Assessment of protein modifications in liver of rats under chronic treatment with paracetamol (acetaminophen) using two complementary mass spectrometry-based metabolomic approaches (original) (raw)
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Journal of Biological Chemistry, 2008
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 3methoxy-APAP glucuronide (VII) and three novel APAP metabolites, including S-(5-acetylamino-2hydroxyphenyl)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 MS 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 CYP2E1mediated 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 unravelling novel mechanisms.
Journal of Chromatography B, 2015
The aims of this study were to develop, validate, and apply a high-performance liquid chromatographyelectrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) method for quantification of protein-derived 3-(cystein-S-yl)-acetaminophen (APAP-Cys) in human serum. Formation of acetaminophen (APAP) protein adducts is thought to be a critical, early event in the development of APAP-induced hepatotoxicity, and quantification of these protein adducts in human serum represents a valuable tool for assessment of APAP exposure, metabolism, and toxicity. In the reported procedure, serum samples were first dialyzed or passed through gel filtration columns to remove APAP-Cys not covalently bound to proteins. Serum eluates were then subjected to enzymatic protease digestion to liberate protein-bound APAP-Cys. Norbuprenorphine-D 3 was utilized as an internal standard (IS). APAP-Cys and IS were recovered from digested serum by protein precipitation with acetonitrile, and sample extracts were analyzed by HPLC-ESI-MS/MS. The method was validated by assessment of intra-and inter-assay accuracy and imprecision on two different analytical instrument platforms. APAP-Cys could be accurately quantified from 0.010 to 10 M, and intra-and inter-assay imprecision were <15% on both analytical instruments. APAP-Cys was stable in human serum for three freeze-thaw cycles and for 24 h at ambient temperature. Extracted samples were stable when stored in refrigerated autosamplers for the typical duration of analysis or when stored at −20 • C for six days. Results from process efficiency and matrix effect experiments indicated adequate recovery from human serum and insignificant ion suppression or enhancement. The utility and sensitivity of the reported procedure were illustrated by analysis of clinical samples collected from subjects taking chronic, therapeutic doses of APAP. Applicability to other biological matrices was also demonstrated by measurement of protein-derived APAP-Cys in plasma collected from APAP-treated mice, a common animal model of APAP-induced hepatotoxicity.
Acetaminophen Induced Hepatotoxicity in Wistar Rats—A Proteomic Approach
Molecules, 2016
Understanding the mechanism of chemical toxicity, which is essential for cross-species and dose extrapolations, is a major challenge for toxicologists. Standard mechanistic studies in animals for examining the toxic and pathological changes associated with the chemical exposure have often been limited to the single end point or pathways. Toxicoproteomics represents a potential aid to the toxicologist to understand the multiple pathways involved in the mechanism of toxicity and also determine the biomarkers that are possible to predictive the toxicological response. We performed an acute toxicity study in Wistar rats with the prototype liver toxin; the acetaminophen (APAP) effects on protein profiles in the liver and its correlation with the plasma biochemical markers for liver injury were analyzed. Three separate groups-control, nontoxic (150 mg/kg) and toxic dose (1500 mg/kg) of APAP-were studied. The proteins extracted from the liver were separated by 2-DE and analyzed by MALDI-TOF. The differential proteins in the gels were analyzed by BIORAD's PDQuest software and identified by feeding the peptide mass fingerprint data to various public domain programs like Mascot and MS-Fit. The identified proteins in toxicity-induced rats were classified based on their putative protein functions, which are oxidative stress (31%), immunity (14%), neurological related (12%) and transporter proteins (2%), whereas in non-toxic dose-induced rats they were oxidative stress (9%), immunity (6%), neurological (14%) and transporter proteins (9%). It is evident that the percentages of oxidative stress and immunity-related proteins were up-regulated in toxicity-induced rats as compared with nontoxic and control rats. Some of the liver drug metabolizing and detoxifying enzymes were depleted under toxic conditions compared with non-toxic rats. Several other proteins were identified as a first step in developing an in-house rodent liver toxicoproteomics database.
The Journal of Toxicological Sciences
Toxicological responses to acetaminophen (APAP) overdose were evaluated in human hepatocytes transplanted chimeric mice using 2-dimensional gel electrophoresis (2DE)-based proteomics and (1)H-nuclear magnetic resonance (NMR)-based metabonomics. Huge variations, which were supported by histopathological findings, were observed in proteins expression in chimeric mice liver. The proteomic analysis of the livers showed that the proteins involved in the pathways of lipid/fatty acid metabolism, glycolysis and energy metabolism/production were affected. In addition, oxidative stress-related proteins showed altered expression. The metabonomic analysis of urine and plasma revealed alterations of endogenous metabolites, which were the intermediates involved in the tricarboxylic acid (TCA) cycle. Those findings were already confirmed in normal mice. We hypothesized that the mechanism of APAP-induced effects on chimeric mice liver was in accordance with the mechanism observed in normal mice. Th...
Journal of Chromatography B, 2008
Urinary metabolic perturbations associated with acute and chronic acetaminophen-induced hepatotoxicity were investigated using nuclear magnetic resonance (NMR) spectroscopy and ultra performance liquid chromatography/mass spectrometry (UPLC/MS) metabonomics approaches to determine biomarkers of hepatotoxicity. Acute and chronic doses of acetaminophen (APAP) were administered to male Sprague-Dawley rats. NMR and UPLC/MS were able to detect both drug metabolites and endogenous metabolites simultaneously. The principal component analysis (PCA) of NMR or UPLC/MS spectra showed that metabolic changes observed in both acute and chronic dosing of acetaminophen were similar. Histopathology and clinical chemistry studies were performed and correlated well with the PCA analysis and magnitude of metabolite changes. Depletion of antioxidants (e.g. ferulic acid), trigonelline, S-adenosyll-methionine, and energy-related metabolites indicated that oxidative stress was caused by acute and chronic acetaminophen administration. Similar patterns of metabolic changes in response to acute or chronic dosing suggest similar detoxification and recovery mechanisms following APAP administration.
Toxicology and Applied Pharmacology, 2013
At therapeutic doses, acetaminophen (APAP) is a safe and effective analgesic. However, overdose of APAP is the principal cause of acute liver failure in the West. Binding of the reactive metabolite of APAP (NAPQI) to proteins is thought to be the initiating event in the mechanism of hepatotoxicity. Early work suggested that APAP-protein binding could not occur without glutathione (GSH) depletion, and likely only at toxic doses. Moreover, it was found that proteinderived APAP-cysteine could only be detected in serum after the onset of liver injury. On this basis, it was recently proposed that serum APAP-cysteine could be used as diagnostic marker of APAP overdose. However, comprehensive dose-response and time course studies have not yet been done. Furthermore, the effects of co-morbidities on this parameter have not been investigated. We treated groups of mice with APAP at multiple doses and measured liver GSH and both liver and plasma APAP-protein adducts at various timepoints. Our results show that protein binding can occur without much loss of GSH. Importantly, the data confirm earlier work that showed that protein-derived APAP-cysteine can appear in plasma without liver injury. Experiments performed in vitro suggest that this may involve multiple mechanisms, including secretion of adducted proteins and diffusion of NAPQI directly into plasma. Induction of liver necrosis through ischemia-reperfusion significantly increased the plasma concentration of proteinderived APAP-cysteine after a subtoxic dose of APAP. While our data generally support the measurement of serum APAP-protein adducts in the clinic, caution is suggested in the interpretation of this parameter.
Metabolomics Analysis of Urine Samples from Children after Acetaminophen Overdose
Metabolites
Acetaminophen (APAP), a commonly used over-the-counter analgesic, accounts for approximately fifty percent of the cases of acute liver failure (ALF) in the United States due to overdose, with over half of those unintentional. Current clinical approaches for assessing APAP overdose rely on identifying the precise time of overdose and quantitating acetaminophen alanine aminotransferase (ALT) levels in peripheral blood. Novel specific and sensitive biomarkers may provide additional information regarding patient status post overdose. Previous non-clinical metabolomics studies identified potential urinary biomarkers of APAP-induced hepatotoxicity and metabolites involved pathways of tricarboxylic acid cycle, ketone metabolism, and tryptophan metabolism. In this study, biomarkers identified in the previous non-clinical study were evaluated in urine samples collected from healthy subjects (N = 6, median age 14.08 years) and overdose patients (N = 13, median age 13.91 years) as part of an IRB-approved multicenter study of APAP toxicity in children. The clinical results identified metabolites from pathways previously noted, and pathway analysis indicated analogous pathways were significantly altered in both the rats and humans after APAP overdose. The results suggest a metabolomics approach may enable the discovery of specific, translational biomarkers of drug-induced hepatotoxicity that may aid in the assessment of patients.
Metabolomic markers predictive of hepatic adaptation to therapeutic dosing of acetaminophen
Background: Drug induced liver injury (DILI) remains a prominent global issue and acetaminophen (APAP) overdose represents a common cause of hepatic injury and DILI. Transient alanine aminotransferase (ALT) elevations have been documented while adhering to recommended daily dosing. However, no metabolites have been identified in pre-treatment samples predicting which patients will develop these transient increases. Methods: This was a secondary analysis of samples collected from a parent study describing the course of ALT levels in subjects receiving therapeutic APAP dosing. Two hundred and four subjects recruited from Denver, Colorado received 4 g APAP/daily for at least 16 days. Subjects were grouped by ALT at any monitored time point above 60 units/L (n ¼ 25) vs. no increase (n ¼ 179). Serum samples from days 0, 7, 16, and 31 were run on ultra-high performance liquid chromatography mass spectrometry. We report the metabolomic results of samples analyzed prior to APAP administration and over time. Significant changes in metabolite and demographic variable expressions were explored using t-tests with false discovery rate correction, chi square, and partial least squares discriminant analyses. Results: Within pre-treatment day 0 samples, allantoate and ornithine were significantly elevated in subjects of the ALT elevation group (p ¼ .032). Baseline ALT (p ¼ .011) and alkaline phosphatase (p ¼ .006) were also significant. These metabolites were significant independent of race, ethnicity, gender, or BMI. Conclusions: Allantoate and ornithine are directly involved in pathways related to nitrogen release and urea production. Further investigation into alterations in the glutathione metabolism and urea cycle pathways may lead to a greater understanding of the mechanisms associated with hepatic adaptation for a variety of pharmaceuticals.
Drug Metabolism and Pharmacokinetics, 2011
The widely used analgesic-antipyretic drug acetaminophen (APAP) is known to cause serious liver necrosis at high doses in man and experimental animals. For studies of toxic processes, 1H NMR spectroscopy of biofluids allows monitoring of endogenous metabolite profiles that alter characteristically in response to changes in physiological status. Herein, a 1H NMR metabolomics approach was applied to the investigation of APAP toxicity in rats and the effect of phenobarbital (PB) on APAP-induced hepatotoxicity. Metabolite differences due to hepatotoxicity were observed in 1H NMR spectra of serum and urine, and enhanced APAP hepatotoxicity by pretreatment with PB was clearly shown by a principal components analysis of the spectral data. NMR spectra of APAP-dosed rat urine provided profiles of APAP-related compounds together with endogenous metabolites. By comparison of endogenous and APAP-related metabolite spectra with those from rats pretreated with PB, it was possible to show the importance of oxidative metabolism of APAP to N-acetyl-p-benzoquinone, an essential step in APAP hepatotoxicity.
PLoS ONE, 2012
Drug-induced liver injury (DILI) is the leading cause of acute liver failure. Currently, no adequate predictive biomarkers for DILI are available. This study describes a translational approach using proteomic profiling for the identification of urinary proteins related to acute liver injury induced by acetaminophen (APAP). Mice were given a single intraperitoneal dose of APAP (0-350 mg/kg bw) followed by 24 h urine collection. Doses of $275 mg/kg bw APAP resulted in hepatic centrilobular necrosis and significantly elevated plasma alanine aminotransferase (ALT) values (p,0.0001). Proteomic profiling resulted in the identification of 12 differentially excreted proteins in urine of mice with acute liver injury (p,0.001), including superoxide dismutase 1 (SOD1), carbonic anhydrase 3 (CA3) and calmodulin (CaM), as novel biomarkers for APAP-induced liver injury. Urinary levels of SOD1 and CA3 increased with rising plasma ALT levels, but urinary CaM was already present in mice treated with high dose of APAP without elevated plasma ALT levels. Importantly, we showed in human urine after APAP intoxication the presence of SOD1 and CA3, whereas both proteins were absent in control urine samples. Urinary concentrations of CaM were significantly increased and correlated well with plasma APAP concentrations (r = 0.97; p,0.0001) in human APAP intoxicants, who did not present with elevated plasma ALT levels. In conclusion, using this urinary proteomics approach we demonstrate CA3, SOD1 and, most importantly, CaM as potential human biomarkers for APAP-induced liver injury. Citation: van Swelm RPL, Laarakkers CMM, van der Kuur EC, Morava-Kozicz E, Wevers RA, et al. (2012) Identification of Novel Translational Urinary Biomarkers for Acetaminophen-Induced Acute Liver Injury Using Proteomic Profiling in Mice. PLoS ONE 7(11): e49524.