Hydrazine it has been used widely as an intermediate in industrialsynthetic chemistry and as a rocket fuel (O’Neil, 2006). Hydrazinehas also been found to be a metabolite of some important drugs,such as the antihypertensive drug hydralazine (Blair et al., 1985;Timbrell and Harland, 1979) and the antituberculosis drug iso-niazid (Blair et al., 1985; Iguchi et al., 1977). It has been reportedthat hydrazine induces hepatotoxicity (Scales and Timbrell, 1982;Timbrell et al., 1982; Waterfield et al., 1993), carcinogenicity (IARC,1999), mutagenicity (IARC, 1999), teratology (Toth, 1993) and neu-rotoxicity (Moloney and Prough, 1983). Hydrazine-induced steato-sis has been extensively studied in animal models; however, thedefinitive mechanism of toxicity has not been understood to date.
Metabolomic investigations attempt to detect and profilechanges in metabolites, which reflect changes in metabolic path-ways and may provide information concerning a disease state orthe biological stress of an organism (Lindon et al., 2004; Weckw-erth and Morgenthal, 2005). Metabolomics is increasingly beingapplied to pharmacology and toxicology studies, and in particu-lar, high-resolution 1H nuclear magnetic resonance (NMR)spectrometry-based metabolome analysis study is a well-established technique for detection of the endogenous meta-bolic changes in biofluids such as plasma and urine caused bydrug toxicity or disease processes (Clarke and Haselden, 2008;
Goldsmith et al., 2010; Lindon et al., 2007; Powers, 2009; Serkovaand Niemann, 2006). In fact, NMR-based metabolomicsapproaches were previously used to investigate hydrazine toxic-ity of animals, and it has already been reported that hydrazineinduces alterations of endogenous metabolites (Bollard et al.,2005; Garrod et al., 2005; Kleno et al., 2004; Nicholls et al., 2001;Wang et al., 2003).
However, the majority of metabolic profiling studies using com-bined gas chromatography–mass spectrometry (GC-MS) andchemometric techniques reside in the field of plant metabolomics
*Correspondence to: E. Fukusaki, Department of Biotechnology, Graduate Schoolof Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.E-mail: [email protected]
aDepartment of Biotechnology, Graduate School of Engineering, Osaka University,2-1 Yamadaoka, Suita, Osaka 565-0871, Japan
bSafety Research Laboratories, Dainippon Sumitomo Pharma Co. Ltd, 3-1-98Kasugade-naka, Konohana-ku, Osaka 554-0022, Japan
†The study represents a portion of the dissertation submitted by Kiyoko Bando toOsaka University in partial fulfillment of the requirement for her Ph.D.
Research Article
Received: 21 June 2010, Revised: 16 August 2010, Accepted: 16 August 2010 Published online in Wiley Online Library: 10 December 2010
(Bino et al., 2004; Fiehn et al., 2000; Glinski and Weckwerth, 2006).Although GC-MS has been used for the detection of many meta-bolic disorders since the 1970s (Chace, 2001), the application ofGC-MS-based metabolomics for pharmacology/toxicology is rela-tively underdeveloped compared with NMR or LC-MS. Advantagesof GC-MS include high resolution and reproducibility, as well asthe availability of a reliable electron impact (EI) mass spectrallibrary database for metabolite identification or confirmation.Liquid chromatography (LC/MS) mass spectral libraries are, inmost cases, instrument-dependent, and therefore standard refer-ence LC/MS libraries are unavailable for general use, making theidentification efficiency even lower for metabolites (Bino et al.,2004). Additionally, GC-MS has great advantages for analyzingorganic acids and amino acids, which are often targets in efficacyand/or toxicity studies. Therefore, metabolic profiling usingGC-MS has potential as a powerful tool in toxicological evalua-tions, providing a comprehensive understanding of the responseof biological systems to xenobiotic intervention (Lee et al., 2009;Lee et al., 2007; Pasikanti et al., 2008).
In the present study, we investigated the applicability of aGC-MS-based metabolomics approach for toxicological evalua-tion, and tried to elucidate the mechanism of toxicity of hydra-zine using metabolic profiles derived from GC-MS.
MATERIALS AND METHODS
Chemicals
Hydrazine dihydrochloride and EDTA-2K and were purchasedfrom Kanto Chemical Co. Inc. (Tokyo, Japan). Sodium heparin(Novo-Heparin for injectionTM) was purchased from MochidaPharmaceutical Co. Ltd. (Tokyo, Japan). Saline, a Japanese Phar-macopoeia standard product (Saline PL ‘Fuso’), was purchasedfrom Fuso Pharmaceutical Industries Ltd (Osaka, Japan). Isoflu-rane (IsofluTM) was obtained from Dainippon Sumitomo PharmaCo. Ltd (Osaka, Japan). Chemicals used in GC-MS analysis were asfollows: methoxyamine hydrochloride from Sigma Aldrich Corp.(St Louis, MO, USA), pyridine from Wako Pure Chemical IndustriesLtd (Osaka, Japan), chloroform and methanol from KishidaChemical Co. Ltd (Osaka, Japan), and N-methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA) from GL Science Inc. (Tokyo, Japan).
Animals
Male Crl:CD(SD) rats (SPF) were purchased from Charles RiverJapan Inc. (Hino Breeding Facility, Japan). Two rats per cage werehoused in aluminum wire mesh cages or rats were housed indi-vidually in metabolic cages during the collection of urine, andmaintained in an air-conditioned animal room (temperature
24.0 � 2.0 °C, relative humidity 55.0 � 15.0%) with a 12 h light–dark cycle (fluorescent lighting from 8:00 to 20:00) and more than10 air changes per hour. The rats received pelleted basal diet(CRF-1; Oriental Yeast Co. Ltd, Chiba, Japan) and tap water adlibitum. However, diet was removed during urine collection. Afterquarantine/acclimatization for 7 days, hydrazine was adminis-tered to rats at 7 weeks of age (body weight 228–278 g at 7 weeksof age).
Animal Study Design
Animals were randomly assigned to six groups (eight animals/group), and three of the groups were allocated to a samplinggroup for sampling at 24 h post-dosing, while the other threegroups were allocated to a sampling group for sampling at 48 hpost-dosing. The dosage levels of hydrazine dihydrochloridewere 0, 120 and 240 mg kg-1. The high dosage was selected as adosage level expected to show marked hepatotoxicity, and a halfdose of the high dose was set as a low dose to observe thedose-dependence of toxicity.
Hydrazine dihydrochloride was dissolved in saline, and dosingformulation volumes were calculated based on body weights ofanimals (5 ml kg-1). Saline was administered to rats of the0 mg kg-1 groups (control groups). The dosing formulations weredrawn into polypropylene syringes, and were promptly adminis-tered once orally via intubation.
Figure 1 shows a urine and plasma sampling design. Animalswere housed in metabolic cages for 8 h from 16 to 24 h post-dosing and from 40 to 48 h post-dosing, and then urine sampleswere collected. During urine pooling in metabolic cages, theurine collection tubes were frozen on dry ice. Pooled urinesamples were thawed in a refrigerator after the collection period,all urine samples were centrifuged at 540g for 5 min at 5 °C, anda portion of supernatant was used for measurement of creatinineconcentration. Creatinine concentrations of urine samples weremeasured by a sarcosine oxidase–peroxidase method (JCA-BM1650, JEOL, Japan). Remaining urine samples were dividedinto sub-samples, frozen rapidly using liquid N2, and stored in adeep freezer (-80 °C) until GC-MS analysis.
Blood samples were drawn via the abdominal aorta under isof-lurane anesthesia from animals at 24 and 48 h post-dosing. Bloodsamples were divided into two sub-samples. One was treatedwith EDTA-2K and the other was treated with sodium heparin asanticoagulant, then both were centrifuged at 2150g for 10 min at5 °C, and supernatants (plasma) were divided into sub-samples,frozen rapidly using liquid N2, and stored in a deep freezer(-80 °C) until analysis.
Figure 1. Two sampling groups were designed: 24 h and 48 h post-dosing groups. Urine samples were pooled for 8 h from 16 to 24 h post-dosing andfrom 40 to 48 h post-dosing. Just after pooling urine, blood was drawn from animals, and plasma samples were obtained. Then, animals were necropsiedand liver samples were obtained.
The present study was conducted in compliance with the ‘LawConcerning the Protection and Control of Animals, with PartialAmendments (Law No. 68, Jun. 22, 2005, Japan)’ and in-houseguidance.
Blood Biochemistry
Plasma, obtained by treatment with heparin, was used for deter-mining the flowing blood biochemistry parameters: aspartateaminotransferase (AST), alanine aminotransferase (ALT), alkalinephosphatase (ALP), lactate dehydrogenase (LDH), total bilirubin(T-Bil), g -glutamyl transpeptidase(g -GTP), creatine kinase (CK),total cholesterol (T-Cho), phospholipids (PL), triglycerides (TG),glucose (Glu), urea nitrogen (BUN), creatinine (Cre), inorganicphosphorus (P), calcium (Ca), sodium (Na), potassium (K) andchloride (Cl). Measurements were performed using a clinical bio-chemistry analyzer (JCA-BM1650, JEOL, Japan).
Histopathology
All animals were necropsied, and liver was removed and fixed in10% neutral buffered formalin. Liver specimens were trimmed,embedded in paraffin wax, sectioned and stained with hema-toxylin and eosin (H&E) for histopathological examination. Thesections were also stained by periodic acid–Schiff (PAS) methodwith or without diastase digestion.
Sample Preparation for GC-MS Analysis
Mixed solvent (250 ml; CHCl3:CH3OH:H2O, 1 : 2.5 : 1, v/v) was mixedinto 50 ml of urine or plasma samples with 90 ml of 0.2 mg ml-1
solution of ribitol, which was used as an internal standard. Thesamples were shaken for 30 min at 37 °C and centrifuged at 16000g for 3 min at 4 °C. A 250 ml portion of the supernatant wastransferred to an Eppendorf tube with a pierced cap. The sampleswere dried first in a vacuum centrifuge dryer and then a freezedryer. For derivatization, 100 ml of methoxylamine hydrochloridein pyridine (20 mg ml-1) – the first derivatizing agent – wasadded to the samples. The mixture was incubated at 30 °C for90 min. The second derivatizing agent, i.e. 50 ml N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA), was added, and themixture was incubated at 37 °C for 30 min. A 1 ml sample wasinjected in split mode (urine, 25 : 1, v/v; plasma, 10 : 1, v/v).
GC-MS Analysis
Chromatography was performed on an 6890N gas chromato-graph system (Agilent Co., Palo Alto, CA, USA) equipped with afused-silica capillary column (internal diameter, i.d., 30 m ¥0.25 mm) coated with a CP-SIL 8 CB low-bleed film (thickness,0.25 mm; Varian Inc., Palo Alto, CA, USA) that was connected to aPegasus III TOF mass spectrometer (LECO, St Joseph, MI, USA)equipped with an autosampler 7683B series injector (Agilent Co.,Palo Alto, CA, USA). The injection temperature was 230 °C. Thehelium gas flow rate through the column was 1 ml min-1. Thecolumn temperature was isothermally maintained at 80 °C for2 min and then raised to 330 °C at the rate of 15 °C min-1; thetemperature was isothermally maintained at 330 °C for 6 min. Thetemperatures of the transfer line and the ion source were 250 and200 °C, respectively. Ions were generated at an electron impact
(EI) energy of 70 kV, and 20 scans s-1 were recorded over the massrange of m/z 85–500. The acceleration voltage was appliedafter a 428 and 250 s solvent delay on urine and plasma samples,respectively.
Analysis of GC-MS Data
Baseline correction and automatic peak detection with Chro-maTOF (LECO, St Joseph, MI, USA) software were performed witha peak width of 1.333. Peaks with signal-to-noise (S/N) ratioslower than 10 were rejected, and raw chromatographic data(Pegasus file, *.peg) were converted into ANDI files (AnalyticalData Interchange Protocol, *.cdf ). ANDI formatted data can beconverted and transferred among different mass spectral equip-ment. The ANDI formatted data were read into LineUp (InfometrixInc., Bothell, WA, USA) and PiroTrans (GL Science Inc. Tokyo,Japan), where alignment of chromatogram and normalization ofintensity were performed on the basis of ribitol peak in plasmasamples. Meanwhile, chromatogram alignment was performedon the basis of a particular peak (retention time: around 8.8 min),which had appeared in chromatograms of all urine samples(probably a peak derived from derivatization reagents), andintensities of each peak were normalized by creatinine concen-trations of each animal, because 2-aminoadipate, which wasremarkably elevated in urine by hydrazine treatment, co-elutedwith ribitol in urine samples.
The generated peak lists were imported into SIMCA-P+(version 12; Umetrics, Umeå/Malmö, Sweden) for multivariatestatistical analysis. Principal component analysis (PCA) was con-ducted to find alterations of metabolic profiling induced byhydrazine treatment.
Identification of Metabolites and Semi-quantification
Remarkably altered metabolites and associated metabolitesinduced by hydrazine were identified and relative intensitieswere calculated. Metabolites were identified by mass spectralpatterns and retention times on the basis of an in-house chemicallibrary; 82 and 67 metabolites were identified in the urine andplasma samples, respectively. Total intensities of each metabolitepeak in plasma were normalized by the total intensity of theribitol peak, while total intensities of each metabolite peak inurine were normalized by the intensity of unique mass (m/z 319)of ribitol peak and creatinine concentration. The unique mass(m/z 319) of ribitol was not included in the mass spectrum of2-aminoadipate, which co-eluted with ribitol in urine samplesof the hydrazine treatment groups, and therefore the intensity ofthe unique mass was considered an appropriate normalizationfactor.
RESULTS
Toxicological Examination
Table 1 shows prominent alterations in parameters of the bloodbiochemistry in rats treated with hydrazine. Several parametersremarkably changed in the hydrazine-treated groups; thesechanges were dose-dependent at 24 and 48 h post-dosing.Changes at 48 h post-dosing were less than those at 24 hpost-dosing, which indicated that 48 h post-dosing was on the
convalescent phase. Typical conventional hepatotoxicity biomar-kers, aspartate aminotransferase (AST) and alanine aminotrans-ferase (ALT), decreased in the hydrazine-treated group. Thesephenomena were previously reported (Waterfield et al., 1993).Hydrazine and its analogs have been shown to lead to sequestra-tion of the aminotransferase cofactor pyridoxal 5-phosphate(PLP), inhibiting aminotransferase activity (Cornish, 1969; Light-cap and Silverman, 1996), which may explain the reduced levelsof ALT and AST in the rat, despite the observation of liver toxicity,thus conventional hepatotoxicity biomarkers cannot predicthydrazine induced hepatotoxicity. A few lipid parameters (i.e.total cholesterol, phospolipid, and triglyceride) and total proteindecreased dose-dependently, which may suggest metabolicturnover of lipid and protein were lower in the liver by hydrazinetreatment. In addition, the level of BUN slightly increased at 24 hpost-dosing in a dose dependent manner, which may suggestkidney hypofunction.
The incidences of the histopathological findings in the liver arepresented in Table 2, and representative photographs are pro-
vided in Figure 2. Figure 2(A and B) shows centrilobular to mid-zonal areas of the liver in control and hydrazine-treated rats,respectively. In Fig. 2(A), normal hepatocytes surrounding thecentral vein, which is located in the center of the picture, can beobserved. In hydrazine-treated rats, fatty degenerations charac-terized by fine cytoplasmic vacuoles in hepatocytes were seen inthe midzonal areas (Fig. 2B). In addition, anisonucleosis andsingle cell necrosis of the hepatocytes were observed (Fig. 2C, D).Figure 2(C) shows the nuclei of hepatocytes which are irregular insize and Fig. 2(D) shows isolated extracellular, eosinophilicbodies, which are characteristic in single cell necrosis. These find-ings were seen to occur in a dose dependent manner and moreprominent at 24 h post-dosing than at 48 h post-dosing, whichindicated 48 h post-dosing was on the convalescent phase, in thesame manner as with blood biochemistry parameters. Moreover,hydrazine-treated rats showed minimal to mild increases of PAS-positive granules in hepatocytes of the centrilobular areas(Fig. 2E, F). These granules were digested by diastase (datanot shown), revealing that they consisted of glycogen. All the
Table 1. Alterations in the parameters of blood biochemistry in rats treated with hydrazine
AST, aspartate aminotransferase; ALT, alanine aminotransferase; TP, total protein; TC, total cholesterol; PL, phospholipid; TG, triglyc-eride; BUN, urea nitrogen.*Significantly different from the 0 mg kg-1 group (24 h) (P < 0.05); **significantly different from the 0 mg kg-1 group (24 h) (P < 0.01).†Significantly different from the 0 mg kg-1 group (48 h) (P < 0.05); ††significantly different from the 0 mg kg-1 group (48 h) (P < 0.01).
Table 2. Treatment-related histopathological findings of the liver in rats treated with hydrazine
histopathological findings indicated the toxic effects of hydra-zine, which induced steatosis, as in preliminary reports (Scalesand Timbrell, 1982; Timbrell et al., 1982).
Metabolic Profiling of Urine and Plasma after Treatmentwith Hydrazine
First, metabolome analysis was performed via non-targetapproach; all peak data of GC-MS chromatograms were usedfor multivariate analysis after alignment and normalization.Figure 3(A) shows a score plot of principal component analysis(PCA) of urine data. Each group was clearly clustered dependingon dosages or sampling time after hydrazine treatment; spots of240 mg kg-1 groups were farther from those of 0 mg kg-1 groupsthan were those of 120 mg kg-1 groups. Furthermore, spots of48 h post-dosing groups were closer to those of 0 mg kg-1 groupsthan were those of 24 h post-dosing groups; in particular, spots of120 mg kg-1 group at 48 h post-dosing group were plotted at thesame area of those of 0 mg kg-1 groups. These results suggestedthat metabolic profiling showed dose-dependent toxicity andrecovery induced by hydrazine. The corresponding loading plotrevealed metabolites that were responsible for group separation(Fig. 3C), and those metabolites were identified by mass spectraand retention times on the basis of an in-house chemical library.Highly contributing metabolites found to be elevated in thehydrazine-treated groups were 2-aminoadipate and b-alanine,
whereas highly contributing metabolites found to be decreasedin the hydrazine-treated groups were 2-oxoglutarate and citrate.
The PCA score plot of plasma metabolic profiling showed doseand time dependency as did urine metabolic profiling (Fig. 3B);however, group separation was less clear than in those of plasma.The corresponding loading plot revealed glucose decreased inthe hydrazine treatment group (Fig. 3D); however, individualvariation in glucose levels was large, which may have led to lessgroup separation on the score plot. Highly contributing metabo-lites other than glucose found to be elevated in the hydrazine-treated groups were lactate and tyrosine.
Alterations of Metabolites Associated withGlutathione Metabolism
Next, remarkably altered metabolites and associated metabolitesinduced by hydrazine were identified, and relative intensities ofeach metabolite were calculated. The mapping of these data intogeneral biochemical pathways as illustrated in the Kyoto Encyclo-pedia of Genes and Genomes (KEGG) revealed significant biologi-cal impact of alterations of metabolic profiling by hydrazinetreatment. We found that the glutathione metabolism pathwaywas the most noteworthy in relation to hydrazine-induced toxic-ity. Figure 4 shows glutathione metabolism pathway and relativeintensities of associated metabolites. Intermediate metabolites ofglutathione, namely cystein, glutamate and glycine in the urine
Figure 2. Histopathology of the liver 24 h after dosing. No abnormality was detected in control rats (0 mg kg-1) (A). In hydrazine-treated rats(240 mg kg-1), fatty degenerations in the midzonal areas (B), anisonucleosis (C) and single cell necrosis (arrowhead) (D) were observed. H&E. In contrastto the control rats (0 mg kg-1) (E), hydrazine-treated rats (240 mg kg-1) showed increases in PAS-positive granules in hepatocytes of the centrilobularareas (F).
and/or plasma were elevated by hydrazine treatment at 24 hpost-dosing. 5-Oxoproline, a product of glutathione metabolism,also slightly increased, which is an indicator for glutathione bio-synthesis flux and oxidative stress (Lu, 2009; Metges et al., 1999,2000; Yu et al., 2002). These alterations indicated hydrazine-induced oxidative stress, although glutathione (GSH) and glu-tathione disulfide (GSSG) could not be directly measured. At 48 hpost-dosing, levels of the metabolites had recovered: particularly,the levels in the120 mg kg-1 group were generally comparable tothose in the 0 mg kg-1 group. In addition, ascorbate, a typicalanti-oxidant (Foyer, 2001; Sies, 1997) was also remarkablyelevated in the urine of 240 mg kg-1 groups, and the levels at 48 hpost-dosing were almost same as those at 24 h post-dosing. Thismay suggest continuous response to hydrazine-induced oxida-tive stress. The levels of putrescine also were elevated, which mayrelate with alterations in the urea cycle, as described below.
Alterations of Metabolites Associated with the Tricarboxylicacid (TCA) Cycle
One of the dramatic changes observed in the present study wasthe reduction of levels of the TCA cycle metabolites. Figure 5
shows the TCA cycle and alterations of relative intensities ofintermediate metabolites. At 24 h post-dosing, almost interme-diate citrate, 2-oxoglutarate, succinate and fumarate, werefound to be decreased dose-dependently in urine and/orplasma. Changes of some metabolites, which were detected inboth urine and plasma, were more dramatic in urine than inplasma. These results indicated that hydrazine may cause alter-ation in the TCA cycle. On the contrary, the levels of pyruvateand lactate in plasma and urinary glucose were elevated in thehydrazine treatment groups. At 48 h post-dosing, levels ofthe metabolites had recovered: particularly, the levels in the120 mg kg-1 group were generally comparable to those in the0 mg kg-1 group.
Alterations of Metabolites Associated with Urea Cycle
Participants in the urea cycle were at higher levels after hydrazinetreatment when compared with 0 mg kg-1 groups, including cit-rulline and ornithine in urine or plasma at 24 h post-dosing(Fig. 6). Accordingly, the levels of associate metabolites, namelyfumarate and urea, also increased. These results indicatedup-regulation of the urea cycle. The levels of putrescine andGABA showed increases by hydrazine treatment.
Figure 3. Score plots and the corresponding loading plots of principal component analysis. PCA score plot derived from GC-MS spectra of urine (A) andplasma (B) samples. Corresponding loading plots of PC 1 and PC 2 in urine (C) and plasma (D). Twenty-four hour groups: 0 mg kg-1, solid circles;120 mg kg-1, solid triangles; 240 mg kg-1, solid squares. Forty-eight hour groups: 0 mg kg-1, open circles; 120 mg kg-1, open triangles; 240 mg kg-1, opensquares.
Alterations of Metabolites Associated withLysine Metabolism
Amino acid metabolisms were altered by hydrazine treat-ment: among them, the most remarkable changes wereobserved in the lysine metabolism (Fig. 7). The levels oflysine and cadaverine were increased by hydrazine treatment inurine: these changes persisted at 48 h post-dosing. Further-more, the level of 2-aminoadipate was remarkably increased inboth plasma and urine, although it was not detected in urine orplasma of 0 mg kg-1 groups. This result suggested that lysineexcretion and degradation were accelerated.
DISCUSSION
We investigated the applicability of a GC-MS-based metabolom-ics approach for toxicological evaluation. Before identification ofmetabolites, all peak data of chromatograms obtained by GC-MSwas included in principal component analysis, a typical unsuper-vised statistical method. The result showed clear group separa-tion of spots, depending on dosages of hydrazine and samplingtimes after treatment. In particular, results for a group thatshowed most prominent toxicity in the toxicological examina-tion, the 240 mg kg-1 group at 24 h post-dosing, were spotted in
Figure 4. Alterations of metabolites in the glutathione and associated metabolism pathway. Vertical axis of graphs is relative intensity (mean � SD) ofeach metabolite peak. Total intensities of each metabolite peak in plasma were normalized by the total intensity of ribitol peak, while those in urine werenormalized by the intensity of unique mass (m/z 319) of ribitol peak and creatinine concentration. C, 0 mg kg-1; L, 120 mg kg-1; H, 240 mg kg-1.
the farthest area from 0 mg kg-1 groups. On the contrary, resultsfor a group that showed less toxicity (indications of recovery) inthe toxicological examination, the 120 mg kg-1 group at 48 hpost-dosing, were spotted in the almost same area of 0 mg kg-1
groups. GC-MS analysis data reflected hydrazine-induced toxicitywith extreme sensitivity and related well to toxicological param-eters. The results of the present study suggested that non-targeted fingerprinting derived from chromatogram data ofGC-MS has good potential for applicability in efficacy and toxicityscreening, classification, or ranking.
Previous metabolic profiling using NMR (Bollard et al., 2005;Kleno et al., 2004; Nicholls et al., 2001; Wang et al., 2003) showedresults with similar tendency, but metabolic profiling by GC-MSrevealed toxicity and recovery more clearly in the present study.One of the possible reasons is that GC-MS has higher sensitivityand selectivity than NMR, and that GC-MS has great advantages
for analyzing organic acids and amino acids, which dramaticallychanged with hydrazine treatment. The latter point is importantfor applicability of metabolomics to pharmacology and toxicol-ogy. In fact, we were able to find that 2-aminoadipate andb-alanine increased, and that 2-oxoglutarate and citratedecreased in the urine using the corresponding loading plot, andthus we could understand alterations of organic and amino acidmetabolism and obtain possible biomarkers that can predicthydrazine-induced toxicity.
We indentified compounds by mass spectral patterns andretention times on the basis of an in-house chemical library withhigh accuracy, but even without an in-house library, compoundscan be identified by comparison with an open mass spectrallibrary database that can be commercially obtained, such as theNational Institute of Standards and Technology GC library. Datacollections for LC-MS mass spectral libraries are in progress, such
Figure 5. Alterations of metabolites in the TCA cycle and associated metabolism pathway. Vertical axis of graphs is relative intensity (mean � SD) ofeach metabolite peak. Total intensities of peak of each metabolite in plasma were normalized by the total intensity of ribitol peak, while those in urinewere normalized by the intensity of unique mass (m/z 319) of ribitol peak and creatinine concentration. C, 0 mg kg-1; L, 120 mg kg-1; H, 240 mg kg-1.
Figure 6. Alterations of metabolites in the urea cycle and associated metabolism pathway. Vertical axis of graphs is relative intensity (mean � SD) ofeach metabolite peak. Total intensities of peak of each metabolite in plasma were normalized by the total intensity of ribitol peak, while those in urinewere normalized by the intensity of the unique mass (m/z 319) of ribitol peak and creatinine concentration. C, 0 mg kg-1; L, 120 mg kg-1; H, 240 mg kg-1.
Figure 7. Alterations of lysine degradation pathway. Vertical axis of graphs is relative intensity (mean � SD) of each metabolite peak. Total intensitiesof the peak of each metabolite in plasma were normalized by the total intensity of ribitol peak, while those in urine were normalized by the intensity ofunique mass (m/z 319) of ribitol peak and creatinine concentration. C, 0 mg kg-1; L, 120 mg kg-1; H, 240 mg kg-1.532
as the Mass Bank (http://www.massbank.jp/), the KNApSAcK(http://kanaya.naist.jp/KNApSAcK/) and the Human MetabolomeDatabase (HMDB) (http://www.hmdb.ca/), while LC-MS librariesare not yet available for general use, because mass spectral datasometimes depend on instruments or analysis conditions. There-fore, in terms of abundance and availability of mass spectrallibrary data for compound identification, compared with LC-MS,GC-MS has a greater advantage for applicability of metabolomicsto the pharmacology or toxicology, which need biological inter-pretation and discussion of results of metabolome analysis.
Hydrazine is a typical hepatotoxicity compound; however, itstoxicity mechanism has not been completely elucidated. In thepresent study, we could discuss mechanism of hydrazine-induced toxicity based on semi-quantification data of manyidentified metabolites. Most notable changes were observed inthe glutathione metabolism pathway. Glutathione (GSH) is atripeptide (L-g-glutamyl-L-cysteinyl-glycine) that is synthesizedin the cytosol from the precursor amino acids glutamate, cys-teine and glycine. It is the most abundant redox molecule andis critical in maintaining redox status in cells. GSH plays a keyrole in the detoxification of reactive oxygen species, reactivenitrogen species and xenobiotic compounds in cells. In liver,depletion of GSH has been shown to induce apoptosis of hepa-tocytes. GSH homeostasis is an important mechanism in medi-ating the pathogenesis of many liver diseases (Han et al., 2006;Yuan and Kaplowitz, 2009). While glutathione was not directlymeasured in the plasma and urine samples in the present study(it is typically detected in cell or tissue samples), higher levelsof metabolites in the glutathione biosynthetic pathway (i.e.glutamate, cysteine, and glycine) and associated metaboliteswere found in the present study. This indicated that thispathway was accelerated, most likely due to higher cellulardemand for glutathione to combat oxidative stress. The synthe-sis of GSH is up-regulated during oxidative stress and inflamma-tion (Biswas and Rahman, 2009). In addition, higher levels ofascorbate were observed. Ascorbate participates in a variety ofenzymatic reactions (e.g. collagen and catecholamine synthesis)as an electron donor and it is one of the most important water-soluble antioxidants of mammalian tissues (Levine and Morita,1985; Levine, 1986; Meister, 1994; Rose and Bode, 1993; Winkleret al., 1994). The GSH and ascorbate can mutually regulate thesynthesis of each other (Banhegyi et al., 1997): GSH deficiencyproduced by the in vivo administration of buthionine sulfox-imine increases ascorbate synthesis in mice (Martensson andMeister, 1992). Additionally, the glutathione-depleting agentsinvestigated (buthionine sulfoximine, menadione, diamide andacetaminophen) increased glycogen breakdown and ascorbateproduction in isolated murine hepatocytes (Braun et al., 1996).Based on these findings, oxidative stress and GSH deficiency areconsidered to be primary modes of action of hydrazine-inducedtoxicity.
Additionally, many TCA cycle intermediates decreased afterhydrazine treatment. It is indicated that hydrazine inhibited activ-ity of enzymes that mediate reactions in the TCA cycle, suchas citrate synthase (E.C. 2.3.3.1), isocitrate dehydrogenase(E.C. 1.1.1.4.2) and succinate dehydrogenase (E.C. 1.3.5.1). Ojimaet al. reported that the activity of citrate synthase and mRNAexpressions of the enzymes in TCA cycle were repressed by abouthalf under oxidative stress conditions, as compared with thoseunder normal conditions (Ojima et al., 2008). It is considered thathydrazine-induced-oxidative stress caused down-regulation ofthe TCA cycle.
In the present study, plasma pyruvate and lactate levels alsoincreased. Lactate is produced through anaerobic glycolysis inthe liver tissue. The buildup of lactate in cells can be caused bylactate overproduction or underutilization (Luft, 2001). Overpro-duction of lactate, also termed type A lactic acidosis, occurs whenthe body must regenerate ATP without oxygen (tissue hypoxia).Underutilization involves removal of lactate by oxidation orconversion to glucose. Liver disease, inhibition of gluconeoge-nesis, pyruvate dehydrogenase deficiency and uncoupling ofoxidative phosphorilation are the most common causes ofunderutilization. Ojima et al. also suggested that the oxidative-stress-suffering cells switch the metabolic pathway into a ‘sup-pressed aerobiosis’, possibly for lowering the generation ofreactive oxygen species (Ojima et al., 2008). Therefore, down-regulation of the TCA cycle may lead to underutilization of pyru-vate and lactate. Thus, general down-regulation of energymetabolism might lead to alterations of sugar or lipid metabo-lism, which might cause histopathological changes (i.e. fattydegeneration and glycogen accumulation) in the liver.
Increase of urinary glucose was observed in the hydrazinetreatment group, although glucose levels in the plasma showedlittle change. Under normal conditions, urinary glucose is notdetected, because glomerular filtrated glucose is reabsorbed inthe renal tubule in the kidney (Guyton and Hall, 2006). In addi-tion, the level of BUN, a conventional biomarker of kidney func-tion, increased at 24 h post-dosing in blood biochemistry.Therefore, elevation of urinary glucose levels suggested kidneymalfunction, rather than TCA cycle alterations.
Furthermore, up-regulation of the urea cycle was observed inthis study. The urea cycle is a cycle of biochemical reactions thatproduces urea from ammonia in the liver. Biosynthesis of aminoacids was up-regulated by hydrazine treatment, glutathionemetabolism, and lysine metabolism, so the urea cycle might beexpected to be up-regulated in order to remove excess ammoniaderived from elevated amino acids. In addition, the amino groupsof amino acids that have been used as metabolic fuel are con-verted into urea through the urea cycle, which occurs mainly inthe liver.
The levels of putrescine showed increases by hydrazine treat-ment. It is plausible that the putrescine elevation is accompaniedby up-regulation of urea cycle. On the other hand, previousstudies showed that putrescine exhibits a protective effectagainst acute liver injury caused by other hepatotoxins such ascarbon tetrachloride, D-galactosamine and cadmium (Nagoshiet al., 1994; Nishiguchi et al., 1990; Tzirogiannis et al., 2004).Polyamines have been reported to inhibit lipid peroxidation andoxidative damage caused by ferric iron and free radicals by actingas scavengers, to stabilize biological membranes and to modu-late the production of secondary messengers and inflammatorymediators as well as the activity of enzymes and ionic carriers(Heby, 1981; Schuber, 1989; Tadolini et al., 1984). Thus, anotherexplanation for elevated putrescine may be an adaptive cellularresponse for damaged hepatocytes.
Hydrazine treatment had very dramatic impacts on aminoacids and amino acid metabolism; in particular, lysine metabo-lism was remarkably affected. The levels of 2-aminoadipateincreased in both urine and plasma, as previously reported inNMR-based metabolomics studies (Holmes et al., 2000; Nichollset al., 2001; Perry et al., 1981). It is likely that hydrazine inhibitsactivity of 2-aminoadipate aminotransferase (EC 2.6.1.39), whichis responsible for the catalysis of the reversible transamination of2-aminoadipate and 2-oxoglutarate to form 2-oxoadipate and
glutamate, because hydrazine reduces the activity of aminotrans-ferases by removal cofactors, as previously described. Elevationof 2-aminoadipate may be related to hydrazine-induced neuro-toxicity, because 2-aminoadipate causes apoptosis of primaryastrocytes in the brain or interferes with neurotransmitters(Chang et al., 1997; Haugstad and Langmoen, 1997; Nishimuraet al., 2000).
CONCLUSION
We demonstrated that a GC-MS-based metabolomics approachcould reveal alterations of metabolic profiles, which related wellto dose-dependent toxicity and recovery induced by hydrazinetreatment. Additionally, many identified metabolites could bediscussed in terms of the biochemical metabolic pathway basedon the alterations, and from this we found that oxidative stressplays an important role in the etiology of hydrazine-inducedhepatotoxicity. Many other biochemical pathways, such asenergy metabolism and amino acid metabolism, can be associ-ated with liver malfunction, and thus we can simultaneouslyoverviewed living organisms through large amounts of detailedinformation about biochemical metabolism. The GC-MS-basedmetabolomic approach will be a useful tool for pharmacologyand toxicology, in screening, elucidating modes of action, andbiomarker discovery.
Acknowledgment
The authors would like to thank K. Horii, S. Mikami and E. Saijo ofDainippon Sumitomo Pharma. Co. Ltd (Japan), and M. Oyanagi ofSumika Technoservice Co. Ltd (Japan) for conducting animalexperiments.
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