a means of obtaining vastly improved tissue specific information (22-25). By spinning solid or semisolid samples at the magic angle (θ ) 54.7°) in the NMR probe, chemical shift anisotropies are minimized and dipolar couplings, which scale by (3cos2 θ - 1)/2, go to zero. Thus, line broadening effects, which are characteristic of spectra obtained from semisolid tissues, are minimized (26). To date, the technology has been applied to characterize the biochemical profile of biological tissues such as renal cortex and
Garrod et al.
papilla (22), red blood cells (23), liver (24), and brain (25) and to differentiate malignant and benign tissue based on variations in lipid profiles of the MAS spectra (27, 28). 1 H MAS NMR spectroscopy of liver samples from hydrazine-treated rats has shown that there is a marked elevation of tissue levels of triglycerides, particularly unsaturated fatty acids, including an increase in an ω3 type fatty acid (29). Additionally, depletions of the relative concentrations of glycogen, glucose, choline, and TMAO were noted, together with increased alanine concentrations consistent with the inhibition of alanine transaminase (29). In the current study, the metabolic consequences of hydrazine dosing were investigated in the liver, renal cortex, hippocampus, and substantia nigra of Sprague-Dawley (SD) rats using MAS NMR spectroscopy with a view to furthering the understanding of the aetiology of toxicity and the sequence of biochemical events.
Experimental Procedures Animal Handling. All work in this study was carried out in accordance with procedures under the U.K. Animal (Scientific Procedures) Act, 1986. Male SD rats (240-260 g, n ) 18) were obtained from Charles River (United Kingdom). Animals were dosed orally with either sterile water (n ) 9) or a single dose of hydrazine dihydrochloride in sterile water (n ) 9) at 90 mg/kg (10 mL/kg). The dose level was chosen from the literature to cause steatosis in SD rats (5, 12). Animals were housed by treatment group in plastic cages over paper-lined trays and were allowed access to food and drinking water ad libitum for the duration of the study. Environmental conditions were set at 21 ( 2 °C and 55 ( 10° relative humidity, with a cycle of 12 h each light/dark cycle. Three hydrazine-treated and three control animals were killed via CO2 anaesthesia at 24, 48, and 168 h p.d. The liver was removed from each animal and perfused with 0.9% NaCl in D2O via the hepatic portal vein to remove the paramagnetic effects of residual haemoglobin in the vasculature. Samples from both the left lateral and the median lobe were taken for analysis. The kidneys were removed from each animal immediately postmortem and perfused by injection of 0.9% NaCl in D2O into the renal artery via the descending aorta to remove residual blood. Sections of renal cortex were then taken for analysis. The brain was sectioned into five coronal slices, and the hippocampus and substantia nigra were removed for analysis. All tissue samples were snap-frozen in liquid nitrogen and stored at -70 °C until analyzed. 1H MAS NMR Spectroscopic Analysis of Intact Tissue. Samples were placed in zirconium oxide 4 mm diameter rotors (Bruker Analytische GmbH, Rheinstetten, Germany) and analyzed by 1H MAS NMR spectroscopy at 400.13 MHz on a Bruker DRX 400 instrument at a spin rate of 4,200 Hz and a temperature of 303 K. Typically, 128 transients were collected into 32K data points with a spectral width of 6000 Hz, using a standard one-dimensional (1D) pulse sequence with presaturation irradiating in the mixing period (100 ms) and the relaxation delay (2 s) and with an acquisition time of 3.2 s. The Carr-PurcellMeiboom-Gill (CPMG) spin-echo pulse sequence (29) (total T2 relaxation delay of 80 ms) was used to acquire spin-echo 1H MAS NMR spectra on all samples in order to attenuate macromolecular components, such as lipid resonances in tissues. For assignment purposes, two-dimensional (2D) 1H-1H total correlation spectroscopy (TOCSY) spectra were acquired on selected samples using the MLEV sequence for the spin-lock of 60 ms, with eight transients per increment for 200 increments, collected into 2K data points using a spectral width of 4000 Hz. The data were zero filled by a factor of 2 and a sine-bell apodization function applied in both dimensions prior to Fourier transformation. One-dimensional experiments were performed prior to and at the end of each 2D experiment in order to determine whether biological degradation had occurred. Reso-
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Figure 2. Stack plot of 400 MHz 1H MAS NMR spectra of liver tissue from a control rat and rats treated with hydrazine at 24, 48, and 168 h p.d. livers, and brains were processed, wax-embedded sections (5 µm) were cut, stained with hematoxylin and eosin, and examined by light microscopy.
Results
Figure 1. Haemotoxylin and eosin sections of (A) control rat liver and liver sections from rats treated with hydrazine, (B) 24 h p.d. (key: bile duct A, hepatic arteriole; B, smooth muscle; C, Kupffer cells; D, hepatocytes; and E), and (C) 48 h p.d. (key: A, focal hepatocyte necrosis; and B, polymorphic nuclei). nances were assigned based on literature data (12, 23, 31) and on comparison of 1H chemical shifts and spin-spin coupling constants with those of model compounds measured in phosphate buffer at pH 7.4. Spectra were referenced to the CH3 group of lactate at δ 1H ) 1.33. Histology. The right kidney, the liver (excluding the left lateral lobe), and the brain were dissected free of fat and connective tissue and weighed and processed for histology. Representative samples of renal and hepatic tissue for histology were fixed in 10% buffered formyl saline. After the kidneys,
Histopathological Effects of Hydrazine in the Liver. Comparison of hematoxylin and eosin sections obtained from control (Figure 1A) and hydrazine-treated rats (Figure 1B-C) indicated several distinctive features of hydrazine toxicity. At 24 h p.d. (Figure 1B), minimal to mild hepatocyte alteration was observed in all animals. This change was characterized by homogeneous acidophilic cytoplasm with fine vacuolation. At 48 h p.d. (Figure 1C), the vacuolation was prominent in periportal zones and, in two animals, was associated with multiple scattered foci indicating hepatocyte necrosis with polymorph infiltration. By 168 h p.d., mild or moderate hepatocyte alteration in 2/5 animals was indicated by increased basophilia or acidophilia with cystolic vacuolation. No overt signs of toxicity were observed in either the kidney or the neuroanatomical regions examined, although single cell necrosis was apparent in the kidneys of two rats at 24 h p.d. 1 H MAS NMR Spectroscopic Analysis of Liver Tissue. Control liver 1H MAS NMR spectra were characterized by strong resonances from triglycerides, glucose, glycogen, and a range of organic and amino acids (Figure 2 and Table 1). The dominant changes observed in the liver following hydrazine administration shown in Figure 2 included increases in the signal intensities for triglycerides and alanine, together with decreases in hepatic glycogen/glucose oligomers at 24 h p.d. The marked increase in concentrations of unsaturated fatty acids and an ω3 type fatty acid confirmed the observa-
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Table 1.
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Chemical Shifts and Assignments for Endogenous Metabolites Perturbed in Tissues after Treatment of Rats with Hydrazinea 1H
metabolite N-acetyl aspartate
alanine β-alanine 2-aminoadipate
aspartate
choline creatine
GABA R-glucose
β-glucose
glutamate
glutamine
glycogen
glycogen isoleucine
lactate lysine
myo-inositol
phosphatidylcholine phosphocholine
moiety
multiplicity
N-acetyl CH3 RCH βCH β′CH RCH βCH3 CH2 CH2 RCH βCH2 γCH2 δCH2 RCH βCH β′CH N(CH3)3 CH2 (NH) CH2 (OH) CH2 CH3 RCH2 βCH2 γCH2 C1H C2H C3H C4H C5H half CH2-C6H half CH2-C6H C1H C2H C3H C4H C5H half CH2-C6H half CH2-C6H RCH βCH2 γCH2 RCH βCH2 γCH2 C1H (R1f4) C1H (R1f4)′ C2H C3H C4H C5H C6H RCH βCH δCH3 δCH RCH βCH3 RCH βCH2 γCH2 δCH2 CH2 C1H C2H C3H C4H C5H C6H
s dd dd dd q d t t t m m t
(CH3) 3-N+ N-CH2 -PO -CH 3 2
m m m s s t q t d dd dd dd ddd dd dd d t t dd dd dd dd t m t t m m d d dd dd q q d m m t d q d t m m q t dd t dd t t t s s m m
chemical shifts (δ) 2.01 4.38 2.67 2.48 3.79 1.47 2.56 3.20 3.74 1.84 1.63 2.25 3.89 2.69 2.80 3.20 3.51 4.07 3.92 3.04 2.30 1.91 3.01 5.24 3.56 3.70 3.40 3.83 3.72 3.85 4.65 3.25 3.47 3.40 3.47 3.78 3.90 3.76 2.06 2.36 3.76 2.13 2.39 5.41 5.40 3.63 3.98 3.66 3.86 3.87 3.65 1.96 0.91 0.99 4.11 1.33 3.77 1.91 1.45 1.71 3.03 3.53 4.06 3.53 3.62 3.28 3.62 3.32 3.22 3.60 4.18
increase (v) or decrease (V) after hydrazine exposure and organ/biofluid of effect B
L, K, B, U, P L, K, U K, U, P
K, B
L, K, B B, U, P
B
L, K
L, K
K, B
K
L
K
L, K, B K
K, B
L, K L, B
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Table 1 (Continued) 1H
metabolite proline
taurine threonine
triglycerides
triglycerides
tyrosine
valine
chemical shifts (δ)
increase (v) or decrease (V) after hydrazine exposure and organ/biofluid of effect
moiety
multiplicity
RCH β′CH βCH δ′CH γCH2 N-CH2 S-CH2 RCH βCH γCH3 COO(H) CH3-(CH2)n -(CH2)n CH3-CH2-CH2 CH2-CH2-CO CHdCH-CH2-CH2 CH2-H2-CO CHdCH-CH2-CHdCHCH2-O-COR CH)CH-CH2-CH)CHCH3-CH2-CHdCH (ω-3 type fatty acid) RCH βCH β′CH C3,5Hring C2,6Hring RCH βCH γCH3 γ′CH3
t m m t m t t d m d
4.15 2.08 2.36 3.36 2.01 3.26 3.42 3.60 4.28 1.32
t
0.87 1.29 1.3 1.59 2.03 2.23 2.80 4.11-4.32 5.30
L, K, B, P L, K L L, K L, K L, K L L L, L
dd dd dd d d d m d d
3.97 3.14 3.06 6.90 7.20 3.61 2.28 0.97 1.02
K, P, U
K
L, K, B, U K, U, P
K, P
a Key: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; L, liver; K, kidney; B, brain; U, urine (taken from ref 12); and P, plasma (taken from ref 12).
Figure 3. 1H-1H MAS NMR TOCSY spectrum (400.13 MHz) of liver from a control rat (red) overlaid with a matched spectrum obtained from a rat 48 h after the administration of hydrazine (black).
tions noted by Wang et al. (29). In addition, a decrease in the intensity of signals corresponding to taurine was observed 24 h p.d. Examination of the 1H-1H TOCSY spectra (Figure 3) showed the presence of resonances at δ2.56 and δ3.20 from β-alanine in the hydrazine-treated rat but not in the control animals. These resonances were not readily observed in any of the 1D spectra due to extensive peak overlap in this spectral region. In animals
sampled at 168 h p.d., the 1H MAS NMR spectra were visually very similar to the controls. From comparison of the 1D experiments performed prior to and at the end of each 2D experiment, it was established that no significant changes in metabolite profile, due to degradation, had taken place during the time the liver tissue was in the rotor. 1 H MAS NMR Spectroscopy of Brain. 1H MAS NMR spectra acquired of tissue from the hippocampus (Figure 4) and substantia nigra (not shown) in the control rat brain were dominated by amino acids, organic acids, and osmolytes, including lactate, alanine, GABA, Nacetyl aspartate, and myo-inositol (Table 1). From analysis of the 1H MAS NMR spectra of tissues over the time course studied, it was apparent that there was a small increase in the relative concentrations of GABA, aspartate, and creatine with a decrease in myo-inositol and N-acetyl aspartate in the hippocampus after dosing. All of these alterations had returned to predose levels by 168 h p.d. (Figure 4). No biochemical changes were observed in the substantia nigra by 1H MAS NMR spectroscopic analysis following hydrazine dosing. 1 H MAS NMR Spectroscopic Analysis of Renal Tissue. The 1H MAS NMR spectra of control renal cortex were dominated by a range of organic and amino acids, glucose, TMAO, choline, and triglycerides (Figure 5). At 24 h after dosing, small changes in the triglyceride ratios, the ratios of triglyceride to lactate, were seen and these persisted at 48 h (not shown). Increased concentrations of alanine, β-alanine, and tyrosine, together with decreased levels of myo-inositol and taurine, were characteristic of renal cortical samples 24 h after hydrazine
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Figure 4. Stack plot of 400 MHz 1H MAS NMR spectra of hippocampus samples from a control rat and rats treated with hydrazine at 24, 48, and 168 h p.d.
Figure 5. 1H MAS NMR spectra (400 MHz) of renal cortex from a control rat and a rat treated with hydrazine 24 h p.d.
treatment. In addition, resonances at δ1.63, δ2.25, and δ3.74 were observed and assigned to 2-aminoadipate (Figure 5) from examination of the 2D TOCSY and from literature data (12). Correlation of these resonances together with the signal at δ1.84 was confirmed in the 1 H-1H TOCSY spectra (Figure 6). From comparison of the 1D experiments performed prior to and at the end of each 2D experiment, it was established that no significant degradation of renal tissue had occurred during the 2D acquisition.
Discussion Hydrazine-induced metabolic perturbations in liver, kidney, and brain have been detected via the application of 1H MAS NMR spectroscopy for analysis of the intact tissue. The increase in the concentration of alanine in the liver, renal cortex, and hippocampus after hydrazine
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Figure 6. 1H-1H MAS NMR TOCSY spectrum (600 MHz) from the renal cortex of a rat 24 h after hydrazine administration.
dosing was consistent with the known inhibitory effect of hydrazine on aminotransferase activity, affecting ALT in this particular case. The elevations in tissue are in agreement with reported increases in urinary alanine after hydrazine exposure (4, 12), other MAS NMR studies of hepatotoxicity (29), and in vitro studies of hydrazine toxicity in isolated hepatocytes (3). The reduction in hepatic glycogen/glucose oligomers concurs with results obtained in studies conducted by Amenta and Johnson (11) and Wang et al. (29). It was suggested that the reduction in glycogen was not a reflection of the injury to the cellular mechanism relating to glycogen synthesis but that it may have been caused by alterations to the mechanism of glycogen storage or glycogen release via direct action of hydrazine on hepatic cells or as a result of reduced food consumption causing mobilization of glycogen (11). It has been suggested that the accumulation of triglycerides and, in particular, unsaturated fatty acids observed with hepatotoxininduced steatosis is a consequence of a deficiency in the production of the apolipoprotein complex responsible for transporting triglycerides out of the liver (32). In the current work, a decrease in taurine concentration was observed in both liver and renal cortical samples at 24 and 48 h p.d. Urinary taurine has been reported to increase after the administration of toxic levels of hydrazine (12) and is also seen after toxic insult by other hepatotoxins (4, 5). The elevated excretion of taurine has been suggested to occur via a number of mechanisms, including altered membrane permeability of hepatocytes (which would correlate with the observed decrease in taurine in hepatic tissue), interference with bile salts or bile production, increased synthesis of taurine in response to the toxic challenge, inhibition of uptake in the kidney, or competition with β-alanine for uptake in the
Metabonomic Analysis of Hydrazine Toxicity in the Rat
kidney. In this study, β-alanine was shown to increase in the liver at 48 h p.d., which supported published data (33) and may result from enhanced pyrimidine catabolism (4). Taurine is extensively reabsorbed by the β-amino acid uptake system in the kidney (34), and so, an increase in β-alanine production might cause an increase in the competition for uptake between β-alanine and taurine. Concomitant changes in the urinary excretion of β-alanine and taurine have been shown to be markers of hepatotoxicity (5). Although creatinuria has previously been documented following the onset of hydrazine toxicity (12), the levels of creatine in the liver and kidney were not significantly altered in this study. A decrease in myo-inositol concentration in both the hippocampus and the renal cortex was observed after hydrazine treatment. Myo-inositol acts as an osmoregulator in many tissues; hence, the decrease in myo-inositol observed here may be a response to a change in osmolality (35, 36). Videen et al. reported a severe reduction in myo-inositol (49%), choline metabolites (36%), and N-acetyl aspartate (11%) concentrations in the human brain during episodes of hypernatremia (36). Likewise, TMAO and other choline metabolites, which also act as osmolytes, were depleted in renal cortex and hippocampus samples. The decreased hippocampal concentrations of myo-inositol, choline-related metabolites, and N-acetyl aspartate observed in the current study may, thus, be indicative of a perturbation in ion regulation and membrane disruption. One of the major urinary biomarkers of hydrazine toxicity in the rat is 2-AA (12), and although it was not detected in hippocampal spectra, it could be detected in millimolar concentrations in the kidney cortex of hydrazine-treated rats. The increased levels of 2-AA in the kidney are likely to have resulted as an indirect effect of hydrazine on the activity of aminotransferases. It has been suggested previously that the increase in 2-AA may result from reduced activity of 2-aminoadipate aminotransferase (2-AAT) since the cofactor required for function of all aminotransferases, pyridoxal 5′-phosphate (PLP), is sequestered by hydrazine (12, 37). This hypothesis is supported by the observation in the current work of an elevated alanine concentration indicating a reduction in activity for ALT and would support the fluctuations in GABA through an effect on GABA aminotransferase. In 1H MAS NMR spectroscopic investigations into the biochemical effects of hydrazine on the brain, GABA was found to increase at 48 h p.d. A hydrazine-induced increase in GABA in the brain has been recorded previously (3), where rats dosed with hydrazine showed a significant decrease in the enzyme GABA aminotransferase, the first enzyme in the degradation pathway for GABA. There was no reported increase in the GABAsynthesizing enzyme glutamate decarboxylase (3). Because 2-AA is a known glial toxin, an elevation of this compound in the periphery has been suggested to have the potential for neurological effects (12). The exact role of 2-AA in hydrazine toxicity remains to be elucidated; however, it would appear that the kidney may play a role in the production of this metabolite. In addition to the hydrazine-induced decrease in Nacetyl aspartate and myo-inositol in the hippocampus, increases in the concentration of aspartate and creatine were observed in this region of the brain at 24 h p.d. The increase in aspartate and decrease in myo-inositol has not previously been observed following hydrazine admin-
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istration. Aspartate is a ubiquitous amino acid transmitter mediating fast excitatory responses in the CNS, and it has been shown to cause excitotoxic death when extracellular concentrations are elevated (38). The increase in aspartate in these studies may aid in explaining the cause of CNS effects observed in some studies following hydrazine administration (39). Hydrazine produces a wide range of metabolic perturbations across several organs and tissues, which has been reflected in previous analyses of biofluids. However, urinary biomarkers can be misleading without an understanding of their origin. MAS NMR spectroscopy of intact tissues provides a link between histopathological evidence of lesions and toxin-induced urinary biomarkers, thereby enabling specific and general metabolic events to be deconvolved. Some urinary metabolites, such as β-alanine, taurine, tyrosine, and creatine, can be linked to hepatotoxicity whilst 2-AA is more specifically related to renal metabolism. In other cases, changes in the concentrations of urinary metabolites reflect global changes in metabolism, such as alaninuria caused by inhibition of alanine transaminase and depletion of the tricarboxylic acid intermediates, suggesting mitochondrial dysfunction. Hydrazine is known to deplete ATP both in vivo and in isolated hepatocytes and to interfere with mitochondrial ATP production (4, 40). In a previous study, electron microscopy of the livers from hydrazinedosed rats revealed swollen mitochondria in the midzonal hepatocytes (10). Alternatively, reductions in TCA cycle intermediates in the urine may be nonspecific biochemical changes, due in part to a general increase in energy metabolism and reduced food intake, rather than relating to tissue specific toxicity. Previous studies into hydrazine toxicity, in pair-fed male rats, where animals were weight matched, resulted in alterations in the levels of citrate, 2-oxoglutarate, hippurate, and creatinine in urine samples from control rats where food was restricted (41). The increase in creatine observed in the urine after hydrazine treatment may in part result from reduced food intake and body weight loss (42). Yet other alterations in urinary profile, e.g., hippurate depletion, are more likely to reflect the influence of hydrazine on gut microflora rather than endogenous processes. MAS NMR spectroscopy presents a robust means of assigning biomarkers to specific metabolic events or locations.
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