Identification of Novel Toxicity-associated Metabolites by Metabolomics and Mass Isotopomer Analysis of Acetaminophen Metabolism in Wild-type and Cyp2e1-null Mice
2007; Elsevier BV; Volume: 283; Issue: 8 Linguagem: Inglês
10.1074/jbc.m706299200
ISSN1083-351X
AutoresChi Chen, Kristopher W. Krausz, Jeffrey R. Idle, Frank J. Gonzalez,
Tópico(s)Pharmacogenetics and Drug Metabolism
ResumoCYP2E1 is recognized as the most important enzyme for initiation of acetaminophen (APAP)-induced toxicity. In this study, the resistance of Cyp2e1-null mice to APAP treatment was confirmed by comparing serum aminotransferase activities and blood urea nitrogen levels in wild-type and Cyp2e1-null mice. However, unexpectedly, profiling of major known APAP metabolites in urine and serum revealed that the contribution of CYP2E1 to APAP metabolism decreased with increasing APAP doses administered. Measurement of hepatic glutathione and hydrogen peroxide levels exposed the importance of oxidative stress in determining the consequence of APAP overdose. Subsequent metabolomic analysis was capable of constructing a principal components analysis (PCA) model that delineated a relationship between urinary metabolomes and the responses to APAP treatment. Urinary ions high in wild-type mice treated with 400 mg/kg APAP were elucidated as 3-methoxy-APAP glucuronide (VII) and three novel APAP metabolites, including S-(5-acetylamino-2-hydroxyphenyl)mercaptopyruvic acid (VI, formed by a Cys-APAP transamination reaction in kidney), 3,3′-biacetaminophen (VIII, an APAP dimer), and a benzothiazine compound (IX, originated from deacetylated APAP), through mass isotopomer analysis, accurate mass measurement, tandem mass spectrometry fragmentation, in vitro reactions, and chemical treatments. Dose-, time-, and genotype-dependent appearance of these minor APAP metabolites implied their association with the APAP-induced toxicity and potential biomarker application. Overall, the oxidative stress elicited by CYP2E1-mediated APAP metabolism might significantly contribute to APAP-induced toxicity. The combination of genetically modified animal models, mass isotopomer analysis, and metabolomics provides a powerful and efficient technical platform to characterize APAP-induced toxicity through identifying novel biomarkers and unraveling novel mechanisms. CYP2E1 is recognized as the most important enzyme for initiation of acetaminophen (APAP)-induced toxicity. In this study, the resistance of Cyp2e1-null mice to APAP treatment was confirmed by comparing serum aminotransferase activities and blood urea nitrogen levels in wild-type and Cyp2e1-null mice. However, unexpectedly, profiling of major known APAP metabolites in urine and serum revealed that the contribution of CYP2E1 to APAP metabolism decreased with increasing APAP doses administered. Measurement of hepatic glutathione and hydrogen peroxide levels exposed the importance of oxidative stress in determining the consequence of APAP overdose. Subsequent metabolomic analysis was capable of constructing a principal components analysis (PCA) model that delineated a relationship between urinary metabolomes and the responses to APAP treatment. Urinary ions high in wild-type mice treated with 400 mg/kg APAP were elucidated as 3-methoxy-APAP glucuronide (VII) and three novel APAP metabolites, including S-(5-acetylamino-2-hydroxyphenyl)mercaptopyruvic acid (VI, formed by a Cys-APAP transamination reaction in kidney), 3,3′-biacetaminophen (VIII, an APAP dimer), and a benzothiazine compound (IX, originated from deacetylated APAP), through mass isotopomer analysis, accurate mass measurement, tandem mass spectrometry fragmentation, in vitro reactions, and chemical treatments. Dose-, time-, and genotype-dependent appearance of these minor APAP metabolites implied their association with the APAP-induced toxicity and potential biomarker application. Overall, the oxidative stress elicited by CYP2E1-mediated APAP metabolism might significantly contribute to APAP-induced toxicity. The combination of genetically modified animal models, mass isotopomer analysis, and metabolomics provides a powerful and efficient technical platform to characterize APAP-induced toxicity through identifying novel biomarkers and unraveling novel mechanisms. Acetaminophen (APAP) 3The abbreviations used are: APAPacetaminophenNAPQIN-acetyl-p-benzoquinone imineCys-APAP3-cysteinylacetaminophenNAC-APAP3-N-acetylcysteinylacetaminophenGS-APAP3-glutathionylacetaminophenAPAP-Gacetaminophen-O-glucuronideAPAP-Sacetaminophen-O-sulfateSAMPS-(5-acetylamino-2-hydroxyphenyl)mercaptopyruvic acidGSSGoxidized glutathioneP450cytochrome P450PCAprincipal components analysisMS2tandem mass spectrometryLC-MSliquid chromatography-mass spectrometryUPLCultra-performance liquid chromatographyTOFMStime-of-flight mass spectrometryALTalanine aminotransferaseASTaspartate aminotransferaseHRPhorseradish peroxidaseROSreactive oxygen species. 3The abbreviations used are: APAPacetaminophenNAPQIN-acetyl-p-benzoquinone imineCys-APAP3-cysteinylacetaminophenNAC-APAP3-N-acetylcysteinylacetaminophenGS-APAP3-glutathionylacetaminophenAPAP-Gacetaminophen-O-glucuronideAPAP-Sacetaminophen-O-sulfateSAMPS-(5-acetylamino-2-hydroxyphenyl)mercaptopyruvic acidGSSGoxidized glutathioneP450cytochrome P450PCAprincipal components analysisMS2tandem mass spectrometryLC-MSliquid chromatography-mass spectrometryUPLCultra-performance liquid chromatographyTOFMStime-of-flight mass spectrometryALTalanine aminotransferaseASTaspartate aminotransferaseHRPhorseradish peroxidaseROSreactive oxygen species. overdose causes acute liver and kidney failure (1Larson A.M. Polson J. Fontana R.J. Davern T.J. Lalani E. Hynan L.S. Reisch J.S. Schiodt F.V. Ostapowicz G. Shakil A.O. Lee W.M. Hepatology. 2005; 42: 1364-1372Crossref PubMed Scopus (1446) Google Scholar, 2Boutis K. Shannon M. J. Toxicol. Clin. Toxicol. 2001; 39: 441-445Crossref PubMed Scopus (57) Google Scholar). Because of its clinical importance, APAP-induced acute toxicity has become an indispensable model for studying drug-induced liver and kidney injury. Over the past 40 years, numerous efforts have been undertaken to understand the molecular mechanism of this toxicological event. Results from those studies indicated that the toxicity is initiated by P450-mediated reactions that convert APAP to the reactive electrophile, N-acetyl-p-benzoquinone imine (NAPQI), causing glutathione depletion and covalent binding (3Dahlin D.C. Miwa G.T. Lu A.Y. Nelson S.D. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 1327-1331Crossref PubMed Google Scholar). Subsequent damage to mitochondria, cell membranes, and nuclei, as well as the disruption of cell death- and survival-related signaling pathways, lead to massive necrosis and apoptosis (4Cohen S.D. Hoivik D.J. Khairallah E.A. Plaa G.L. Hewitt W.R. Second Ed. in: Toxicology of the Liver. Taylor & Francis, Washington, DC1998: 159-186Google Scholar). Besides the P450-catalyzed oxidation reactions, detoxicating reactions including sulfation, glucuronidation, and glutathione conjugation also significantly contribute to the biotransformation of APAP and NAPQI. The balance between these activation and detoxication routes can largely determine the consequences of APAP treatment. Several P450s, including CYP1A2, CYP2A6, CYP2E1, and CYP3A, have been identified as APAP-metabolizing enzymes. Among them, CYP2E1 was widely accepted as the major isoform responsible for the bioactivation of APAP based on several convincing pieces of evidence. Firstly, in vitro enzyme kinetic assays showed that purified CYP2E1 enzyme possessed low Km and high Vmax values for the formation of NAPQI and the bioactivation of APAP by liver microsomes was largely inhibited by CYP2E1 antibody (5Raucy J.L. Lasker J.M. Lieber C.S. Black M. Arch. Biochem. Biophys. 1989; 271: 270-283Crossref PubMed Scopus (461) Google Scholar, 6Patten C.J. Thomas P.E. Guy R.L. Lee M. Gonzalez F.J. Guengerich F.P. Yang C.S. Chem. Res. Toxicol. 1993; 6: 511-518Crossref PubMed Scopus (360) Google Scholar). Secondly, there is a clear link between enhanced sensitivity to APAP hepatotoxicity and chronic alcoholism, which significantly increases the CYP2E1 levels in liver (7Lieber C.S. Physiol. Rev. 1997; 77: 517-544Crossref PubMed Scopus (775) Google Scholar). Thirdly, APAP-induced hepatic necrosis mainly occurs in the centrilobular region, where CYP2E1 is highly expressed (8Hart S.G. Cartun R.W. Wyand D.S. Khairallah E.A. Cohen S.D. Fundam. Appl. Toxicol. 1995; 24: 260-274Crossref PubMed Scopus (70) Google Scholar). Finally, Cyp2e1-null mice are highly resistant to APAP, compared with wild-type mice (9Lee S.S. Buters J.T. Pineau T. Fernandez-Salguero P. Gonzalez F.J. J. Biol. Chem. 1996; 271: 12063-12067Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar), whereas reintroduction of the human CYP2E1 gene into Cyp2e1-null mice reestablished the toxic response to APAP (10Cheung C. Yu A.M. Ward J.M. Krausz K.W. Akiyama T.E. Feigenbaum L. Gonzalez F.J. Drug Metab. Dispos. 2005; 33: 449-457Crossref PubMed Scopus (138) Google Scholar). Therefore, the Cyp2e1-null mouse line is an ideal model as the negative control for investigating the mechanism and biomarkers of the APAP-induced toxicity.Metabolomics, a methodology for measuring small-molecule metabolite profiles and fluxes in biological matrices, following genetic modification or exogenous challenges, has become an important component of systems biology, complementing genomics, transcriptomics, and proteomics (11Nicholson J.K. Wilson I.D. Nat. Rev. Drug Discov. 2003; 2: 668-676Crossref PubMed Scopus (936) Google Scholar, 12Fernie A.R. Trethewey R.N. Krotzky A.J. Willmitzer L. Nat. Rev. Mol. Cell. Biol. 2004; 5: 763-769Crossref PubMed Scopus (625) Google Scholar). Combining advanced analytical instrumentation for comprehensive metabolite measurement with robust chemometric computation for detecting subtle changes in a large dataset, metabolomics techniques have been utilized to identify biomarkers and unravel pathophysiological mechanisms in many scientific fields, including xenobiotic metabolism (13Chen C. Gonzalez F.J. Idle J.R. Drug Metab. Rev. 2007; 39: 581-597Crossref PubMed Scopus (189) Google Scholar) and toxicology (14Nicholson J.K. Connelly J. Lindon J.C. Holmes E. Nat. Rev. Drug Discov. 2002; 1: 153-161Crossref PubMed Scopus (1729) Google Scholar). Recently, APAP-induced toxicity has also been investigated using metabolomics approaches. Two NMR-based studies have depicted the general changes caused by the APAP-elicited disruption of carbohydrate and lipid metabolism (15Coen M. Ruepp S.U. Lindon J.C. Nicholson J.K. Pognan F. Lenz E.M. Wilson I.D. J. Pharm. Biomed. Anal. 2004; 35: 93-105Crossref PubMed Scopus (155) Google Scholar, 16Coen M. Lenz E.M. Nicholson J.K. Wilson I.D. Pognan F. Lindon J.C. Chem. Res. Toxicol. 2003; 16: 295-303Crossref PubMed Scopus (237) Google Scholar), and a capillary electrophoresis-MS-based study identified ophthalmic acid as a general biomarker of oxidative stress following glutathione depletion (17Soga T. Baran R. Suematsu M. Ueno Y. Ikeda S. Sakurakawa T. Kakazu Y. Ishikawa T. Robert M. Nishioka T. Tomita M. J. Biol. Chem. 2006; 281: 16768-16776Abstract Full Text Full Text PDF PubMed Scopus (545) Google Scholar).To obtain more insights into the underlying mechanism of APAP-induced toxicity and the role of CYP2E1, metabolite profiling and metabolomics of APAP-treated wild-type and Cyp2e1-null mice were conducted in this study. Profiling of major known APAP metabolites as well as glutathione and hydrogen peroxide assays revealed the potential role of CYP2E1-mediated oxidative stress in APAP-induced toxicity. Metabolomic analysis resulted in the identification of four toxicity-associated APAP metabolites that should find utility in monitoring the emergence of acetaminophen toxicity.EXPERIMENTAL PROCEDURESReagents–APAP, APAP-glucuronide, p-aminophenol, α-ketoglutarate, hydrogen peroxide, horseradish peroxidase, high-performance liquid chromatography-grade water, acetonitrile, and formic acid were purchased from Sigma-Aldrich. [Acetyl-2H3]APAP and 3-methoxy-APAP were purchased from Syn-Fine Research (Richmond Hill, Ontario, Canada). 2,3,5,6-[2H4]APAP and Cys-APAP were purchased from Toronto Research Chemicals (North York, Ontario, Canada). GS-APAP and NAC-APAP standards were kindly provided by Professor Bernhard Lauterburg, University of Bern, Switzerland.Animal Treatments and Sample Collection–The Cyp2e1-null mouse line (Cyp2e1–/–) was described previously (9Lee S.S. Buters J.T. Pineau T. Fernandez-Salguero P. Gonzalez F.J. J. Biol. Chem. 1996; 271: 12063-12067Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar). Female wild-type (Cyp2e1+/+) and Cyp2e1-null mice on the 129/Sv strain background, from 2 to 3 months of age, were used in this study. All animals were maintained in an NCI, National Institutes of Health animal facility under a standard 12-h light/12-h dark cycle with food and water ad libitum. Handling and treatment procedures were in accordance with animal study protocols approved by the NCI Animal Care and Use Committee. APAP was dissolved in saline solution at doses ranging from 10 to 400 mg/kg and administered by intraperitoneal injection to mice. Control mice were treated with blank saline solution. Serum samples were collected by retro-orbital bleeding. 24-h urine samples were collected by housing mice in glass metabolic bowls (Jencons, Leighton Buzzard, UK), 1-, 2-, and 4-h urine samples were collected by combining urine in a metabolic bowel, and urine was harvested from bladder emptying during retro-orbital bleeding. After CO2 euthanization, liver and other tissue samples were harvested. All samples were stored at –80 °C before analysis.Assessment of APAP-induced Toxicity–APAP-induced liver injury was evaluated by measuring the catalytic activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in serum. Briefly, 1 μl of serum was mixed with 200 μl of AST or ALT assay buffer (Catachem, Bridgeport, CT) in a 96-well microplate, and the oxidation of NADH to NAD+ was monitored at 340 nm for 5 min.APAP-induced kidney injury was evaluated by measuring the blood urea nitrogen in serum. Briefly, 2 μl of serum was mixed with 200 μl of urea assay buffer (BioAssay Systems, Hayward, CA) in a 96-well microplate, and the reaction was monitored at 520 nm after a 30-min incubation at room temperature.LC-MS Analysis of Glutathione–The levels of GSH and oxidized glutathione (GSSG) in liver and GSH in mitochondria were measured by LC-MS/MS. Samples for total GSH and GSGS measurement were prepared by homogenizing liver in 10 volumes of 5% 5-sulfosalicylic acid. Precipitated protein was removed by centrifugation at 10,000 × g for 10 min, and supernatant was diluted by deionized water prior to LC-MS analysis. Samples for mitochondrial GSH measurement was prepared by homogenizing liver in 10 volumes of Mito buffer (0.2 mm EDTA, 0.25 m sucrose, 10 mm Tris-HCl, pH 7.8). Cytosol fraction was removed by centrifugation at 10,000 × g for 20 min, and the pellet was resuspended in Mito buffer. Nuclear fraction was removed by centrifugation at 1,000 × g for 10 min. Mitochondrial fraction was precipitated by spinning supernatant at 18,000 × g for 10 min. Mitochondrial GSH was extracted by mixing the mitochondrial pellet with 5% 5-sulfosalicylic acid. After removing protein by centrifugation, the supernatant was diluted with deionized water and transferred to a sample vial for LC-MS analysis. Samples from whole liver homogenate or mitochondrial fraction were injected into a high-performance liquid chromatography system (PerkinElmer Life Sciences) using a Synergi Polar-RP column (Phenomenex, Torrance, CA, 50 × 2.1 mm inner diameter). The flow rate through the column at ambient temperature was 0.2 ml/min with a gradient (methanol:water:acetonitrile, containing 0.1% formic acid) from 5:85:10 to 5:40:55 in a 5-min run. The column was equilibrated for 1.5 min before each injection. API 2000™ mass spectrometer (Applied Biosystems, Foster City, CA) was operated in the turbo ion spray mode with positive ion detection. The turbo ion spray temperature was maintained at 350 °C, and a voltage of 5.5 kV was applied to the sprayer needle. Nitrogen was used as the turbo ion spray and nebulizing gas. Detection and quantification were performed using the multiple reactions monitoring mode, with m/z 308.0/75.9 for GSH and m/z 613.1/355.1 for GSSG.Hydrogen Peroxide Assay–Hydrogen peroxide (H2O2) level in liver was determined by the ferrous thiocyanate assay (18Palumbo A. Astarita G. d'Ischia M. Biochem. J. 2001; 356: 105-110Crossref PubMed Google Scholar). Samples were prepared by homogenizing liver in 10 volumes of 5% 5-sulfosalicylic acid. Precipitated protein was removed by centrifugation at 10,000 × g for 10 min. The H2O2 level in supernatant was determined by measuring the absorbance at 492 nm after reacting with 3.2 mm ferrous ammonium sulfate and 180 mm potassium thiocyanate.UPLC-QTOFMS Analysis of Urine and Serum–A 5-μl aliquot of diluted urine and serum samples was injected into a Waters UPLC-QTOFMS system (Milford, MA). An Acquity UPLC™ BEH C18 column (Waters) was used to separate chemical components, including APAP and its metabolites at 35 °C. The mobile phase flow rate was 0.5 ml/min with an aqueous acetonitrile gradient containing 0.1% formic acid over a 10-min run. The QTOF Premier™ mass spectrometer was operated in the positive electrospray ionization mode. Capillary voltage and cone voltage were maintained at 3 kV and 20 V, respectively. Source temperature and desolvation temperature were set at 120 °C and 350 °C, respectively. Nitrogen was used as both cone gas (50 liters/h) and desolvation gas (600 liters/h), and argon was used as collision gas. For accurate mass measurement, the QTOFMS was calibrated with sodium formate solution (range m/z 100–1000) and monitored by the intermittent injection of the lock mass sulfadimethoxine ([M+H]+ = 311.0814 m/z) in real-time. Mass chromatograms and mass spectral data were acquired and processed by MassLynx software (Waters) in centroid format.Determination of APAP Metabolite Profile in Urine–Urine samples were collected for 24 h after intraperitoneal injection of 10, 200, and 400 mg/kg APAP to wild-type and Cyp2e1-null mice. Samples for LC-MS analysis were prepared by mixing 50 μl of urine with 200 μl of 50% aqueous acetonitrile and centrifuging at 18,000 × g for 10 min to remove protein and particulates. Supernatants were injected into the UPLC and metabolites separated by a gradient ranging from water to 95% aqueous acetonitrile containing 0.1% formic acid over a 10-min run. After data acquisition in QTOFMS, chromatograms and spectra of urine samples were processed by MetaboLynx software (Waters). APAP and its four major metabolites (Cys-APAP, NAC-APAP, APAP-G, and APAP-S) were identified through accurate mass measurement, comparison with authentic standards, and analysis of the MS2 fragmentation pattern, and their peak areas were quantified to represent the signal intensities. Urinary metabolite profiles of APAP in wild-type and Cyp2e1-null mice were compared based on relative peak area in the total peak area of APAP and its four major urinary metabolites.Quantitation of APAP and Determination of APAP Metabolite Profiles in Serum–Serum samples were collected at 1, 2, 4, and 8 h after intraperitoneal injection of 400 mg/kg APAP. One volume of serum was deproteinated by 20 volumes of 66% aqueous acetonitrile. After 10-min centrifugation at 18,000 × g, supernatant was injected into UPLC-QTOFMS for quantitation with a gradient of mobile phase ranging from water to 99% aqueous acetonitrile containing 0.1% formic acid over a 10-min run. To prepare the calibration curve, an APAP standard was mixed with blank serum and diluted 20 times by 66% aqueous acetonitrile. A linear range from 0.1 μm to 50 μm of APAP (r = 0.99) was achieved by using dynamic range enhancement function of QTOF Premier. Serum APAP concentrations were determined by the integration of peak area and fitting with a calibration curve using QuantLynx software (Waters).To examine the metabolite profile of APAP in serum of wild-type and Cyp2e1-null mice, chromatograms and spectra of 1-, 2-, and 4-h serum samples acquired by UPLC-QTOFMS were processed by MarkerLynx software (Waters). APAP and its major serum metabolites (GS-APAP, Cys-APAP, NAC-APAP, APAP-G, and APAP-S) were identified through accurate mass measurement, comparison with authentic standards, and analysis of MS2 fragmentation pattern. Relative abundances of APAP and its metabolites were determined by normalizing the single ion counts of each metabolite versus the total ion counts of each serum sample.Principal Components Analysis (PCA)–Chromatographic and spectral data of wild-type and Cyp2e1-null mice were deconvoluted by MarkerLynx software. A multivariate data matrix containing information on sample identity, ion identity (retention time and m/z) and ion abundance was generated through centroiding, deisotoping, filtering, peak recognition, and integration. The intensity of each ion was calculated by normalizing the single ion counts versus the total ion counts in the whole chromatogram. The data matrix was further exported into SIMCA-p™ software (Umetrics, Kinnelon, NJ) and transformed by mean-centering and Pareto scaling, a technique that increases the importance of low abundance ions without significant amplification of noise. Principal components were generated by PCA analysis to represent the major latent variables in the data matrix and were described in a scores scatter plot. Potential APAP metabolites were identified by analyzing ions contributing to the principal components and to the separation of sample groups in the loadings scatter plot.Mass Isotopomer Analysis of Novel APAP Metabolites and Structural Elucidation–24-h urine samples were collected after treating wild-type mice with 400 mg/kg [acetyl-2H3]APAP or 400 mg/kg 2,3,5,6-[2H4]APAP by intraperitoneal injection, and then analyzed by UPLC-QTOFMS. Data from [acetyl-2H3]APAP treatment were compared with those from unlabeled APAP treatment through a PCA-based metabolomic analysis. The identity of individual ion as APAP metabolite was validated by comparing extracted ion chromatograms from three treatments. The structure of each APAP metabolite was elucidated by tandem mass spectrometry (MS2) fragmentation with collision energy ramping from 15 to 35 eV.In Vitro Transamination Reaction of Cys-APAP–Liver and kidney were homogenized in microsomal buffer (50 mm potassium phosphate, 320 mm sucrose, 1 mm EDTA, pH 7.4) containing 100 μm pyridoxal-5-phosphate (1:10, w/v). The S9 fraction was prepared by centrifuging tissue homogenate at 9,000 × g for 10 min. The transamination reaction was conducted by incubating tissue homogenate or S9 supernatant with 5 mm α-ketoglutarate and 1 mm Cys-APAP in a phosphate-buffered saline solution at 37 °C for 30 min. The reaction was terminated by adding equal volumes of acetonitrile and the reaction products were further analyzed by LC-MS.Peroxidase-mediated Metabolism of APAP and Cys-APAP–Analytical reactions were performed according to Potter's method (19Potter D.W. Miller D.W. Hinson J.A. J. Biol. Chem. 1985; 260: 12174-12180Abstract Full Text PDF PubMed Google Scholar). Briefly, 0.1 m potassium phosphate (pH 7.4), 1 mm APAP or Cys-APAP, and 2.5 units/ml horseradish peroxidase were preincubated at 25 °C for 2 min before adding 1 mm H2O2. The reaction mix was incubated at 25 °C for 5 min. The reaction was terminated by adding equal volume of ice-cold 90% aqueous methanol containing 2 mm ascorbate. Reaction products were further analyzed by LC-MS.Statistics–Experimental values are expressed as mean ± S.D. Statistical analysis was performed with two-tailed Student's t-tests for unpaired data, and a p value of <0.05 was considered as statistically significant.RESULTSAPAP Metabolite Profiles in Wild-type and Cyp2e1-null Mice–Elevation of serum aminotransferase activities is a reliable indicator of hepatic injury (20Amacher D.E. Regul. Toxicol. Pharmacol. 1998; 27: 119-130Crossref PubMed Scopus (173) Google Scholar). 24 h after 200 mg/kg and 400 mg/kg APAP treatments, the activities of serum ALT and AST in wild-type and Cyp2e1-null mice were examined. Consistent with a previous report (9Lee S.S. Buters J.T. Pineau T. Fernandez-Salguero P. Gonzalez F.J. J. Biol. Chem. 1996; 271: 12063-12067Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar), serum ALT and AST activities in wild-type mice were dramatically increased by both treatments but their activities in Cyp2e1-null mice were unaffected by 200 mg/kg and slightly increased by 400 mg/kg treatment (Fig. 1, A and B). In addition, the effect of APAP treatment on kidney function of wild-type and Cyp2e1-null mice was determined by measuring blood urea nitrogen levels. After a 400 mg/kg treatment, the blood urea nitrogen level in wild-type mice was much higher than its level in Cyp2e1-null mice and wild-type controls. These results indicated that not only does CYP2E1 play an important role in APAP-induced hepatotoxicity but it also contributes to the APAP-induced nephrotoxicity.Major urinary metabolites of APAP (I) have been identified previously as 3-cysteinyl-APAP (Cys-APAP: II), N-acetylcysteinyl-APAP (NAC-APAP: III), APAP-O-glucuronide (APAP-G: IV), and APAP-O-sulfate (APAP-S: V) (21Bales J.R. Sadler P.J. Nicholson J.K. Timbrell J.A. Clin. Chem. 1984; 30: 1631-1636Crossref PubMed Scopus (125) Google Scholar). Although APAP-G and APAP-S are formed by direct conjugation to facilitate the elimination and excretion of APAP, Cys-APAP and NAC-APAP are the products of multiple reactions, including P450-mediated metabolic bioactivation and subsequent glutathione conjugation. Because CYP2E1 is one of the major enzymes capable of converting APAP to NAPQI, the metabolite profiles of APAP in wild-type and Cyp2e1-null mice were compared by LC-MS profiling of APAP and four major metabolites in urine. Chemical identities of urinary ions corresponding to APAP, Cys-APAP, NAC-APAP, and APAP-G were confirmed by use of authentic standards. The ion corresponding to APAP-S was identified by accurate mass measurement and analysis of the MS2 fragmentation pattern (data not shown). The urinary metabolite profile of APAP was defined by calculating the relative abundances of APAP and four major metabolites in the whole APAP metabolite cluster (22Chen C. Meng L. Ma X. Krausz K.W. Pommier Y. Idle J.R. Gonzalez F.J. J. Pharmacol. Exp. Ther. 2006; 318: 1330-1342Crossref PubMed Scopus (52) Google Scholar). Surprisingly, with the 400 mg/kg treatment, which is the LD50 for wild-type but a nontoxic dose for Cyp2e1-null mice (9Lee S.S. Buters J.T. Pineau T. Fernandez-Salguero P. Gonzalez F.J. J. Biol. Chem. 1996; 271: 12063-12067Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar), the urinary metabolite profiles of APAP in wild-type and Cyp2e1-null mice were quite comparable except for a slightly higher abundance of Cys-APAP in wild-type mice and slightly lower abundances of APAP-G and APAP-S in Cyp2e1-null mice (Fig. 2A). This marginal difference in APAP metabolism was in clear contrast to the dramatic differences in the toxic response to 400 mg/kg APAP treatment between wild-type and Cyp2e1-null mice (Fig. 1). To clarify this observation, urinary metabolite profiles of APAP in 200 mg/kg (a low toxic dose for wild-type) and 10 mg/kg (a non-toxic dose comparable to the therapeutic dose in human) treatments were further examined. Distinct from 400 mg/kg treatment, a 200 mg/kg dose led to significantly higher abundance of urinary Cys-APAP and lower abundance of APAP in wild-type mice than those in Cyp2e1-null mice (Fig. 2B). Furthermore, following 10 mg/kg APAP treatment, the differences between wild-type and Cyp2e1-null mice in APAP metabolism became even more apparent with a much higher abundance of Cys-APAP in wild-type mice and much higher abundances of APAP, APAP-G, and APAP-S in Cyp2e1-null mice (Fig. 2C).FIGURE 2Distribution of APAP and its major metabolites in urine and serum of wild-type and Cyp2e1-null mice. Metabolite profiles of APAP in 24-h urine samples of wild-type and Cyp2e1-null mice were examined by LC-MS analysis of APAP and four major metabolites (Cys-APAP, NAC-APAP, APAP-G, and APAP-S), and defined by their relative abundances (area % ± S.D.) that were calculated by normalizing individual peak area with total peak area of APAP and four metabolites (n = 8; *, p < 0.05 and **, p < 0.01). A, urinary metabolite profile of wild-type and Cyp2e1-null mice after 400 mg/kg APAP treatment. B, urinary metabolite profile after 200 mg/kg APAP treatment. C, urinary metabolite profile after 10 mg/kg APAP treatment. D, serum concentrations of APAP in wild-type and Cyp2e1-null mice at 1, 2, 4, and 8 h after 400 mg/kg APAP treatment (n = 4).View Large Image Figure ViewerDownload Hi-res image Download (PPT)To confirm the results from urinary metabolite profiling, the distribution of APAP and its major metabolites in serum during the first 8 h after 400 mg/kg treatment was examined. Comparison of APAP pharmacokinetics in wild-type and Cyp2e1-null mice indicated that significant difference in serum concentrations of APAP did not occur until 4 h of treatment (Fig. 2D). Subsequent analysis of the LC-MS signal abundances of APAP metabolites in serum revealed that at 1 h after dosing, serum metabolite profiles of wild-type and Cyp2e1-null mice were comparable while at 2 h and 4 h, higher abundances of 3-glutathionyl-APAP (GS-APAP) and Cys-APAP in wild-type mice were observed (supplemental Table S1). Overall, these results suggested that, when exposed to high concentrations of APAP, wild-type mice only have a slightly higher APAP-oxidizing capacity than Cyp2e1-null mice.APAP-induced Oxidative Stress in Wild-type and Cyp2e1-null Mice–GSH is an essential thiol antioxidant for protecting cells from oxidative stress.
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